Extended Data Fig. 5: EHDs sense PM curvatures and play important roles in JPH’s membrane curvature targeting.
From: Plasma membrane curvature regulates the formation of contacts with the endoplasmic reticulum
a, EHD4-GFP, GFP-CAVIN1, GFP-CAV1, and GFP-CAV2 in U2OS on nanobars. Right: end-to-side ratios quantification. Cell number: n = 14 (CAV2), n = 15 (all others). ***P = 0.0001, **P = 0.0015; ****P < 0.0001. b, Protein transcript levels in U2OS cells (normalized transcripts per million, nTPM). CLTC encodes clathrin heavy chain. Genes in gray: not identified in the JPH2 interactome but closely related genetically or functionally. EHD3, CAVIN2, CAVIN3, and CAVIN4 were not knocked down due to low TPM in U2OS cells. c, Western blots confirming shRNA knockdown of CAV1, CAV2, EHD1, EHD2, clathrin heavy chain, and BIN1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. d, Images of U2OS cells co-expressing EHD4-mCherry with EHD1-GFP (Top) or with EHD2-GFP (Bottom). Enlarged views are from yellow boxes. e, Images of U2OS cells expressing GFP-JPH3 (green) and EHD4-mCh (magenta) on nanobars. Enlarged views are from yellow boxes. f, Images of EHD2 or EHD4 immunostaining in iPSC-CMs or rat embryonic CMs on nanopillars. Bottom: enlarged yellow-box regions. Scale bar: 10 µm (top), 2.5 µm (bottom). g, Images of EHD4 immunostaining in iPSC-CMs on nanobars. Middle: enlarged yellow-box regions and averaged fluorescent signals of all nanobars from multiple cells. n = 16 cells. Right: end to side ratios of EHD4 compared to BFP-CAAX expressed in iPSC-CM on nanobars. Cell number: CAAX = 23, EHD4 = 16. ****P < 0.0001. h, Images of JPH2 immunostaining in iPSC cells transfected with shRNA of scramble (top) or EHD1/2/4 (bottom). Left to right: Bright field image, fluorescent protein indicating shRNA transfection, and JPH2 staining. Bottom: averaged nanobar images of JPH2 staining from multiple cells. Right: end-to-side ratio. Cell number: Scramble = 24, shEHD1/2/4 = 26. ****P < 0.0001. Scale bars: 10 µm (whole cell), 5 µm (enlarged region), 2.5 µm (average), unless otherwise mentioned. Experiments in (h) were independently replicated two times with similar results, and others were independently replicated for three times with similar results. Data are mean values +/− SD for (a,g,h). Kruskal–Wallis test corrected with Dunn’s multiple-comparison test was used to assess significance in (a). Unpaired two-tailed Welch’s t test was used in (g). Two-tailed Mann-Whitney test was used in (h). Source numerical data and unprocessed blots are available in source data Source data.
