Journal: bioRxiv

Article Title: Rab12 is a regulator of mitophagy and mitochondrial homeostasis

doi: 10.64898/2026.03.29.715103

Figure Lengend Snippet: mt-mKeima/YFP-Parkin HeLa cells were reverse transfected with siRNAs against the notated Rab GTPase family members for 24 h. Following transfection, cells were treated with vehicle or 30 μM FCCP for 4h to induce mitophagy. Cells were immediately live-cell imaged on a confocal microscope for mt-mKeima-based mitophagy analysis to analyze impact of Rab target knockdown on mitophagic flux. Results are graphed as average mt-mKeima (pH 4-5) positive foci per cell, normalized to respective plate scramble (Scr) siRNA controls. All plates also included an siRNA against PINK1 as a positive control for mitophagy impairment. Candidates with a ≥50% increase are presented in blue, and those with a ≥50% decrease in mitophagy readout are presented in red. n = 3 replicates (except Rab2B and Rab15: n = 2 replicates). Data are presented as mean ± SEM.

Article Snippet: A working mix of Lipofectamine RNAiMAX transfection reagent (Fisher Scientific 13-778-150) and Opti-MEM reduced serum medium (Thermo Scientific 31985070) was prepared by diluting 300 μL RNAiMAX in 9.7 mL OptiMEM.

Techniques: Transfection, Microscopy, Knockdown, Positive Control

Journal: bioRxiv

Article Title: Rab12 is a regulator of mitophagy and mitochondrial homeostasis

doi: 10.64898/2026.03.29.715103

Figure Lengend Snippet: ( A ) RNA was collected for qRT-PCR analysis of Rab12 mRNA expression in mt-mKeima/YFP-parkin HeLa cells following 72 h transfection with siRNA against Rab12 (siRab12) or the scramble control (siScr). Internal control for normalization was GAPDH. **** p < 0.0001 determined by unpaired t-test. n = 3 biological replicates with three technical replicates each. ( B ) Representative western blot of lysates collected from mt-mKeima/YFP-parkin HeLa cells transfected with siScr or siRab12 assessed for Rab12 levels and β-actin as a loading control. ( C ) Quantification demonstrates Rab12 protein knockdown with Rab12 siRNA by the same protocol. **** p < 0.0001, as determined by unpaired t-test. n = 3 biological replicates. ( D ) Representative 60X confocal images of YFP-Parkin (green), mt-mKeima at neutral pH (cyan), and mt-mKeima at lysosomal pH (magenta) and ( E ) quantification of mt-mKeima mitophagy analysis demonstrated increased levels of mitophagy induced by treatment with 30 μM FCCP with Rab12 knockdown. ** p < 0.01, **** p < 0.0001, as determined by two-way ANOVA with Bonferroni’s multiple comparisons test. n = 3 biological replicates. Data are presented as mean ± SEM.

Article Snippet: A working mix of Lipofectamine RNAiMAX transfection reagent (Fisher Scientific 13-778-150) and Opti-MEM reduced serum medium (Thermo Scientific 31985070) was prepared by diluting 300 μL RNAiMAX in 9.7 mL OptiMEM.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Western Blot, Knockdown

Journal: bioRxiv

Article Title: Rab12 is a regulator of mitophagy and mitochondrial homeostasis

doi: 10.64898/2026.03.29.715103

Figure Lengend Snippet: ( A ) mt-mKeima/YFP-Parkin HeLa were transfected with siRNA against Rab12 (siRab12) or a scramble control siRNA (siScr) for 72 h. Mito DNA DX analysis of mtDNA lesion frequency showed that Rab12 knockdown caused a significant decrease in mtDNA lesions relative to siScr transfection. n = 3 biological replicates. * p < 0.01, as determined by unpaired t-test. ( B ) No differences in steady state of mtDNA copy number was observed between conditions. n = 3 biological replicates. Data are presented as mean ± SEM.

Article Snippet: A working mix of Lipofectamine RNAiMAX transfection reagent (Fisher Scientific 13-778-150) and Opti-MEM reduced serum medium (Thermo Scientific 31985070) was prepared by diluting 300 μL RNAiMAX in 9.7 mL OptiMEM.

Techniques: Transfection, Control, Knockdown