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. 2020 May 21;11:2549. doi: 10.1038/s41467-020-16312-7

Fig. 5. Drp1-K38A overexpression phenocopies PGAM5−/− mitochondrial phenotype and cell senescence.

Fig. 5

a Tom20 immunostaining in WT and PGAM5−/− ARPE-19 cells infected with adenovirus expressing Ad-LacZ or Drp1-K38A mutant, showing that Ad-Drp1-K38A mutant mimics PGAM5−/− hyperfusion phenotype. Scale bar = 20 µm. n = 4. b Quantification of mitochondrial branches in a using ImageJ. n = 4, *p < 0.05, **p < 0.005, analyzed by two-way ANOVA Tukey’s multiple comparisons test. Error bars, mean ± s.e.m. c ATP production in WT ARPE-19 cells overexpressing Ad-LacZ or Ad-Drp1-K38A. n = 10 biologically independent samples, ****p < 0.0001, two-tailed unpaired t-tests, error bars, mean ± s.d. d ATP production in PGAM5 KO cells overexpressing Ad-LacZ or Ad-Drp1-K38A. n = 10 biologically independent samples, ****p < 0.0001, two-tailed unpaired t-tests, error bars, mean ± s.d. e Expression of DRP1, phosphor-S6 and S6 in Ad-LacZ or Ad-Drp1-K38A overexpressing HRPE cells at 7 days after infection. Samples were collected at 48 h after the last medium change for western blot. β-Actin was used as loading control. n = 3. f SA-gal staining of HRPE cells 5 weeks after Ad-GFP or Ad-Drp1-K38A infection (MOI = 100). Scale bar = 500 µm. Boxed region represents the magnified picture in the figure. n = 3. g Expression of Drp1, Lamin B1, MMP3 and P16Ink4a protein at 8 weeks after infection. Samples were collected at 48 h after the last medium change for western blot. α-Tubulin was used as loading control. n = 4. h Quantification of bands in g. n = 4, **p = 0.0018 (Lamin B1), **p = 0.0026 (MMP3), ***p = 0.0004, two-tailed unpaired t-tests, error bars, mean ± s.d. i mRNA level of IL6, MMP3 and TNFα were measured by qRT-PCR at 8 weeks after infection as shown in f. Samples were collected at 48 h after the last medium change. n = 3, *p = 0.0338, **p = 0.0057, ***p = 0.0001, two-tailed unpaired t-tests, error bars, mean ± s.e.m. For assays in the figure, n represents the number of biologically independent experiments unless otherwise specified. Images were captured under same settings, and representative images were shown. Source data are available as a Source Data file.