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).

Fig 3: Cell culture media impacts on mitochondrial dynamics. (A) Live-cell super-resolution imaging evaluating mitochondrial dynamics in iPSDM differentiated with M-CSF and cultured using X-VIVO15 (top panel) or HPLM (bottom panel). Scale bar: 10 mm and 2 mm (zoom-in). (B) Schematic showing the 4D analysis sequence. (C–E) Dot plots show the quantification of mitochondrial mean branch length, number of branches, and volume over 20 time points (30 s timeframe) from one out of three independent experiments using iPSDM as described in as in panel A. (F, G) Histogram showing the quantification of PEX14-positive peroxisome spots per cell and representative images of iPSDM differentiated with M-CSF or GM-CSF, cultured in the indicated media and stained for PEX-14. n = 3 independent experiments. Scale bar: 20 mm

Fig 4: Calcium deficiency induces peroxisomal protein degradation in a time-dependent manner. A Immunoblot analysis of AML12 cells in calcium-deficient medium for indicated durations. Whole cell lysates were reacted with anti-catalase, anti-Pex14, anti-PMP70, anti-Acox1, anti-Pex3, anti-Pex1, anti-Pex19, anti-Pex11β, and anti-β-actin for protein expression analyses. B AML12 cells in calcium-deficient medium for indicated durations were fixed and immunostained with anti-PMP70 (red), shown as representative fluorescence image. Scale bar represents 25 µm. C Quantification of PMP70 puncta represents the number of peroxisomes per cell. Data are expressed as means ± S.D. (n = 3, independent experiments, 30 cells were analyzed in each experiment), * p < 0.05. D Immunoblot analysis of AML12 cells in calcium-deficient medium for indicated durations. Whole cell lysates were reacted with anti-VDAC, anti-Calnexin, and anti- β-actin

Fig 5: Calcium deficiency-induced pexophagy is regulated by ROS-mediated TFEB activation. A Immunoblot analysis results of AML12 cells in calcium-deficient medium with or without NAC treatment. Whole cell lysates were reacted with anti-catalase, anti-Pex14, anti-PMP70, and anti-β-actin. B Immunofluorescence of mRFP-GFP-SKL cells in calcium-deficient medium for indicated durations with or without NAC treatment. Scale bar represents 25 µm. C Quantification of red fluorescence represent pexophagy. Data are expressed as means ± S.D. (n = 3, independent experiments, 30 cells were analyzed in each experiment), * p < 0.05. D AML12 cells in calcium-deficient medium with or without chloroquine treatment are immunoblotted. Whole cell lysates were reacted with anti-catalase, anti-Pex14, anti-PMP70, and anti-β-actin. E Cytosolic and nuclear fractions from AML12 cells with or without calcium for 24 h. Also includes treatment of NAC and chloroquine. Immunoblot the fractions with anti-TFEB. For the cytosolic and nuclear fractions, alpha-tubulin and CREB antibodies, respectively, were utilized as the controls