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. 2018 Jun 13;7:e35012. doi: 10.7554/eLife.35012

Figure 9. FRMD8 is required for iRhom2/TACE regulation in human iPSC-derived macrophages and mice .

(A) Schematic representation of the differentiation protocol of iPSCs into macrophages based on (van Wilgenburg et al., 2013). Scale bars = 10 μm. (B) Lysates of iPSC-derived macrophages (on day seven after harvest from EBs) were immunoblotted for ADAM17, FRMD8, and actin. Western blots from three experiments were quantified using ImageJ with actin serving as the loading control. (C) 25,000 iPSC-derived macrophages were either left unstimulated or stimulated with 50 ng/ml LPS for 4 hr. TNFα concentration in the cell supernatants was measured by ELISA and then normalised to the protein concentration in macrophage cell lysates to adjust the cytokine release for potential differences in cell numbers. Each experiment was performed in biological triplicates. Data from three independent experiments were statistically analysed using a Mann-Whitney test; ***=p value<0.001; ****=p value<0.0001. (D, E) Lysates from tissues derived from Frmd8-/- or Rhbdf2-/- and their wild-type littermates were immunoblotted for ADAM17, FRMD8, iRhom2 and actin. Blots from three experiments using three different littermates of Frmd8-/- and Frmd8+/+ mice were quantified using ImageJ with actin serving as the loading control.

Figure 9.

Figure 9—figure supplement 1. Generation of FRMD8 knockout iPSCs and iPSC-derived macrophages.

Figure 9—figure supplement 1.

(A) Sequencing of the genomic DNA isolated from clonal FRMD8 KO iPSCs shows a 1-nt insertion (clone 1) and a 7-nt and 10-nt deletion (clone 2). The targeting sequence of the sgRNA is shown in bold; small letters indicate the sequence within the intronic region; the protospacer adjacent motif (PAM) sequence underlined. (B) Parental wild-type and FRMD8 KO iPSC lines were karyotyped by SNP array. Detected copy number variations are indicated in red (DNA copy number loss in the indicated region) and green (DNA copy number increase). The AH017-13 iPSC line used was derived from a female donor, therefore the Y chromosome is marked in red (loss of Y chromosome DNA). (C) 25,000 iPSC-derived macrophages were either left unstimulated, stimulated with 50 ng/ml LPS, or with 50 ng/ml LPS and simultaneously with 2 μM GI or 2 μM GW for 4 hr. TNFα concentration in the cell supernatants was measured by ELISA and then normalised to the protein concentration in macrophage cell lysates to adjust the cytokine release for potential differences in cell numbers. Each experiment was performed in biological triplicates. Data from three independent experiments were statistically analysed using a Mann-Whitney test; ns = p value>0.05; ****=p value<0.0001. (D) TNFα mRNA levels relative to actin mRNA levels were measured by TaqMan PCR in WT and FRMD8 KO iPSC-derived macrophages without stimulation and after stimulation with 200 ng/ml LPS for 0.5 hr.
Figure 9—figure supplement 2. Generation of Frmd8 knockout mice.

Figure 9—figure supplement 2.

(A) Schematic representation of the insertion of a lacZ/neomycin cassette into the Frdm8 locus in the ES cells used to generate Frmd8-/- mice. (B) Offspring of Frmd8+/- × Frmd8+/- (HET x HET) crosses listed by genotype: Frmd8+/+(WT), Frmd8+/- (HET), and Frmd8-/- (KO). Two Frmd8 mouse strains were bred (both in BL6 background): one with the entire lacZ/neomycin cassette inserted and one strain in which the neomycin resistance gene has been removed from the cassette. (C) Lysates from skin derived from Frmd8-/-, Rhbdf2-/- mice and their wild-type littermate were immunoblotted for iRhom2 and actin.