Fig 2: MRPL12 influences the opening of MPTP via ANT3(A–F) qRT?PCR and Western blotting were performed to detect MRPL12 and ANT3 expression in HK-2 cells transfected with MRPL12-silencing shRNA or MRPL12-overexpressing plasmids.(G and H) The opening of the mPTP in HK-2 cells was detected by a CoCl2-calcein fluorescence quenching assay. After CoCl2 quenching, calcein fluorescence decreased, indicating MPTP pore opening. Scale bars = 25 µm.(I and J) Quantification of calcein fluorescence was performed using Image J and is shown in the histogram. The independent experiments above were performed in triplicate. The differences were analyzed by Student’s t test. Data are presented as the mean ± SD, ****p<0.0001.

Fig 3: The MRPL12/ANT3 interaction is diminished in I/R-injured kidneys and H/R-treated HK-2 cells(A and E) Coimmunoprecipitation with endogenous proteins indicated that MRPL12 physically interacted with ANT3. HK-2 cells were treated with H/R or left untreated. Cell lysates were immunoprecipitated with normal IgG, MRPL12 (A), or ANT3 (E) antibody. The immunoprecipitates were immunoblotted (IB) with MRPL12 and ANT3 antibodies. The interaction between MRPL12 and ANT3 was diminished by H/R treatment.(B, C, F, and G) Quantifications of MRPL12 (B, F) and ANT3 (C, G) expression as shown in A and E, respectively.(D and H) Quantifications of ANT3 immunoprecipitated with MRPL12 (D) and of MRPL12 immunoprecipitated with ANT3 (H), as shown in A and E, respectively. The coimmunoprecipitated proteins were normalized to ß-actin.(I) Interaction of MRPL12 and ANT3 in HK-2 cells visualized by the Duolink@ proximity ligation assay. Scale bars = 25 µm.(J and K) Renal function of mice was evaluated by serum creatinine (sCr) and blood urea nitrogen (BUN).(L–O) HE staining for patients and mice with AKI compared with controls. Scale bars = 50 or 100 µm. Renal tubule injury was quantitatively analyzed.(P and Q) Representative images of immunohistochemical staining for MRPL12 and ANT3 expression in the human renal cortex. Scale bars = 100 µm.(R) Interaction of MRPL12 and ANT3 in the human renal cortex visualized by the Duolink@ proximity ligation assay. Scale bars = 25 µm. The independent experiments above were performed at least in triplicate. The differences were analyzed by Student’s t test. Data are presented as the mean ± SD, **p<0.01, ***p<0.001 and ****p<0.0001.

Fig 4: MRPL12 attenuates MPTP opening by interacting with ANT3 under H/R conditions(A) HK-2 cells transfected with MRPL12 FL-overexpressing plasmid were subjected to H/R treatment, and the interaction of MRPL12 and ANT3 was visualized by a Duolink@ proximity ligation assay. Scale bars = 25 µm.(B) Mean intensity was calculated by Image J and is shown in the histogram.(C) HK-2 cells transfected with empty vector, MRPL12 FL-overexpressing plasmid or MRPL12 DM3-overexpressing plasmid were subjected to H/R treatment. The opening of the MPTP in HK-2 cells was detected by a CoCl2-calcein fluorescence quenching assay. Scale bars = 25 µm.(D) Quantification of calcein fluorescence was performed using Image J and is shown in the histogram.(E) HK-2 cells transfected with scramble shRNA or MRPL12-silencing shRNA were subjected to H/R treatment. The opening of the MPTP in HK-2 cells was detected by a CoCl2-calcein fluorescence quenching assay. Scale bars = 25 µm.(F) Quantification of calcein fluorescence was performed using Image J and is shown in the histogram. The independent experiments above were performed in triplicate. The differences were analyzed by Student’s t test. Data are presented as the mean ± SD, **p<0.01, ***p<0.001 and ****p<0.0001.

Fig 5: MRPL12 manipulates MPTP opening by influencing ANT3 conformation(A) Domain structure of MRPL12 FL and MRPL12 DMs. MRPL12 DM1: 45–198 amino acids; MRPL12 DM2: 1–45 and 91–198 amino acids; MRPL12 DM3: 1–90 and 129–198 amino acids; MRPL12 DM4: 1–128 amino acids.(B) FLAG and rabbit immunoglobulin (IgG, used as a negative control) immunoprecipitations (IP) of lysates from 293 T cells cotransfected with ANT3-FLAG and MRPL12 FL-HA or the indicated MRPL12 DMs-HA. Immunoprecipitates were immunoblotted (IB) either with HA or FLAG antibodies to examine deletion mutants of MRPL12 or ANT3, respectively.(C and E) The opening of the MPTP in HK-2 cells was detected by a CoCl2-calcein fluorescence quenching assay. After CoCl2 quenching, calcein fluorescence decreased, indicating MPTP pore opening. Scale bars = 25 µm.(D and F) Quantification of calcein fluorescence was calculated by Image J and is shown in the histogram.(G) Western blotting for OXPHOS components in MRPL12 FL-overexpressing or MRPL12 DM3-overexpressing HK-2 cells. The independent experiments above were performed in triplicate. The differences were analyzed by Student’s t test. Data are presented as the mean ± SD, **p<0.01, ***p<0.001 and ****p<0.0001.