Fig 2: MARCH5 interacts with PEX19 and PEX3. (A) IP of MARCH5-Myc performed with HEK293T cells cotransfected with MARCH5-Myc and PEX19-V5. The samples were analyzed by IB with indicated antibodies. (B) HEK293T cells were cotransfected with PEX19-3×FLAG and different MARCH5-mCherry-Myc variants. Cells were collected 36 h after transfection for IP of PEX19-3×FLAG and IB with indicated antibodies. (C) V5-PEX19 or NLS fused V5-PEX19 were coexpressed with MARCH5-Myc or TOMM20-Myc in HeLa cells for 24 h. Cells are stained for Myc and V5 tags. (D) HEK293T cells were transfected with MARCH5-Myc and PEX3-3×FLAG for 30 h. Cells were treated with 50 μM MG132 for 6 h and then harvested for IP and IB with indicated antibodies. (E) HEK293T cells were cotransfected with PEX3-3×FLAG and different MARCH5-mCherry-Myc variants. IP and IB were performed with indicated antibodies. (F) HEK293T cells were transfected with MARCH5-Myc and PEX3-3×FLAG or PEX3(46–373)-3×FLAG for 36 h. Cells were treated with 50 μM MG132 for 6 h and then harvested for IP of MARCH5-Myc and IB with indicated antibodies. (G) Confocal fluorescent images of HeLa cells infected with lentiviruses expressing MARCH5-mCherry and PEX3-YFP. White arrows indicate where mCherry and YFP signals overlap.

Fig 3: Other supplementary information. (A) Schematic of the genome-editing strategy to knock out endogenous MARCH5 in HeLa cells. Exons 1–6 (E1-E6) are indicated. Three designed sgRNAs were tested initially, and the validated sgRNA confirmed by sequencing, as shown. Protospacer adjacent motif sequences are depicted in red. The KO of MARCH5 is confirmed by both Sanger sequencing (left) and IBs (right). (B) WT or MARCH5 KO HeLa cells expressing Ub-GFP-SKL were cultured in DMEM (supplemented with 10% FBS) or HBSS for 24 h. GFP signal was analyzed with FACS. (C) Representative images for Fig 9, D and E. WT or MARCH5 KO HeLa cells were transfected with Ub-GFP-FKBP and FRB-HA-PEX26 for 24 h. Cells were treated with DMSO or rapalog for 24 h, fixed, immunostained for PMP70, and imaged with confocal microscopy. Scale bars: 5 µm. (D) HeLa cells were treated with Torin1 (1 μM) for the indicated time. MARCH5 mRNA levels were detected with quantitative PCR assay. Values are mean ± SD, n = 3 independent experiments. n.s., not significant (P > 0.05) by two-tailed Student’s t test. (E) HeLa cells were transfected with MARCH5-Myc plasmid for 24 h, then with or without Torin1 (1 μM) were added for an additional 24 h before harvesting. Cells were then fixed and immunostained for PMP70 (Alexa Fluor 488, green) and Myc (Alexa Fluor 555, red) and examined by microscopy to detect GFP and RFP signals. Pearson’s correlation coefficients show the colocalization between PMP70 and MARCH5-Myc derived from above. Values are mean ± SD *, P < 0.05 by two-tailed Student’s t test.

Fig 4: miR-21-5p interactes with MARCH5 and regulates its expression.A Binding sites of miR-21-5p and MARCH5 using Starbase 3.0. B Relative luciferase activity from reporter assays in cardiomyocytes co-transfected with mimic-NC/miR-21-5p mimic or inhibitor-NC/miR-21-5p inhibitors along with MARCH5-wt or MARCH5-mut (n = 3). C, D The mimic-NC and miR-21-5p mimic were intraperitoneally administered 24 h before myocardial I/R injury and infarction size (%) and levels of miR-21-5p expression were evaluated (n = 6). E Cardiomyocytes were transfected with mimic-NC and miR-21-5p mimic, followed by induction of H/R. Expression of miR-21-5p was evaluated by qRT-PCR (n = 3). F, G Cardiomyocytes were co-transfected with mimic-NC/miR-21-5p mimic and Vector/MARCH5 or inhibitor-NC/miR-21-5p inhibitor and sh-NC/sh-MARCH5 followed by induction of H/R. MARCH5 expression was evaluated by western blot and qRT-PCR (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 and n.s no significance.

Fig 5: MARCH5 binds and ubiquitinates PMP70. (A) IP was performed with anti-V5 antibody in HEK293T cells transfected with PMP70-V5 and MARCH5-Myc, followed by IB for V5, Myc, and β-actin. (B) HeLa cells were transfected with MARCH5-Myc plasmids for 48 h. After 10 μM MG132 treatment for 6 h, IP was performed with anti-PMP70 antibody, followed by IB for PMP70 and Myc. (C) HEK293T cells were transfected with HA-Ub, PMP70-V5, MARCH5-Myc, and MARCH5(HW)-Myc plasmids for 48 h. After 10 μM MG132 treatment for 6 h, cell lysates were immunoprecipitated with anti-V5 antibody and then immunoblotted with indicated antibodies. (D) HeLa cells were transfected with empty vector (EV), MARCH5-Myc, or catalytically inactive MARCH5-CS-Myc plasmids for 48 h. The cell lysates were immunoblotted with indicated antibodies. (E) The protein levels in D were quantified with ImageJ, then normalized to β-actin. Values are mean ± SD, n = 3 independent experiments. n.s., not significant (P > 0.05) by two-tailed Student’s t test. (F) HeLa cells were transfected with two different shRNAs targeting MARCH5. The cell lysates were immunoblotted with indicated antibodies. (G) The protein levels in F were quantified with ImageJ, then normalized to β-actin. Values are mean ± SD, n = 3 independent experiments. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; n.s., not significant (P > 0.05) by two-tailed Student’s t test.