PKR as a dsRNA sensor in antiviral innate immunity

Abstract

Protein kinase R (PKR), a key double-stranded RNA (dsRNA)-activated sensor, is pivotal for cellular responses to diverse stimuli. This protocol delineates a comprehensive methodological framework employing single luciferase assays, yeast assays, immunoblot assays, and quantitative PCR (qPCR) to discern and validate PKR activities and their downstream impacts on NF-κB-activating signaling pathways. These methodologies furnish a systematic approach to unraveling the role of PKR as a dsRNA sensor and effector in antiviral innate immunity, enabling in-depth analyses of dsRNA sensor activities.

Introduction

Protein kinase R (PKR) is a pivotal dsRNA sensor that is crucially involved in the innate immune response against viral infections. PKR consists of two N-terminal dsRNA-binding domains (dsRBDs), a linker region, and a C-terminal catalytic kinase domain, which allow it to function both as an intracellular sensor for dsRNA and as an effector serine/threonine protein kinase [1,2]. Upon encountering dsRNA, PKR undergoes a series of activation steps. Initially, it recognizes dsRNA through its dsRBDs, leading to dimerization and subsequent autophosphorylation. Once activated, PKR phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), thereby inhibiting cap-dependent translation initiation. This inhibition impedes viral replication and facilitates cellular defense mechanisms [3,4]. Additionally, PKR is implicated in various stress-induced signaling pathways, including interferon (IFN) responses and NF-κB-dependent inflammatory responses [5]. Despite its antiviral functions, PKR activation can be counteracted by several inhibitors through diverse molecular mechanisms, including those utilized by viral products [6,7].

In this Methods section, we detail the methodologies employed to investigate PKR function and activity. First, we performed a single luciferase assay, which quantifies relative translation by detecting luciferase expression as a proxy. Transfection of PKR alone resulted in a reduction in luciferase expression, indicative of PKR activation-mediated inhibition of protein translation. Conversely, co-transfection of PKR with a plasmid encoding an inhibitor led to increase luciferase expression, with higher values corresponding to greater inhibition of PKR activity by the inhibitor (Fig. 1). Furthermore, we describe a yeast assay, in which the toxic effect of PKR expression on yeast growth serves as an indicator of its activity. Poxvirus infections, including the Myxoma virus (MYXV) examined in this study, triggered PKR activation, as evidenced by the phosphorylation of PKR and its downstream target eIF2α through western blot analysis (Fig. 2). To verify the downstream effects of PKR activation on NF-κB proinflammatory signaling pathway, we employed western blotting to quantify key regulators of NF-κB pathways, including IκBα and NF-κB p65 subunit (Fig. 2) [8]. Additionally, quantitative (q)RT‒PCR was performed to detect elicited pro-inflammatory gene responses following PKR transfection, including Interleukin-6 (IL-6) and Tumor Necrosis Factor alpha (TNFα) (Fig. 3) [9]. The procedures for each method are described in this section. Moreover, these methodologies can be adapted and optimized for testing PKR inhibitors, including viral antagonists such as K3L orthologs from poxviruses [7], or for identifying potential effectors or essential signaling pathway components involved in PKR-activated NF-κB mediated proinflammatory signaling pathways [10].

Fig 1. Assessment of Protein Translation Inhibition Induced by PKR Activation Using Firefly Luciferase Reporter.

(a) The pGL3 plasmid serves as the reporter, and the pSG5 vector expresses either PKR or inhibitor. HeLa cells with knocked out (KO) PKR (HeLa-PKR^KO) were utilized for transient transfection assays. (b) Upon transfection with the pGL3 firefly luciferase reporter plasmid and pSG5 empty vector, luciferase activity is measured and normalized (set as 1). Transfection with the PKR-expressing plasmid results in a significant reduction in luciferase activity, indicative of PKR activation-induced translation inhibition. Co-transfection of PKR with a plasmid encoding an inhibitor counteracts PKR-mediated reduction of luciferase, assessed 48 h post-transfection. Refer to the Methods section for a detailed procedure.

Fig 2. DsRNA Activated PKR-mediated eIF2α Pathway and NF-κB Activation during MYXV Infection.

A549 wild-type (A549-WT), A549 PKR knockout (A549-PKR^KO), and HeLa PKR knockout (Hela-PKR^KO) were infected with MYXVΔM029LΔM156R, which lack two known PKR inhibitors, M029 and M156, at an MOI of 3 for 8 h. Total protein lysates were harvested and subjected to Western blot analysis for phosphorylated PKR (P-PKR), phosphorylated eIF2α (P-eIF2α), and key components of the pro-inflammatory pathway NF-κB p65 and IκBα, as described in the Methods section. Mock-infected control lysates (−), collected at 8 h, were included for comparison.

Fig 3. Modulation of NF-κB Activity by PKR Activation.

HeLa cells with knocked out (KO) PKR (HeLa-PKR^KO) were transiently transfected with PKR from European rabbit (E.rabbit PKR) expression plasmid. Total RNA was isolated from the transfected cells 24 h post-transfection and subjected to Quantitative Real-Time PCR (qRT-PCR) analysis for TNFα (a) and IL-6 (b), following the detailed procedure outlined in the Methods section. These proinflammatory genes serve as established markers of NF-κB activity. Error bars represent standard deviations (SDs).

1. Single-Luciferase Assay for PKR Activity

1.1. Materials

Cell Culture Materials

• HeLa-PKR knockout (KO) cells.

• Gibco DMEM, High Glucose, GlutaMAX Supplement (Gibco)

• Fetal bovine serum (FBS), (GE Healthcare)

• Penicillin-Streptomycin (10,000 U/mL) (Gibco)

• Phosphate Buffer Saline (PBS-D) (1x) (Thermo Fisher Scientific)

• Trypsin-EDTA (0.05 %), phenol red (Thermo Fisher Scientific)

Plasmid DNA

• pGL3 plasmid (firefly luciferase reporter, Promega) (50 ng/μL)

• pSG5 empty vector (control) (200 ng/μL)

• pSG5 plasmid encoding PKR (200 ng/μL)

• pSG5 plasmid encoding PKR inhibitor (200 ng/μL)

Transfection reagents

• GenJet-In Vitro DNA Transfection Reagent (SignaGen Laboratories)

Cell Lysis Buffer

• Mammalian Cell Lysis Buffer (GoldBio)

Luciferase Assay Kits

• Single luciferase assay kits (Promega)

Equipment:

• Microtubes (1.5 mL)

• 15 mL centrifuge tubes

• 50 mL centrifuge tubes

• White opaque 96 well plate for Glomax ^® Plate Reader

• Centrifuge (Microtube and 6 °C capable)

• Biosafety cabinet

• Ice bucket

• Pipettes/Pipette Tips (10 μL, 200 μL, 1000 μL)

• Glomax ^® Plate Reader (Promega)

• Counting chamber slides (Invitrogen)

• Countess II automated cell counter (Invitrogen)

• Aspirator

• Plate rocker

1.2. Methods

Plan out your plate, for each experimental group prepare a set of triplicates (3 wells of the same experiment) and prepare a negative control set (transfection with luciferase (50 ng) + empty vector pSG5 (400 ng)) and PKR control set (transfection with luciferase (50 ng) + PKR encoding plasmid (200 ng) + empty vector pSG5 (200 ng)).

1.2.1. Cell Seeding

1. Remove media, wash the cells with PBS and add 1 mL of trypsin to the plate and incubate for 5 min to detach cells.

2. Resuspend cells in 10 mL DMEM (5 % FBS/Antibiotics).

3. Count cells and calculate the number of cells needed for the experiment (50,000 cells/well; 500 μL/well). Dilute the cell suspension accordingly and pipette 500 μL into each well.

4. Shake the plate gently to distribute cells evenly.

5. Incubate plates at 37 °C with 5 % CO₂ overnight (24 h).

1.2.2. Plasmid Transfection

1. Prepare and label 1.5 mL tubes, one tube per each triplicate.

2. For a one-to-one ratio of PKR to inhibitor, pipette inhibitor at a concentration of 200 ng/μL, PKR at 200 ng/μL, and empty vector pSG5 200 ng/μL to appropriate tubes. The amount pipetted should be x3.30 (to account for potential pipetting errors).

   • For a negative control contains 6.60 μL empty vector (pSG5)
   • For PKR control contains 3.30 μL PKR and 3.30 μL empty vector (pSG5)
   • For an experimental triplicate contains 3.30 μL PKR and 3.30 μL Inhibitor.
3. Adjust the volume to 65 μL with serum free DMEM.

   	PKR (200 ng/μL)	PKR inhibitor (200 ng/μL)	pSG5 (200 ng/μL)	Serum-free DMEM	pGL3 (50 ng/reaction)	Genjet (2 μL/1 μg DNA)	Total volume
   Negative control	0 μL	0 μL	6.6 μL	58.4 μL	100 μL	165 μL	330 μL
   PKR control	3.3 μL	0 μL	3.3 μL	58.4 μL	100 μL	165 μL	330 μL
   Experimental PKR + inhibitor	3.3 μL	3.3 μL	0 μL	58.4 μL	100 μL	165 μL	330 μL

4. Dilute the luciferase plasmid in serum free DMEM (luciferase plasmid 165 ng/per 3.3 reactions) adjust the volume to 100 μL with serum free DMEM. Vortex briefly. Add 100 μL of diluted pGL3 luciferase reporter plasmid into each tube. Each tube will have a total volume of 165 μL after addition of luciferase. Vortex briefly.

5. Dilute Genjet transfection reagent (2 μl per 1μg plasmid DNA) in serum free DMEM. Adjust the volume to 165 μL/3.3 reactions with DMEM.

6. Add 165 μL of Genjet transfection reagent into each tube of diluted DNA (Important: do not mix the solutions in the reverse order).

7. Vortex each tube quickly three times.

8. Incubate the samples at room temperature for 15 min.

9. Add 100 μL dropwise into each appropriate well of the 24-well plate. Gently rock the plates after mixing.

10. Incubate at 37 °C with 5 % CO₂ for 48 h.

1.2.3. Cell Lysis and Luciferase Assay

1. Remove the media gently from each well using an aspirator to avoid sucking up cells.

2. Wash each well with 500 μL PBS.

3. Add 220 μL of mammalian lysis buffer into each well and rock the plate on the rocker for 15 min at power level 24.

4. While the plate is incubating, prepare 1.5 mL tubes for each well and label the tubes.

5. Transfer all 220 μL of cells+lysis buffer by pipetting up and down 8–10 times and placing into the appropriately labeled tube. Then spin down the tubes in the centrifuge at 4500xg for 10 min at 6 °C.

6. Pipette 10 μL of supernatant into each well of a 96-well reading plate.

7. Turn on Glomax reader, click on Glomax Discover and begin priming procedure according to the manufacturer’s instructions.

8. Place the 96-well plate in the Glomax plate reader and ensure it is primed and ready. Load luciferin solution into the injection tube loading area. Select an appropriate luciferase assay protocol and start.

9. Export results in Excel format.

1.2.4. Data analysis

To assess the relative luciferase activity and sensitivity to the PKR inhibitor, the following steps were undertaken:

• 1
  Normalization of pSG5 Empty Vector

  • The average value of the pSG5 empty vector luciferase readout was calculated.
  • Each replicate value of the pSG5 empty vector was normalized by dividing it by the average value derived from the pSG5 triplicates, resulting in values close to 1.
• 2
  Normalization of PKR Control

  • Each value of the PKR luciferase readout was divided by the average value derived from the pSG5 triplicates.
  • Low relative luciferase activity indicates PKR activity.
• 3
  Normalization of Experimental PKR + Inhibitor

  • Each value of the luciferase readout for the experimental PKR + inhibitor was divided by the average value derived from the pSG5 triplicates.
  • High relative luciferase activity indicates sensitivity to the PKR inhibitor.

Alternatively, the data can be presented as the relative luciferase activity normalized to PKR control:

• 4
  Normalization to PKR Control:

  • The average value of the PKR luciferase readout was calculated.
  • Each value of the experimental PKR + inhibitor luciferase readout was divided by the average value derived from the PKR triplicates.

*Notes

1. Place the plates and microtubes on ice during the protein preparation.

2. Do not use Opti-MEM to dilute GenJet reagent and DNA, it can disrupt transfection complex.

3. Incubate the GenJet/DNA complex for 15 min at room temperature, do not keep the GenJet/DNA complex longer than 30 min.

2. Yeast Assay for PKR Activity

2.1. Materials

Vectors and Plasmids

• pYX113 vector (R&D Systems)

• pRS305 vector

• pEMBLyex4 vector

• Plasmid pC3853

• Low-copy-number SUI2, URA3 plasmid p919

Reagents

• PCR reagents (primers, Pfu Polymerase)

• LiAcetate/PEG transformation reagents

• SC-Gal minus uracil medium

• SD plates + tryptophan

• TE buffer, pH 7.5

• SD and YEPD media

• 0.1 M LiAc

• Calf thymus DNA

• Water bath at 30 °C and 42 °C

2.2. Methods

2.2.1. Generation of Yeast Strains Expressing PKR

1. Cloning of PKR cDNA

   • Human PKR cDNAs containing N-terminal His6- and Flag tags were cloned and inserted into the yeast expression vector pYX113 (R&D Systems) under a GALCYC1 hybrid promoter, as described previously [11].
   • The GAL-CYC1 promoter and PKR cDNA fragments were subcloned and inserted into the LEU2 integrating vector pRS305.
   • Integration into the leu2 locus of the yeast strain H2557 yields strains expressing PKR.

2.2.2. Yeast Transformation and induction

1. Pick colonies from the master plate (YEPD plate), inoculate each strain in 3 mL of YEPD medium, and incubate at 30 °C overnight with shaking.

2. Make a 1:50 dilution of each culture into 50 mL fresh YEPD medium. Culture the dilution at 30 °C with shaking for about 4 h.

3. Spin 10 mL of the cells down at 3000 rpm @ RT for 5 min. Discard the supernatant and resuspend the pellet with 1 mL of TE (pH 7.5) buffer. Transfer the cells into 1.5 mL Eppendorf tubes (1 strain/tube).

4. Spin down at 3000 rpm @ RT for 3 min. Discard the supernatant and resuspend with 1 mL of 0.1M LiAc. Vortex, spin down at 3000 rpm @ RT for 3 min and discard the supernatant.

5. Resuspend pellets with 200 μL of 0.1M LiAc. Incubate at 30 °C for 15 min, vortex after incubation.

6. Prepare new Eppendorf tubes, 1 transformation/tube. Label properly with strain name and plasmid name. Add 5 μL of calf thymus DNA and 2 μL plasmid into each tube.

7. Add 50 μL of yeast cells into each tube and add 300 μL of 40% PEG. Vortex, incubate at 30 °C for 30 min, vortex.

8. Incubate at 42 °C for 20 min. Pellet the cells and completely remove the supernatant.

9. Resuspend the cells in 300 μL SD medium, plate 100 μL on each plate. Incubate at 30 °C until colonies appear.

10. Transformation verification

    • Four independent clones were picked and colony-purified on SD plates.
11. Induction and Growth Assessment

    • Streak the purified colonies onto either glucose (non-inducing conditions) or galactose-containing medium [synthetic complete medium containing 2 % (wt/vol) galactose and all amino acids without uracil, inducing conditions].
    • Incubate the plates for 4 days at 30 °C.

*Notes

1. Before the transformation, spread 200 μL of tryptophan solution on each SD plate, spread evenly, and let the plates dry.

3. Immunoblot Assay for Detecting Phosphorylated PKR, eIF2α and IκBα, NF-κB p65

3.1. Materials

Cell Culture Materials

• A549 wild-type, A549-PKR^KO and HeLa-PKR^KO cell lines

• Gibco DMEM, High Glucose, GlutaMAX Supplement (Gibco)

• Fetal bovine serum (FBS), (GE Healthcare)

• Penicillin-Streptomycin (10,000 U/mL) (Gibco)

• Phosphate Buffer Saline (PBS-D) (1×) (Thermo Fisher Scientific)

• Trypsin-EDTA (0.05 %), phenol red (Thermo Fisher Scientific)

Viral infection

• MYXVΔM029LΔM156R virus as described previously [12].

Cell Lysis and Protein Extraction

• 1 % sodium dodecyl sulfate (SDS) in PBS

• Sonicator

Protein Quantification

• A NanoDrop One spectrophotometer (Thermo Scientific)

SDS-PAGE and immunoblotting

• 12 % SDS-polyacrylamide gels (FastCastTM Acrylamide Kit Bio-Rad)

• Polyvinylidene difluoride (PVDF) membranes

• Freshly made 10 % ammonium per sulfate (APS)

• Tetramethylethylenediamine (TEMED)

• Filter paper

• Sponge

• 5 % Nonfat dry milk

• Bovine serum albumin (BSA)

• TBST buffer (20 mM Tris, 150 mM NaCl, 0.1 % Tween 20, pH 7.4)

• Enhanced chemiluminescence (ECL) detection system (GE Healthcare)

• Protein ladder (SeeBlueTM Plus2 Pre-Stained Protein Standard)

Required buffers

• 6X Laemmli buffer (freshly add ß-mercaptoethanol (9 % V/V) before use)

• 10x Tris/Glycine/SDS running buffer (Bio-Rad)

• 10x transfer buffer

  • Tris-HCl 25 mM
  • Glycine 200 mM
  • SDS 0.1 % (w/v)
• 1x transfer buffer

  • 1400 mL ddH₂O
  • 200 mL 10x transfer buffer
  • 400 mL Methanol
• 1x TBST buffer

  • 900 mL ddH₂O
  • 100 mL TBS (10x)
  • 1 mL Tween-20

Antibodies

• Primary antibodies against phosphorylated PKR (Abcam: ab32036), rabbit anti-Phospho-eIF2α (Ser51) polyclonal antibodies (Cell Signaling Technology: 9721L), mouse anti-IκBα monoclonal antibodies (mAb) (Cell Signaling Technology: L35A5) and rabbit anti-NF-κB p65 mAb (Cell Signaling Technology: D14E12).

• Secondary antibodies: donkey anti-rabbit or goat anti-mouse (Invitrogen, A16110)

Western Blot Equipment

• Ice bucket

• Heat block fit for 1.5 mL tubes (Benchmark)

• Vertical electrophoresis system (Mini-PROTEAN System Bio-Rad)

• Methanol-based wet transfer apparatus (Bio-Rad)

• Fastcast glass plates/casting cassette (Bio-Rad)

• Gel combs (Bio-Rad)

• Gel loading pipette tips

• Power supply (Bio-Rad)

• Transfer chamber & cassettes (Bio-Rad)

• Cold pack

• Rocker

• Plastic bag

• Tweezers

• Membrane wash boxes

• Plastic wrap

• Serological pipettes

• Centrifuge

• Roller

• Scissor

• iBright visualizing system (Invitrogen)

3.2. Methods

3.2.1. Cell Seeding and Infection

1. Seed 4 × 10^5 indicated cells in 6-well plates in a total volume of 2 mL media (DMEM containing 10 % FBS, 100 U PenStrep) per well.

2. Incubate the cells in 37 °C, 5 % CO₂ incubator overnight.

3. Infect the cells with MYXVΔM029LΔM156R at a multiplicity of infection (MOI) of 3 for 8 h.

3.2.2. Cell Lysis and Protein Extraction

1. After infection, place the cell culture on ice, remove the media, and wash the cell with PBS.

2. Lyse the cells with 1 % SDS in PBS (500 μL/well), scrape the cells with plastic cell scraper, and transfer the lysates into 1.5 mL microcentrifuge tubes.

3. Sonicate the cell lysates at 50 % amplitude for 10 s twice in a 4°C cold room.

4. Centrifuge the cell lysates 13,000 xg for 5 min at 4 °C and transfer the supernatant to the clean microcentrifuge tubes.

5. Determine the protein concentrations of each cell lysate using a NanoDrop One spectrophotometer.

6. Calculate the amount of protein to load (25 μg/well), adjust the volume of the protein with 1 % SDS.

7. Add 6X Laemli buffer to the protein and boil the mixture 95 °C for 5 min.

3.2.3. Loading and running the protein in SDS-PAGE gel

1. Prepare the 12 % SDS-polyacrylamide gels according to the manufacturer’s instruction.

2. Load equal amount of protein samples onto 12 % SDS-polyacrylamide gels using the gel loading pipet along with protein marker.

3. Run the gel in 1x Tris/Glycine/SDS running buffer for 1–2 h at 120 V or until the blue line reaches the green bar.

3.2.4. Transferring protein from the gel to the PVDF membrane

1. Prepare the 1x transfer buffer and soak 2 sponges and 2 filter papers per gel in transfer buffer.

2. Cut PVDF membrane to the size of the gel and activate the PVDF membrane by soaking in 100 % methanol for 1 minute.

3. Transfer the gel to the PVDF membranes using a methanol-based wet transfer apparatus as follows.

   1. Place the gel holder cassette in the tray with black plastic side down and emerged into the buffer, and clear plastic side up away from the buffer.
   2. Sponge
   3. Filter paper
   4. Gel
   5. Membrane (remove any air bubble with roller)
   6. Filter paper
   7. Sponge

4. Place the cassette inside the transfer chamber in a way that the black plastic side of the cassette is next to the black side of transfer chamber.

5. Place the cold pack in the tank.

6. Fill the tank with 1x transfer buffer to the level of the white clip on the transfer cassette.

7. Run at ~70 V for 1 hour.

3.2.5. Membrane blocking and antibody staining

1. Prepare the blocking buffer (5% BSA in TBST).

2. Block the PVDF membrane with the blocking buffer for 1 h at room temperature.

3. Incubate the membrane with primary antibodies overnight at 4 °C (p-PKR dilution 1:1000 in TBST containing 5 % BSA; p-eIF2α dilution 1:1000 in TBST containing 5 % BSA; P65 dilution 1:1000 in TBST containing 5 % BSA; and IκBα dilution 1:1000 in TBST containing 5 % BSA).

4. Wash the membrane with TBST 3 times, 5 min each.

5. Incubate the membrane with the appropriate secondary antibodies (1:8000 dilution in TBST containing 5 % nonfat dry milk).

6. Wash the membrane with TBST 3 times, 10 min each.

7. Follow the kit manufacturer’s instructions for protein detection using an enhanced chemiluminescence (ECL) detection system.

8. Place the membrane in a plastic wrap and remove the excess reagent.

9. Acquire image using an iBright Imaging System.

*Notes

1. Perform the protein preparation on ice.

2. Properly wash the sonicator probe with deionized water in between samples to prevent contamination.

3. Fully dissolve the non-fat dry milk or BSA in TBST to get better image/less spotty background during image development.

4. After denaturing the proteins by boiling the cell lysates in Laemli buffer at 95 °C for 5 min, the lysates can be stored at −20 °C for future use.

5. Ponceau S staining can be done prior to transferring to the PVDF membrane to check the protein.

6. The PVDF membrane can be dried and re-blot for detection other proteins.

7. Store the protein −80 °C for longer storage.

4. Quantitative PCR Assessment of PKR Activation Effects on NF-κB Signaling Pathways

4.1. Materials

Cell Culture and Plasmid

• Cell culture media

• HeLa cells with knocked out (KO) PKR (HeLa-PKR^KO)

• pSG5 plasmid express European rabbit PKR (E.rabbit PKR)

RNA Isolation and Treatment of RNA

• TRIzol Reagent (Invitrogen)

• Direct-zol RNA Miniprep Kits (ZYMO Research R2052)

• RNA Clean & Concentrator-5 (ZYMO Research R1016)

• RNase Inhibitor (40 U/μL)

• Ribonuclease-free DNase I (New England Biolabs)

• Ethanol

Reverse Transcription

• ProtoScript II Reverse Transcriptase (200 U/μL) (New England Biolabs)

• 5x ProtoScript II Buffer

• Anchored Oligo dT (20): TTT TTT TTT TTT TTT TTT TV

qPCR reactions

• OneTaq DNA Polymerase (New England Biolabs)

• 10 mM dNTPs

• MgCl₂ (25 mM)

• DMSO

• 5x OneTaq Standard Reaction Buffer

• EvaGreen^® Dye, 20x in Water (Biotium: 89138–982)

• The following primers were used for the target genes:

  • TNFα-qPCR-F primer: 5’-AGCCCATGTTGTAGCAAACC-3'
  • TNFα-qPCR-R primer: 5’-TGAGGTACAGGCCCTCTGAT-3'
  • IL-6-qPCR-F primer: 5’-TACCCCCAGGAGAAGATTCC-3'
  • IL-6-qPCR-R primer: 5’-TTTTCTGCCAGTGCCTCTTT-3'
  • 18S-qPCR-F primer: 5’-AGGAATTGACGGAAGGGCAC-3'
  • 18S-qPCR-R primer: 5’-GGACATCTAAGGGCATCACA-3'

PCR cycling

• Real-time PCR detection system (Bio-Rad)

Melt Curve Analysis

• Melting curve analysis software.

Data Analysis:

• Statistical analysis software

4.2. Methods

4.2.1. Cell Seeding and Transfection

1. Seed 4 × 10^5 HeLa-PKR^KO cells in 6-well plates and allow cells to adhere and grow overnight.

2. Transfected HeLa-PKR^KO with plasmid pSG5 express European rabbit PKR for 24 h.

4.2.2. Cell Lysis and RNA Isolation (ZYMO Research R2052)

1. Lyse cells directly in a culture dish or well or resuspend pelleted cells in an appropriate volume of Trizol and mix thoroughly.

2. Add an equal volume of ethanol (95–100 %) to the sample lysed in Trizol and mix thoroughly.

3. Transfer the mixture into a Zymo-SpinTM IICR Column in a Collection Tube and centrifuge at 10,000–16,000 × g for 30 s. Transfer the column into a new collection tube and discard the flow-through.

4. Add 400 μL Direct-zol^™ RNA PreWash to the column and centrifuge at 10,000–16,000 × g for 30 s. Discard the flow-through and repeat this step.

5. Add 700 μL RNA Wash Buffer to the column and centrifuge at 10,000–16,000 × g for 2 min to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube.

6. To elute RNA, add 10–50 μL of DNase/RNase-Free Water directly to the column matrix and centrifuge at 10,000–16,000 × g for 30 s.

4.2.3. DNase I Reaction

1. Prepare the DNase I Reaction on ice

   • RNA: ~ 10 μg RNA
   • DNase I Reaction Buffer (10x): 10 μL
   • DNAse I (RNase-free): 1 μL (2 units)
   • Nuclease-free H₂O: to 100 μL

2. Incubate at 37 °C for 10 min.

3. Add 1 μL of 0.5 M EDTA (to a final concentration of 5 mM).

4. Heat inactivates at 75 °C for 10 min.

4.2.4. RNA Clean (ZYMO Research R1016)

1. Add 2 volumes RNA Binding Buffer to each sample and mix. Example: Mix 100 μL buffer and 50 μL sample.

2. Add an equal volume of ethanol (95–100 %) and mix. Example: Add 150 μL ethanol to 150 μL sample+binding buffer.

3. Transfer the sample to the Zymo-Spin^™IC Column in a Collection Tube and centrifuge at 10,000–16,000 × g for 30 s. Discard the flow-through.

4. Add 400 μL RNA Prep Buffer to the column and centrifuge at 10,000–16,000 × g for 30 s. Discard the flow-through.

5. Add 700 μL RNA Wash Buffer to the column and centrifuge at 10,000–16,000 × g for 30 s. Discard the flow-through.

6. Add 400 μL RNA Wash Buffer to the column and centrifuge at 10,000–16,000 × g for 2 min to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube.

7. Add 15–40 μL DNase/RNase-Free Water directly to the column matrix and centrifuge at 10,000–16,000 × g for 1 minute.

   Note: Alternatively, for highly concentrated RNA use ≥6 μL elution. The eluted RNA can be used immediately or stored at −80°C.

4.2.5. Reverse Transcription (cDNA Synthesis)

1. Mix RNA sample and anchored Oligo dT (20) in a sterile RNase-free microfuge tube.

   • Total RNA: 1 μg
   • Oligo dT (20) (50 μM): 2 μL
   • 10 mM dNTP: 1 μL
   • RNase-free ddH₂O: to a total volume of 12.8 μL

2. Denature the RNA/primer mixture by incubating at 65 °C for 5 min. Spin briefly and immediately places on ice.

3. Prepare the reverse transcription reaction mix:

   • 5x ProtoScript II Buffer: 4 μL
   • 0.1 M DTT: 2 μL
   • ProtoScript II RT (200 U/μL): 1 μL
   • RNase Inhibitor (40 U/μL): 0.2 μL

4. Incubate the 20 μL cDNA synthesis reaction at 42 °C for one hour. Then, inactivate the enzyme by heating at 65 °C for 20 min.

5. Dilute the original cDNA (1000 ng/20 μL) to 25 ng/10 μL. Use 10 μL (25 ng) of diluted cDNA as a template for qPCR experiments.

4.2.6. Quantitative PCR (qPCR)

1. Preparation of the Reaction Mix

   • The qPCR mixture for each target gene was prepared.
   • For each reaction, the following components were combined in a total volume of 20 μL:

     • 10 mM dNTPs: 0.5 μL
     • MgCl₂ (25 mM): 2 μL
     • 5x OneTaq Standard Reaction Buffer: 4 μL
     • DMSO: 1 μL
     • EvaGreen (20x): 1 μL
     • Forward primer (10 mM): 0.6 μL
     • Reverse primer (10 mM): 0.6 μL
     • OneTaq DNA Polymerase: 0.2 μL
     • cDNA: 10 μL
2. PCR cycling

   • PCR cycling was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad) under the following conditions:

     • Initial denaturation at 95 °C for 2 min
     • 40 cycles of denaturation at 95 °C for 5 s, annealing and extension at 60 °C for 8 s TNFα (12 s for IL-6)
     • The fluorescence data were acquired at the end of each cycle.

3. Melt curve analysis

   65 °C to 95 °C with a heating rate of 0.5 °C/second

4.2.7. Data Analysis

Relative expression levels of the tested mRNAs were determined utilizing 18S rRNA as an internal reference. The process involved:

• Calculation of ΔCt values: Each sample's ΔCt values were computed by subtracting the Ct value of the internal reference gene (18S rRNA) from the Ct value of the target gene.

• Calculation of ΔΔCt values: ΔΔCt values were obtained by subtracting the ΔCt of the control sample from the ΔCt of the experimental sample.

• Determination of relative expression levels: Relative expression levels were derived using the comparative Ct (2^−ΔΔCt) method.

• Calculation of average values and standard deviations: Three replicate amplifications were performed, and average values along with standard deviations were computed.