Drosophila Unfolded Protein Response (UPR) assays in vitro and in vivo

Summary/Abstract

Wild type or mutant proteins expressed beyond the capacity of a cell’s protein folding system could be detrimental to general cellular function and survival. In response to misfolded protein overload in the endoplasmic reticulum (ER), eukaryotic cells activate the Unfolded Protein Response (UPR) that helps cells restore protein homeostasis in the endoplasmic reticulum (ER). As part of the UPR, cells attenuate general mRNA translation and activate transcription factors that induce stress-responsive gene expression.

UPR signaling draws research interest in part because conditions that cause chronic protein misfolding in the ER, or those that impair UPR signaling underlie several diseases including neurodegeneration, diabetes and cancers. Model organisms are frequently employed in the field as the UPR pathways are generally well-conserved throughout phyla. Here, we introduce experimental procedures to detect UPR in Drosophila melanogaster.

1. Introduction

1.1. The three branches of UPR signaling are conserved in Drosophila.

In eukaryotic cells, ribosomes synthesize proteins in three distinct compartments: the cytoplasm, the endoplasmic reticulum (ER) and the mitochondrial matrix. Most secretory and membrane proteins are synthesized in the ER, where they undergo protein folding co-translationally. The ER contains chaperones and other proteins that assist with protein folding and maintain protein homeostasis (proteostasis) in this organelle. However, there are conditions that interfere with proper protein folding in the ER, such as the disruption of proper chaperone function or the expression of mutant proteins with impaired protein folding properties (1. 2). As these conditions impair a cell’s ability to fold other proteins with various cellular roles, they could be detrimental to overall cellular function and survival. Eukaryotic cells have thus evolved signaling pathways that change gene expression in response to the overload of unfolded or misfolded proteins, referred to as the Unfolded Protein Response (UPR).

The UPR pathways operate in diverse organisms ranging from yeast to humans, and the basic mechanisms of signaling are generally well conserved throughout evolution (3, 4, 5, 6, 7, 8, 9). In many metazoan species that include humans and Drosophila, UPR consists of three parallel signaling branches (2, 3, 9), initiated by 1) Inositol-Requiring Enzyme-1 (IRE1), 2) Pancreatic eIF-2α kinase (PERK or PEK), and 3) Activating Transcription Factor 6 (ATF6). Drosophila is now widely used for UPR research as the rich genetic resources make it a facile tool to study the physiological and pathological roles of UPR. Multiple tools have been developed to assess UPR signaling activity in vivo and in cultured Drosophila cells (10, 11, 12). Here, we describe such commonly used and readily available tools for the IRE1 and PERK pathways.

1.2. Studying UPR in vivo using Drosophila eye imaginal discs.

Our laboratory has extensively used the larval eye imaginal disc, which is the precursor tissue to the adult eye, to examine how cells respond UPR activating mutant protein expression (Fig. 1A, B) (5, 6, 11, 12). The Drosophila eye is a well-studied organ with a wide array of cell-type specific and temporal drivers, and extensive cellular markers. The added bonus of the eye being a dispensable organ allows for misexpression of potentially toxic mutant proteins without perturbing the rest of the animal’s viability (13). We have examined the expression of a number of proteins that are prone to misfolding using the eye imaginal disc model. The α1-antitrypsin NHK allele underlies α1-antitrypsin deficiency in humans, and misexpression of this misfolding-prone protein in the eye imaginal disc causes mild UPR activation (14, 15). A more robust UPR activation is observed when Rhodopsin 1 (Rh1), either wildtype or mutant (Rh1^G69D), is misexpressed in this eye imaginal disc using the GMR-GAL4 driver (Fig. 1C) (16, 17, 18, 19, 20). Rh1 is a light detecting protein normally expressed at a later developmental stage when photoreceptors differentiate to specialize in the maturation of high levels of Rh1. In contrast, larval eye discs cells are equipped to fold neither wildtype nor mutant Rh1, thereby strongly activating the UPR. Such misexpression also induces massive cell death in the developing eye, resulting in a glassy eye phenotype. This phenotype is attributable to excessive ER stress, as co-expression of Hrd1, a gene that degrades misfolded ER proteins, almost completely suppresses the eye phenotype (21). Our group and other have developed many tools to monitor activation of the UPR pathways mediated by IRE1 and PERK, which are described below.

Fig.1. The Drosophila eye imaginal disc model and available genetic tools.

(A) Schematic of Drosophila larva: eye discs are located in the anterior and are attached to larval brain, which in turn is attached to the mouth hook. Fat body is located through the anterior to the posterior.

(B) Schematic of the cell types in the eye imaginal disc. Gray shading marks the posterior region where photoreceptor precursor cells are found. This is also the region where the GMR-GAL4 driver is active.

(C) Schematic of GAL4-UAS gene expression system: Tissue specific promotors such as GMR drive the expression of a yeast transcription factor, GAL4. The gene (or reporter) of interest is placed downstream of UAS-sequences which are transcriptionally induced by GAL4.

1.3. Detecting Drosophila IRE1 activity.

IRE1 is a transmembrane endoribonuclease that becomes activated after sensing misfolded proteins in the ER. Once activated, IRE1 excises 26 nucleotides from X-box-binding protein 1 (Xbp1) mRNA, thereby causing a frame-shift in the Xbp1 coding sequence (22, 23, 24). Activated IRE1 mediates the splicing of Xbp1 mRNA, thereby causing a frame-shift in the Xbp1 coding sequence (Fig. 2A). This results in the synthesis of an active Xbp1 isoform (spliced Xbp1, Xbp1s) that induces chaperones and other ER quality control genes.

Fig. 2. Reporters for IRE1 activation.

(A) The IRE1/Xbp1 signaling pathway: Spliced Xbp1 mRNA is translated to make active transcription factor Xbp1, which acts on its own promotor to increase Xbp1 mRNA levels. This autoregulation is captured by the Xbp1P>DsRed reporter where the Xbp1 gene regulatory elements are placed upstream of DsRed.

(B) Schematic of the Xbp1-egfp HG UPR reporter. eGFP is inserted after the Xbp1 coding sequence such that when IRE1 is active and splices the Xbp1-egfp mRNA, it results in the expression of an Xbp1-eGFP fusion protein. Eye imaginal discs dissected from wandering third instar larva expressing the UAS-Xbp1-eGFP reporter and misfolding rhodopsin (UAS-Rh1^G69D) driven by GMR-GAL4. The tissues are stained for the eGFP reporter (magenta) and Rh1^G69D (green), with nuclei counterstained with DAPI (cyan).

(C) Eye imaginal discs from the wandering third instar larva showing induction of the Xbp1P>DsRed reporter (magenta) in response to UAS-Rh1^G69D (green) expression driven by GMR-GAL4. DAPI (cyan) counterstains nuclei.

We took advantage of this splicing event by developing a transgenic reporter where the sequence for enhanced Green Fluorescent Protein (eGFP) is placed downstream of the Xbp1 splice site (Fig. 2A). In unstressed cells without active IRE1, eGFP is not in frame with the preceding Xbp1 sequence. In ER stressed cells, activated IRE1 cleaves the Xbp1 splice site in the reporter mRNA, which causes the eGFP coding sequence to become in frame with that of Xbp1, resulting in eGFP expression (Fig. 2A). We have generated two versions of the Xbp1-eGFP reporter: The first one has the eGFP sequence placed almost immediately downstream of the splice site, and does not include the entire Xbp1 sequence (18); In the second reporter (Xbp1-eGFP HG), the eGFP is inserted after the entire Xbp1 coding sequence (Fig. 2A) (5). We find that the latter transgenic reporter is more sensitive to IRE1 activation, indicating that sequences outside the splice site may also contribute to Xbp1 mRNA splicing efficiency. To aid robust expression of the reporter mRNA and to allow for maximal flexibility in tissue-specific expression, the Xbp1-eGFP HG reporter was placed into the pUAST backbone that allows expression through a GAL4 driver (Fig. 2A). In the eye imaginal disc, this is accomplished by using GMR-GAL4. In this genetic background, misexpression of mutant rhodopsin using UAS-Rh1^G69D results in specific eGFP signal indicative of IRE1 activation and Xbp1 splicing (Fig. 2B).

The XBP1 transcription factor turns on the expression of multiple stress response genes, but notably also regulates itself (Fig. 2A) (23, 25). The increase in Xbp1 mRNA in response to IRE1 activation can be measured in vivo the Xbp1P>DsRed transgenic reporter (26). This reporter was constructed by placing DsRed downstream of regulatory sequence upstream of the Xbp1 coding sequence, including part of the adjacent gene CG9406 (26). The expression pattern of this reporter during early development is consistent with Xbp1 in situ hybridization data (18, 26). This reporter is reliably induced in response to Rh1^G69D in the eye imaginal discs, similar to Xbp1-eGFP HG described above (Fig. 2C).

In addition to fluorescent protein-based reporters described above, transcriptional changes downstream of IRE1 activation can also be measured by qRT-PCR using primers to detect spliced Xbp1 or total Xbp1. This method of measuring IRE1 activation is more precisely quantitative, but also relies on high quality mRNA preparations from small tissues. Sample limitations can of course be overcome by either scaling up where possible, or utilizing Drosophila cultured cells.

1.4. Detection of the PERK branch of UPR.

PERK is a transmembrane kinase that senses misfolded proteins in the ER. Upon activation by ER stress, PERK phosphorylates eukaryotic translation initiation factor 2 subunit-α (eIF2α) (27, 28) (Fig. 3A). Such phosphorylation reduces the availability of initiator methionyl-tRNA to ribosomes, thereby attenuating overall cellular translation and reducing the burden on the protein folding system of stressed cells. Phosphorylation of eIF2α at Ser51 by PERK can be detected in Drosophila tissues and cultured cells using a phospho-specific antibody both by immunostaining and western blotting. To enhance confidence in our immunostaining results with phosphor-eIF2α antibody, we use FLP-FRT mosaic clonal analysis to generate patches of tissues that lack eIF2α kinase of interest. Figure 3B shows such analysis of PERK mutant clones (marked negatively with DsRed) in eye imaginal discs misexpressing Rh1^G69D.

Fig.3. The PERK/ATF4 pathway and available reporters.

(A) Schematic of the PERK/ATF4 signaling cascade.

(B) FLP/FRT mediated clonal analysis of wildtype (top panel) or Perk mutants (Perk^e01744, bottom panel) marked by the absence of DsRed (yellow). Wandering third instar larva bearing these mutant clones and expressing Rh1^G69D (green) driven by GMR-GAL4 show P-eIF2α staining (magenta). Note that the Perk^e01744 mutant clone cells (outlined by white dashed line, see notes 4.3) do not label with anti-phospho eIF2α antibody. DAPI stains cell nucleus (cyan).

(C) In response to eIF2α phosphorylation, the expression of ATF4 and its downstream genes occur, which could be visualized through ATF4 (crc)>DsRed (UPR source is tunicamycin). DsRed (magenta) is regulated by ATF4 (crc) 5’ leader, which contains multiple upstream Open Reading Frames (uORF). These regulatory uORFs allow the main ORF to increase translation in response to eIF2α phosphorylation. DAPI stains cell nucleus (cyan).

(D, E) Thor>lacZ lines (D: UPR source is Rh1^G69D) and 4EBPⁱ-DsRed (E: UPR source is tunicamycin). (D) lacZ (magenta) is inserted at 5’ UTR region (3,478,408~3,478,615). (E) DsRed (magenta) coding sequence is combined with Thor (4EBP) intron where includes ATF4 binding site. DAPI stains cell nucleus (cyan).

While most mRNA translation is reduced upon PERK activation, there are a subset of mRNAs whose translation paradoxically increases under these conditions (29, 30). The best studied among such mRNAs is that of Activating Transcription Factor 4 (ATF4), also referred to as cryptocephal (crc) in Drosophila due to the phenotypic appearance of fly ATF4 mutants (Fig. 3A). Translational induction of the ATF4 mRNA relies on upstream Open Reading Frames (uORFs) in ATF4’s leader (5’UTR) sequence (29, 30). To monitor such translational regulation, we utilize the ATF4-5’UTR-DsRed reporter (31), which is constructed by placing the 5’UTR of ATF4 upstream of the DsRed reporter. To ensure ubiquitous expression, the reporter is driven by a Tubulin promoter. Similar to other UPR reporters described above, ATF4-5’UTR-DsRed responds (though quite weakly in our hands) to misexpression of Rh1^G69D (31). Though DsRed expression is seen throughout development in many tissues (31), we typically use this reporter in the Drosophila third instar larval fat body where we see very high expression in response to Tunicamycin feeding (Fig. 3C).

ATF4 targets multiple stress response genes including protein folding chaperones that help alleviate stress imposed by misfolded proteins (32). Our group and others have extensively characterized a highly induced ATF4 transcriptional target, 4E-BP (eIF4E-Binding Protein) or Drosophila Thor, which itself regulates mRNA translation during UPR and other conditions (11, 33, 34, 35, 36). ATF4-mediated regulation of Thor transcription is attributed to four ATF4 binding sites in the Thor intron (11). ATF4 transcriptional activity can be monitored using the Thor-lacZ reporter which relies on a P-element insertion in the Thor locus, thereby acting as an enhancer-trap reporter (37). LacZ expression is robustly induced in response to Rh1^G69D misexpression in eye imaginal discs (Fig. 3D) (11). It is important to note that in addition to transcriptional regulation by ATF4, Thor is extensively regulated by the transcription factor FoxO (38, 39). To distinguish the effects of ATF4 regulation versus regulation by FoxO, we generated a 4E-BPⁱ-DsRed, which as the name suggests utilizes the 4E-BP intronic sequence upstream of DsRed (11). Similar to the ATF4-5’UTR’-DsRed reporter described above, this reporter is expressed in high levels in the fat body and its’ expression increases in response to Tunicamycin feeding (Fig. 3E). However, unlike the ATF4-5’UTR-DsRed reporter which requires immunostaining to be visualized, 4E-BPⁱ-DsRed expression is readily visible under a fluorescent microscope (11). Mutating the ATF4 binding sites in 4E-BPⁱ-DsRed results in substantial loss of DsRed expression in fat tissues, affirming the specificity of this reporter as a readout of ATF4 transcriptional activity (11). Despite its effective use in studying ATF4-specific 4E-BP regulation in the fat body, a major drawback of the 4E-BPⁱ-DsRed reporter is that it is not detectably induced in eye imaginal discs in response to ER stress. Similar to Xbp1, ATF4 transcriptional activity can also be measured by qRT-PCR against Thor.

1.5. Studying UPR in Drosophila cultured cells.

While in vivo experiments yield the most accurate picture of cellular events, they may present technical challenges. In these situations, Drosophila cultured cells become a viable alternative, where UPR can be activated using pharmacological or molecular biology methods. A variety of Drosophila cell lines are available for study including S2, Kc167, L1, etc., and a comprehensive list can be found here: https://dgrc.bio.indiana.edu/cells/Catalog. Significant advantages offered by cultured cells over in vivo experiments include maximal control over growth conditions and ease of scalability.

Schneider 2 (S2) cells are one of the most popularly used Drosophila cell lines resembling a macrophage-like lineage but are actually derived from a primary culture of Drosophila embryonic cells. The advent of CRISPR-Cas9 gene editing has opened up a vast array of possibilities for altering target gene expression in S2 cells (40). However, classical methods of gene depletion by transfecting/bathing dsRNA can be similarly effective and useful. Similar to cultured mammalian cells, UPR can be induced in S2 cells by treating with ER-stress inducing chemicals such as Tunicamycin or Thapsigargin (41). Tunicamycin, a drug derived from Streptomyces lysosuperificus, blocks protein N-linked glycosylation by inhibiting the UDP-HexNAc: polyprenol-P HexNAc-1-P family enzymes, causing an increase in mis- or unfolded proteins in the ER. Thapsigargin works by specifically blocking the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA), which uptakes Ca²⁺ into ER lumen. Inhibition of SERCA results in reduced activity of Ca²⁺-dependent chaperones required for proper protein folding, thus causing ER stress and inducing UPR. It is worth noting incubation of dissected tissues in S2 cell culture media containing ER stress-inducing chemicals in lieu of misexpression of misfolding proteins also results in the activation of several UPR reporters described above.

The most common method of detecting UPR activation in S2 cells is using qRT-PCR to detect spliced or total Xbp1 and 4E-BP. However, plasmids for the fluorescent reporters described above can also be transfected into S2 cells and visualized by immunostaining or western blotting post-lysis. In addition to the above reporters, wildtype and uORF mutant ATF4-5’UTR-Firefly-luciferase reporters (ATF4-5’UTR-Fluc, Fig. 3A) allow for precise quantification of translation regulation events at the ATF4 5’UTR (31).

1.6. Drosophila ATF6.

ATF6 encodes a basic leucine zipper (bZIP) transcription factor that is tethered to the ER membrane under homeostasis (42). In response to ER stress, ATF6 translocates to the Golgi apparatus where it is proteolytically cleaved, resulting in its translocation to the nucleus where ATF6 induces gene expression (43, 44). While Drosophila encodes an ATF6 homolog (8, 45), the physiological role of this gene in mediating UPR has not been characterized in detail.

2. Materials.

2.1. Fly stocks (see Table 1 for details)

Table 1:

Fly stocks used for the protocols.

Stock name	Chromosome	Reference	Stock numer
GMR-GAL4	2		BDSC 9146
UAS-Rh1G69D	2	36
UAS-xbp1(fl)-eGFP		20
xbp1P>DsRed		41
ATF4-5’UTR-DsRed	2	44
Thor-lacZ	2	50	BDSC 9558
4E-BPi-DsRed	2, 3	25
4E-BPi-mut-DsRed	2, 3	25

1. GAL4 driver expressed in the desired tissue type, e.g. GMR-GAL4 for eye imaginal discs

2. UAS-Rh1^G69D

3. UPR reporters: UAS-Xbp1-eGFP HG, Xbp1P>DsRed, ATF4-5’UTR-DsRed, Thor-lacZ

2.2. Reagents for UPR detection through immunohistochemistry

1. Standard fly media vials

2. Temperature-controlled incubator

3. Phosphate buffered saline (PBS)

4. Tunicamycin (1 mg/ml stock)

5. Dry yeast (active)

6. 35 mm tissue culture dishes

7. Watch glass or silicon dish for dissection

8. Two dissection forceps (size D)

9. Fixative: 4 % paraformaldehyde, 1X PBS (pH 7.3~7.8)

10. Sterilized microcentrifuge tubes

11. PBT: 0.2 % TritonX-100, PBS (see notes 4.1)

12. Mounting media: 50 % glycerol or Vectashield (Vector laboratories) or similar

13. Slide glass, coverslips

14. Sealing agent: nail polish or similar

2.3. Reagents for UPR detection through RT-PCR

1. Dissected eye imaginal discs ~20 pairs

2. RNAse-free microfuge tubes

3. RNAse-free reagents

4. TRIzol (Life Technologies) or other acidified phenol solution

5. Chloroform

6. Isopropanol

7. 70 % ethanol (prepared fresh)

8. Water

9. Reverse transcriptase and buffer: Thermo Maxima H Minus (Thermo Fisher Scientific)

10. dNTPs

11. Random hexamers (Thermo Fisher Scientific)

12. 96-well plate and sealing film compatible with available qRT-PCR machine

13. Power SYBR Green PCR Master Mix

14. 5 μM equimolar mix of forward and reverse gene specific primers (see Table 3 for list).

Table 3:

q-RT-PCR primers used for the protocols.

Target gene	Forward primer	Reverse primer	Reference
Xbp1 (unspliced)	CCG AAT TCA AGC AGC AAC AGC	TAG TCT AGA CAG AGG GCC ACA ATT TCC AG	58
Xbp1 (total)	ATA CGC ATC CTC GTC GAA CAT GGA TGA	TCA TCT AGA AAA ACT CAG ATC AAA CTG	58
4E-BP	TAA GAT GTC CGC TTC ACC CA	CGT AGA TAA GTT TGG TGC CTC C	49

2.4. Reagents of S2 cell cultures.

1. Schneider 2 (S2) cells (Thermo Fisher Scientific)

2. Schneider’s medium (Thermo Fisher Scientific) supplemented with 10 % fetal bovine serum and 1 % penicillin/streptomycin.

3. 25 °C humid incubator without CO₂

4. Sterile 96-well culture dish, serological pipettes, microfuge tubes

5. Hemocytometer or other cell counter

6. Plasmids encoding UPR reporter plasmids (Table 4)

7. Custom designed dsRNA: more details here: https://fgr.hms.harvard.edu/dsrna-synthesis

8. Transfection reagent: Effectene (Qiagen)

9. S2 cell lysis buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 % Nonidet P-40, pH 7.8, protease inhibitor cocktail

10. TBST: 1x TBS, 0.5 % Tween20

11. Dual-Glo Luciferase assay system (Promega)

Table 4:

Reporter plasmids used for the protocols.

Reporter name	Plasmid backbone	Reference
UAS-xbp1(fl)-eGFP	pUAST	20
xbp1P>DsRed	pPelican	41
ATF4-5’UTR-DsRed	pCasper4	44
ATF4-5’UTR-Fluc	pGL3	44
Rluc	pRL	44
4E-BPi-DsRed	pRed-Hstinger	25
4E-BPi-mut-DsRed	pRed-Hstinger	25

3. Methods.

3.1. Detection of UPR reporters through immunohistochemistry.

1. For experiments in the eye imaginal disc, prepare necessary stocks to generate flies containing GMR-GAL4, UAS-Rh1^G69D, and the UPR reporter of choice (see notes 4.2, 4.3). Incubate crosses at 25 °C for 5 days to allow development to the wandering third instar larval stage.

2. For experiments in the fat body, prepare necessary stocks containing the UPR reporter of choice (see notes 4.4) and incubate at 25 °C for 4 days to allow development to early third instar larval stage. Prepare yeast paste with a final concentration of 10 μg/ml of Tunicamycin or DMSO as a control. Spread the yeast paste on a sterile 35 mm dish and allow larvae to feed on the drug-infused food for 6 h prior to dissection.

3. Place wandering third instar larva in a watch glass containing PBS and dissect using forceps to isolate eye imaginal discs following the schematic in Figure 4. Place dissected tissues immediately in PBS-containing microfuge tubes on ice (see notes 4.5) (46).

4. Incubate samples in at least enough fixative to cover dissected tissues for 20 min at room temperature. All incubation steps hereon requires gentle agitation.

5. Wash samples with 1 ml PBT three times, for 10 min each with gentle agitation.

6. Incubate samples in primary antibody solution diluted in PBT for 2 h at room temperature. Alternatively, samples can be incubated at 4 °C overnight. See Table 2 for details on antibody catalog numbers and dilution ranges. (see notes 4.6)

7. Wash samples with 1 ml PBT three times, 10 min each.

8. Incubate with fluorophore-conjugated secondary antibodies diluted in PBT for 1 h at room temperature. From this step onward, ensure the samples are covered when possible to avoid photobleaching of fluorophores.

9. Wash samples with 1 ml PBT three times, 10 min each.

10. If you need to visualize the nucleus, incubate with final 600 nM DAPI diluted in PBT for 20 min.

11. Wash samples with 1 ml PBT three times, 10 min each. Remove as much of wash buffer as possible after the last wash, and add 20-30 μl of mounting medium.

12. Transfer tissue samples to glass slide with the mounting medium. Use forceps to remove excess carcass and separate the eye discs from the brain. Using forceps to guide the cover slip, carefully place cover slip over the sample. Seal with nail polish and allow to dry for 15 min at room temperature before visualizing on microscope. Slides can also be stored at 4 °C for several days or at −20 °C for weeks. Please be advised that fluorophore strength weakens with time, so timely visualization is recommended.

Table 2:

Antibodies used for the protocols.

Antibody	Source	Catalog #	Host species	Immunostaining dilution	Western blotting dilution
Phospho-eIF2a	Cell Signaling technologies	9721	Rabbit	1/100	1/1000
GFP	Aves	GFP-1010	Chicken	1/500	-
	Thermo Fisher Scientific	A6455	Rabbit	1/500	1/2000
	Thermo Fisher Scientific	A-11120	Mouse	1/500	1/2000
DsRed	Thermo Fisher Scientific	R10367	Rabbit	1/500	1/2000
lacZ	Developmental studies hybridoma bank	40-1a	Mouse	1/500	-
Rhodopsin	Developmental studies hybridoma bank	4C5	Mouse	1/500	1/5000

3.2. Detection of UPR through RT-PCR.

1. Remove as much of the PBS as possible from the microfuge tube without perturbing the dissected tissues. Add 200 μl of TRIzol and pipette up and down several times to ensure tissue homogenization. If using tissues tougher than eye imaginal discs, a dounce homogenizer might be required to ensure complete homogenization prior to adding TRIzol.

2. Follow the manufacturer’s protocol to isolate total RNA. Resuspend RNA pellet in 20 μl of RNAse-free water and measure the concentration. It is best to proceed with cDNA prep immediately, but RNA can be stored at −80 °C for up to a month.

3.3. cDNA preparation.

1. Use 500-1500 ng of total RNA and 50 ng random hexamers in a reverse transcription reaction according to the reverse transcriptase manufacturer’s protocol.

2. Measure concentration of cDNA and dilute to normalize across samples as necessary.

3. For a 20 μl reaction of RT-PCR, mix 0.5 μg of cDNA, 2 μl of 5 μM primer mix and 10 μl of 2X SYBR green master mix in each well of a 96-well plate. Ideally, triplicate measurements for each sample and each primer are desired. Optimization of template amounts using serial dilution may be required depending on type and quality of starting tissue material.

4. Seal plate, ensure complete mixing of reagents by tapping or vortexing, and resettle reaction mixture by brief centrifugation.

5. Proceed to qRT-PCR reaction set up. All primers in Table 3 are designed to work at 60°C annealing temperature with 2-step amplification.

3.4. Detection of UPR in cultured S2 cells

1. General S2 cell maintenance and propagation can be done as per manufacturer’s instructions.

2. Transfecting UPR plasmids with dsRNA

   1. Using the hemocytometer, count and seed 40 μl of 1x10⁶ cells/ml per well of a 96 well plate.
   2. Add 600 ng dsRNA and incubate for 1 h at 25 °C.
   3. Add 80 μl of fresh media and incubate for 48 h at 25 °C.
   4. Co-transfect UPR reporter plasmid and dsRNA using effectene as per manufacturer’s instructions with the following modifications: 0.2 μg plasmid, 0.1 μg dsRNA, 24 μl EC buffer, 2.6 μl enhancer, 0.8 μl effectene. (see notes 4.7)
   5. 48 h post effectene-transfection, resuspend the cells in the conditioned medium and divide them across two wells. One will serve as a control while the other can be treated with stress-inducing chemicals.
   6. 24 h after splitting cells, treat cells with 5 mM 1,4-Dithiothreitol (DTT) or 2 μM thapsigargin or 10 μg/ml tunicamycin for 4-8 h to trigger UPR.
3. qRT-PCR for measuring induction of UPR target genes (see notes 4.8)

   1. After drug treatment, collect cells by pipetting or scraping in an RNAse-free microfuge tube.
   2. Pellet cells by centrifuging at 1000 g, 3 min and remove growth medium.
   3. Follow instructions in section 6. Ideal starting material for RNA preparation is 2x10⁵ cells, so transfection may need to be scaled as necessary.
4. Western blotting for detection of UPR reporters

   1. Wash S2 cell pellets collected as described above for qRT-PCR twice with ice cold PBS.
   2. Lyse in 50 μl ice-cold lysis buffer for each 0.5x10⁶ cells. Incubate on ice for 30 min, with intermittent vortexing every 5 min.
   3. Remove debris from lysate by centrifuging at 4°C at 12000 g for 10 min. Transfer lysate to clean microfuge tube and add SDS sample buffer.
   4. Perform SDS-PAGE and Western blotting.
   5. Block membrane in 5 % non-fat dry milk in TBST for 1 h at room temperature. All incubation steps henceforth must be done with agitation on a rocker/nutator.
   6. Incubate membrane with primary antibody diluted in TBST for 2 h at room temperature or 4 °C overnight (see Table 2 for antibody details and dilution range).
   7. Wash membrane thrice with TBST, 10 min each.
   8. Incubate membrane with HRP-conjugated secondary antibody diluted in TBST for 1 h at room temperature (see Table 2 for antibody details and dilution range)
   9. Wash membrane thrice with TBST, 10 min each. Rinse with distilled water and proceed to chemiluminescence-visualization.

5. Luciferase activity detection

Using ATF4-5’UTR-Fluc to measure translation regulation by PERK necessitates the equimolar cotransfection of a control Renilla luciferase (Rluc) plasmid (see Table 2). Post-drug treatment, measure firefly and Renilla luciferase activity by following the Dual-Glo luciferase assay manufacturer’s protocol.

4. Notes

1. For immunostaining of fat bodies, we prefer using PBST prepared with Tween-20 as opposed to TritonX-100 since it is a milder detergent and preserves tissue integrity better.

2. GMR-GAL4.UAS-Rh1^G69D/CyO recombinant flies are not viable at 25 °C or room temperature and hence our laboratory maintains this stock in the 20 °C incubator.

3. For genes that are required during development, it may not be feasible to look at UPR activation in a whole fly mutant. An alternative to this is performing mosaic clonal analysis using the FLP/FRT system. This system relies on mitotic recombination directed by an enzyme (flippase or FLP) at specific sites (FRT) to generate patches of mutant cells that are negatively marked by a fluorescent reporter. An example is shown in Figure 3B where Perk mutant clones (Perk^e01744) negatively marked by DsRed are generated using eyeless-FLP (ey-FLP). See (47) for review of this method.

4. Though this protocol describes the use of ATF5-5’UTR-DsRed and 4E-BPⁱ-DsRed in the fat body, all UPR reporters described in Table 1 are expressed at detectable levels in this tissue.

5. When immunostaining for Phospho-eIF2α, signal strength can be greatly improved by dissecting and fixing in PBS containing phosphatase inhibitors (final 50 μM sodium fluoride, 100 μM sodium orthovanadate and 100 μM beta-glycerophosphate). The inhibitors can be dispensed with post-fixation.

6. All fluorescent reporters described in these methods require antibody staining for their detection with the exception of 4E-BPⁱ-DsRed in the fat body. However, this may vary with tissue type and it is recommended to test both with and without antibody for the desired application.

7. There are many protocols available for DNA/dsRNA transfections. We have found that double dsRNA treatments result in the most efficient knockdowns.

8. If you want to measure the IRE1 activity instantly, you can check the unspliced and the spliced Xbp1 mRNA levels by Pst1 digestion method. Unspliced Xbp1 has the Pst1 cleavage site in the IRE1 splice site, but spliced Xbp1 mRNA do not. Therefore, it is possible to distinguish the spliced and unspliced Xbp1 ratio by digested band size (48).