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. 2018 Nov;188(11):2508–2528. doi: 10.1016/j.ajpath.2018.07.025

Figure 2.

Figure 2

Apolipoprotein (APO) L1–miR193a axis in human parietal epithelial cells (PECs). A: Expression of APOL1 protein by the kidney and nonkidney cells. Protein blots of PECs, human embryonic kidney (HEK) cells, differentiated podocytes (PDs), and HepG2 cells were probed for APOL1 and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are displayed. PECs were evaluated for APOL1 phenotype; both PECs and HEK cells lack APOL1 protein expression. B: To differentiate PECs, undifferentiated PECs were incubated in special media for a different period (0, 4, 8, and 14 days) at 37°C. Subsequently, protein blots were probed for the APOL1 and reprobed for GAPDH. Blots from two different lysates are displayed in the top panel. The bottom panel shows cumulative densitometric data. APOL1 expression emerged on day 4 and progressed with time. C: To determine the time course of APOL1 transcription during the transition, RNAs were extracted from cell lysates of the above-mentioned experiment. cDNAs were amplified with a specific primer to APOL1. APOL1 transcription went up during the transition. D: To evaluate the time course of miR193a levels during the transition, miR193a levels were assayed from RNAs extracted in C. Cumulative data are shown in the bar diagram. miR193a levels went down during PEC transition. E: Undifferentiated PECs were transfected with scrambled (SCR) or APOL1-siRNA (25 or 50 nmol/L). RNAs were extracted. cDNAs were amplified with a primer specific for APOL1; cumulative data are shown in a bar diagram. The same RNAs were assayed for miR193a. APOL1 silencing in PECs up-regulates miR193a expression in a dose-dependent manner. F: HepG2 cells were transfected with SCR, at different concentrations (25, 50, and100 nmol/L), or APOL1 siRNA. RNAs were extracted from control (C) and experimental cells. cDNAs were amplified with a primer specific for APOL1. Cumulative data are shown in the bar diagram. RNAs were assayed for miR193a. In HepG2 cells, the lack of APOL1 enhances miR193a levels in a dose-dependent manner. G: To evaluate the effect of APOL1 induction on miR193a expression, vector (Vec) (lentivirus) and APOL1 lentivirus were transduced in PECs. RNAs were extracted from C, Vec, and APOL1-tranduced PECs. cDNAs were amplified with a primer specific for APOL1. miR193a levels were assayed from the extracted RNAs. Cumulative data of APOL1 mRNA are shown in the bottom panel and of miR193a levels in the top panel. APOL1 induction in PECs down-regulated miR193a expression. H: To determine the effect of miR193a down-regulation on the induction of APOL1, undifferentiated PECs (confluent at 33°C) were transfected with empty vector (EV) or miR193a inhibitor (inh) and incubated at 37°C for 48 hours. Proteins were extracted from C-, EV-, and miR193 inh–transfected PECs. Protein blots were probed for APOL1 and reprobed for GAPDH. Blots of three different lysates of control and experimental PECs are shown. Inhibition of miR193a induced the expression of APOL1 in PECs. I: To evaluate the dose-response effect of miR193a inhibition on APOL1 transcription, PECs (undifferentiated, 0 days) were transfected with EV or different concentrations of miR193a inhibitor (plasmid, 25, 50, and 100 nmol/L). After 48 hours of incubation, RNAs were extracted from C and experimental cells. RNAs were assayed for miR193a. cDNAs were assayed with a primer specific for APOL1. miR193a levels are shown in the bottom panel, and APOL1 mRNA levels are shown in the top panel. Inhibition of miR193a enhances the transcription of APOL1 in a dose-dependent manner. J: To examine the time course effect of miR193a inhibition, PECs (undifferentiated, confluent) were incubated in media that contained a specific inhibitor of miR193a (25 nmol/L, plasmid) for different periods (0, 24, 48, and 72 hours). RNAs were extracted, and cDNAs were amplified for APOL1. Inhibition of miR193a enhances the transcription of APOL1 mRNA in a time course manner. K: To assess the presence of APOL1-miR193a axis in nonkidney cells expressing APOL1, HepG2 cells were transfected with EV or different concentrations of miR193a plasmid (100, 200, and 300 nmol/L). RNAs were extracted and assayed for miR193a from C and experimental cells. cDNAs were amplified with a primer specific for APOL1. mRNA193a levels are shown in the bottom panel, and APOL1 mRNA levels are displayed in the top panel. Up-regulation of miR193a down-regulates the transcription of APOL1 in a dose-dependent manner. n = 3 (A, E–K); n = 4 (B and C). ∗P < 0.05 vs D0 (B and D); ∗∗P < 0.01 versus D0 and D4 (B); ∗P < 0.05 versus D0 and D4 (C); ∗∗P < 0.01 versus D0, D4, and D8 (C); ∗∗P < 0.01 versus D0 (D); ∗P < 0.05 versus respective SCR (E); ∗∗P < 0.01 versus respective SCR (E); †P < 0.01 versus respective SCR and 25 nmol/L siRNA APOL1 (E); ∗P < 0.05 versus respective C and SCR (F); ∗∗P < 0.01 versus respective C, SCR, and 25 nmol/L siRNA APOL1 (F); ∗∗P < 0.01 versus APOL1 (G); ∗∗∗P < 0.001 versus C and Vec (G); ∗P < 0.05 versus respective C and EV (I and K); †P < 0.05 versus respective C and EV (K); ∗∗P < 0.01 versus respective C and EV (I); ∗∗∗P < 0.001 versus respective C and EV (I); ∗P < 0.05 versus 0 and 24 hours (J); ∗∗P < 0.01 versus 0 and 24 hours (J); †P < 0.05 versus 48 hours (J); ∗P < 0.05 versus respective C, EV, and 100 nmol/L miR193a (K); ∗∗P < 0.01 versus respective C, EV, and 100 nmol/L miR193a (K). D, day.