Fig 1: The L535Q KASH5 localizes to the mitochondria. U2OS cells grown on coverslips were transfected with an N-terminal GFP-tagged version of wild-type or L535Q KASH5. The subcellular localization was determined by immunostaining with organelle-specific protein markers. (A) Nuclear localization was assessed using an anti-lamin A/C antibody (red). (B) Mitochondria localization was assessed with an anti-TOM20 antibody (red). (C) Peroxisome localization was assessed with an anti-PEX14 antibody (red). (D) Pearson’s correlation coefficient between GFP-KASH5 and organelle markers. KASH5 vs Lamin A/C: N = 24, KASH5 vs TOM20: N = 35 and KASH5 vs Pex14: N = 72. The values indicate − 1: opposing, 0: no and 1 complete colocalization. Scale bar 10 μm. ****P < 0.0001; ***P < 0.0002. The experiment was repeated seven times.
Fig 2: ACBD5-mediated ER-peroxisome contacts underlie HCMV-driven changes to peroxisome size and numbers.A Average peptide abundances (scaled to mean) of ACBD5 (circle) and its ER tether VAP-B (hexagon) during HCMV infection, compared to ACBD4 and VAP-A (light blue lines). Shaded regions are SEM, and p values are by one-way ANOVA to Mock (data is from MCS-PRM quantification as in Fig. 1, N = 6 biological replicates with =3 peptides/protein monitored in each replicate, see Supplementary Data 1 for complete list of peptides). B PLA quantification of endogenous ACBD5-VAP-B interactions. Plotted are PLA signal counts (solid line at mean, dotted lines at quartiles, N = 40 cells/timepoint, **p = 0.01, ***p = 0.001 by two-tailed student’s t-test to Mock). C Movies of ER (cyan) and peroxisomes (white/red) before and 120 hpi, showing whole-cell and zoomed stills (white circles, lower). Scale bars 10 µm. D Fixed fibroblasts in late (72–120 hpi) stages of HCMV infection, labelled for: ER (cyan), peroxisome membranes (red), and HCMV IE1 (magenta). Each channel from a zoomed region (white circles) is shown at right, including an ER-peroxisome overlap mask heat-colored by increasing overlap. Arrows indicate enlarged (yellow) or small (white) peroxisomes, localizing with expanded or tubular ER, respectively. Scale bars 10 µm. E IF analysis of peroxisomes (PEX14 antibody) before and 120 hpi, comparing control to ACBD5 KD and OE (10×10 µm). See Supplementary Fig. 13 for more examples. F Quantification of peroxisome surface area in control (Ctrl, N = 8973 peroxisomes from 15 cells in Mock, N = 11,991 peroxisomes from 15 cells in 120 hpi), ACBD5 KD (N = 16,625 peroxisomes from 15 cells in Mock, N = 33,392 peroxisomes from 28 cells in 120 hpi), and ACBD5 OE (N = 3961 peroxisomes from 15 cells in Mock, N = 5174 peroxisomes from 15 cells in 120 hpi) cells before and 120 hpi (solid line at median, dotted lines at quartiles; **p = 0.01, ***p = 0.001 by two-tailed student’s t-test to Ctrl/timepoint). G. HCMV titers from ACBD5 KDs (two siRNAs) and OEs (1: 250 ng, 2: 500 ng) versus either siRNA or plasmid controls (N = 4, ***p = 0.001 by two-tailed student’s t-test). H Peroxisome counts per cell in control (Ctrl, N = 70 cells in Mock, N = 20 cells in 120 hpi), ACBD5 KD (N = 28 cells in Mock, N = 23 cells in 120 hpi), and ACBD5 OE cells (N = 42 cells in Mock, N = 15 cells in 120 hpi) (solid line at median, dotted lines at quartiles; ***p = 0.0001 by two-tailed student’s t-test to Ctrl/timepoint). I–K. HSV-1 (N = 4 biological replicates, *p = 0.0151 and **p = 0.0014), Infl. A (N = 3, ***p = 0.0005 and ***p = 0.0003), and HCoV-OC43 (N = 8, ***p = 0.0001) titer measurements in control versus ACBD5 OE cells (1: 250 ng, 2: 500 ng, p-values by two-tailed student’s t-test to control).
Supplier Page from Abcam for Anti-PEX14 antibody