Fig. 6: BiP/HSP-4 and IRE-1 regulate autophagy through ER-Phagy receptor Sec-62/C18E9.2. | Nature Communications

Fig. 6: BiP/HSP-4 and IRE-1 regulate autophagy through ER-Phagy receptor Sec-62/C18E9.2.

From: Functionally diversified Caenorhabditis elegans BiP orthologs control body growth, reproduction, stress resistance, aging, and autophagy

Fig. 6

Western blot (a) and quantifications (b, c) of day 1 adult DA2123 (adIs2122[lgg-1p::GFP::lgg-1 + rol-6(su1006)]) grown on indicated siRNA and probed with an anti-GFP (Roche, Cat #11814460001) and anti-alpha tubulin antibody (DSHB, Cat #12G10). Shapes represent biological replicates (n = 3). d Representative confocal images of day 1 adult MAH215 (sqIs11[lgg-1p::mCherry::GFP::lgg-1 + rol-6) on indicated siRNA. Scale bars are 20 μm. GMC101 (dvIs100[unc-54p::A-beta-1-42::unc-54’‘-UTR + mtl-2p::GFP]) paralysis assays at the non-inducive (e) and inducive temperatures (f). HE250 (unc-52(e669su250)) (g) and CB1157 (unc-54(e1157)) (h) paralysis assays grown on indicated siRNA scored 48 h post-transfer to 25 °C as day 1 adults. i Survival curves of WT worms on indicated siRNA. j Body area quantification of WT worms on indicated siRNA and day of adulthood. Western blot (k) and l quantification of co-immunoprecipitation assays of Tm-treated A549 cells pulling on IRE-1 and probed for IRE-1 (Cell Signaling, Cat #3294S; Rabbit IgG, Cell Signaling, Cat #7074S), Sec-62 (Abcam, Cat #EPR9212; Light Chain-Specific Rabbit IgG (Cell Signaling, Cat #D4W3E), and BiP (Proteintech, Cat #66574; Mouse IgG, Cell Signaling Cat #7076S). Number of animals and median lifespan/paralysis data is in Supplementary Tables 4 and 5. For g, h, j, l biological replicates (n = 3) are distinguished by color; triangles are average of four technical replicates (circles). Source data are provided as a Source Data file. b, c, g, h, j Two-way ANOVA followed by Uncorrected fisher’s LSD: b pC18E9.2 siRNA x hsp-4 siRNA = 0.003; pC18E9.2 siRNA = 0.001; phsp-4 siRNA = 0.001. c pC18E9.2 siRNA x hsp-4 siRNA = 0.01; pC18E9.2 = 0.07; phsp-4 siRNA = 0.01. g pC18E9.2 siRNA x hsp-4 siRNA < 0.0001; pC18E9.2 siRNA < 0.0001; phsp-4 siRNA < 0.0001. h pC18E9.2 siRNA x hsp-4 siRNA < 0.0001; pC18E9.2 siRNA < 0.0001; hsp-4 siRNA < 0.0001. j psiRNA x age < 0.0001; psiRNA < 0.0001; page < 0.0001. (e, f, g). Log-rank Mantel-Cox test. p < 0.05 is considered statistically significant. Error bars: s.d.

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