Fig. 4.

Proliferation, apoptosis, and protein synthesis defects upon GON7 and YRDC knockdown. Transient knockdown (KD) of GON7, LAGE3, YRDC, and OSGEP was performed by lentiviral transduction of shRNA in immortalized human podocyte cell lines with a scrambled (non-targeting) shRNA as control. a Cell proliferation was assessed using a colorimetric MTT assay over 7 days, measuring absorbance at 490 nm at days 1, 2, 3, 4, and 7 (mean ± s.e.m. of n = 5 experiments, with each experiment performed in triplicate; two-way ANOVA (p < 0.0001), Dunnett’s multiple comparisons test, n.s. = 0.2031, ***p < 0.0007, ****p < 0.0001). b Cell apoptosis was evaluated by quantification of caspase 3/7 activation on the basis of fluorescence intensity (530/405 nm). Absolute values were normalized to DAPI fluorescence intensity as an internal control and compared to non-targeting shRNA-treated control cells (scrambled) (mean ± s.e.m. of n = 3 experiments with each experiment performed in triplicate; one-way ANOVA (F (4,10) = 21.42, p < 0.0001), Dunnett’s multiple comparisons test, n.s. = 0.0556, **p = 0.0012, ****p < 0.0001). c Protein biosynthesis rates were assessed on the basis of incorporation of HPG, an alkyne-containing methionine analog. After 2 h, alkyne-containing proteins were quantified on the basis of fluorescence intensity (485/535 nm). Absolute values were normalized to DAPI fluorescence intensity as an internal control and compared to control cells (mean ± s.e.m. of n = 3 experiments, with each experiment performed in triplicate, one-way ANOVA (F (4,10) = 16.36, p= 0.0002), Dunnett’s multiple comparisons test, **p = 0.0035, ****p < 0.0003). d Relative expression of GON7, YRDC, LAGE3, and OSGEP transcripts were normalized to that of HPRT in KD podocytes compared to non-targeting shRNA control treated cells (mean ± s.e.m. of n = 5 experiments, with each experiment being performed in triplicate; two-tailed Mann–Whitney test, **p < 0.05). Source data are provided as a Source Data file