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. 2021 May 3;32(5):1114–1130. doi: 10.1681/ASN.2020071076

Figure 8.

Figure 8.

M-Sec re-expression rescued the phenotype of M-Sec–KO podocytes. (A) M-Sec–KO podocytes were transfected with an adenovirus expressing M-Sec to induce M-Sec re-expression (KOAd–M-Sec). WT and M-Sec–KO podocytes transfected with mock vector (WTAd-Mock, KOAd-Mock) were used as controls. The graph shows the percentage of cells connected by TNTs (n=4; *P<0.001 WTAd-Mock versus KOAd-Mock; # P<0.01 KOAd-Mock versus KOAd–M-Sec by ANOVA). (B) The graph shows efficiency of mitochondrial transfer from donor podocytes, containing GFP-labeled mitochondria, to recipient podocytes labeled with CellTracker Blue as assessed by flow cytometry (n=4; *P<0.001 KOAd-Mock versus others). (C) The graph shows the profile of OCR normalized to total protein OD values under basal conditions and after ATP synthase inhibition with oligomycin, uncoupling the electron transporter chain with FCCP, and blocking complex I and III with rotenone and antimycin A (Rot/A). Dashed lines indicate injections of compounds. (D) Basal respiration, ATP-linked respiration, maximal respiration, and reserve capacity were quantified and shown in the graph (n=3; *P<0.001; # P<0.01 WTAd-Mock versus KOAd-Mock; § P<0.05 KO AD–M-Sec versus KO AD-GFP and WTAd-Mock). (E) Apoptosis was assessed by TUNEL assay (green) and nuclei counterstained with DAPI (magnification ×200, bar = 100 µm). The percentage of apoptotic cells is shown in the graph (n=5; *P<0.001 KOAd-Mock versus WTAd-Mock; § P<0.01 KOAd-Mock versus KOAd-M-Sec). (F) Nephrin mRNA expression was measured by real-time PCR and corrected for the expression of the housekeeping gene GAPDH (n=4; *P<0.001 KOAd-Mock versus WTAd-Mock; § P<0.01 KOAd-Mock versus KOAd–M-Sec). (G) Apoptosis was assessed by TUNEL assay (green) in podocytes (CellTracker Blue) exposed to vehicle or AD (0.8 µg/ml) for 24 hours either with and without coculture with vehicle (Podo)/Rotenone-treated (Rot-Podo) donor podocytes stained with Mito Red (magnification ×400, bar = 50 µm). The percentage of apoptotic cells is shown in the graph (n=3; *P<0.001 AD and AD + Rot-Podo versus others).