ha gsk3 beta wt pcdna3  (Addgene inc)

Journal: The Journal of Cell Biology

Article Title: Protein Kinase C promotes peroxisome biogenesis and peroxisome–endoplasmic reticulum interaction

doi: 10.1083/jcb.202505040

Figure Lengend Snippet: PKC regulates peroxisome – ER VAP-dependent interaction through GSK3b inhibition. (A) Quantification of confocal microscopy of peroxisomes stained with anti-PMP70 antibody in WT and VAPB/VAPA KO HeLa cells in control and GFP-VAPB overexpression conditions. Quantification shows the number of peroxisomes per micron square, mean ± SEM, N = 100 cells pooled from three biological repeats, ***P < 0.001, ****P < 0.0001, Kruskal–Wallis test. (B) Confocal microscopy of peroxisomes stained with anti-PMP70 antibody in WT and VAPB/VAPA KO HeLa cells in control Go6983 (5 μM for 48 h). Quantification shows the number of peroxisomes per micron square, mean ± SEM, N = 100, ***P < 0.001, ****P < 0.0001, one-way ANOVA. Western blot confirming the KO is shown. (C) Co-immunoprecipitation of mycACBD5 in control and PKC inhibition conditions (Go6983 5 μM, 24 h); mycACBD5 and GFP or GFP-VAPB were overexpressed in HEK293T cells. Quantification shows the IP to input ratio, mean ± SEM, N = 3, ***P < 0.001, ****P < 0.0001, one-way ANOVA. (D–F) Live cell confocal microscopy of ER and peroxisomes in U2OS cells in control and Go6983 conditions. Quantification shows the ratio of peroxisomes that are not proximal or overlapping with the ER, mean ± SEM, N = 6 pooled from 1,000 peroxisomes in at least 30 cells per condition, **P < 0.01, and a distance traveled by a single peroxisome in 30 s, mean ± SEM, N = 1,000, ****P < 0.0001, unpaired t test with Welch’s correction. (G) Co-immunoprecipitation of GFP-VAPB in control and PKCD-mCherry overexpression conditions in HEK293T cells overexpressing mycACBD5 and GFP-VAPB. Quantification shows the IP to input ratio, mean ± SEM, N = 4, ***P < 0.001, ****P < 0.0001, unpaired t test. (H) Western blot of GSK3β inhibitory S9 phosphorylation in PMA(0.5 μM for 2 h) and Go6983 (5 μM for 4 h) conditions. Quantification shows the ratio of phosphorylated pS9 GSK3β to non-phosphorylated GSK3β, mean ± SEM, N = 4, ***P < 0.001, **P < 0.01, one-way ANOVA. (I) Confocal microscopy of peroxisomes stained with anti-PMP70 antibody in HEK293T control or overexpression of GSK3β S9A mutant in control or PMA (0.5 μM for 4 h) conditions. (I) Quantification shows the number of peroxisomes per micron square, mean ± SEM, N = 100 pooled from three biological repeats, ****P < 0.0001, Welch ANOVA. (J) Model of PKC regulation of peroxisome– ER interaction. Source data are available for this figure: . OE, overexpressing; ns, nonsignificant; coIP, co-immunoprecipitation.

Article Snippet: HA GSK3 beta WT pcDNA3 was a gift from Jim Woodgett (National Institutes of Health, Bethesda, MD, USA; plasmid #14753; Addgene; https://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ). pCDNA3.1-PPARA was a gift from Claes Wadelius (Uppsala University, Uppsala, Sweden; plasmid #169019; Addgene; https://n2t.net/addgene:169019 ; RRID:Addgene_169019) ( ).

Techniques: Inhibition, Confocal Microscopy, Staining, Control, Over Expression, Western Blot, Immunoprecipitation, Phospho-proteomics, Mutagenesis