Fig. 6: PINCH-1 promotes kindlin-2 interaction with PYCR1 and proline synthesis.
From: PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and tumor growth
a PINCH-1 KO A549 cells were infected with 3xFLAG- PINCH-1 (3f-P1) or 3xFLAG (3f) lentivirus. The cytosolic (Cyto) (lane 5, 7, 9, and 11), mitochondrial (Mito) (lane 6, 8, 10, and 12) and total (lane 1, 2, 3, and 4) fractions were analyzed by Western blotting. Kindlin-2 (K2) level in mitochondria was quantified (right, n = 3). b Cells were analyzed by IP and Western blotting. Lane 2, the sample was prepared as that of lane 3 except anti-kindlin-2 antibody was substituted with irrelevant mouse IgG. PYCR1 level was quantified (right, n = 3). c Cells were analyzed by PLA with kindlin-2 and PYCR1 antibodies. Bar, 10 μm. PLA dots were counted (right, n = 15). d Cells were analyzed by Western blotting. PYCR1 level in PINCH-1 KO cells was quantified and compared to that of A549 cells (right, n = 5). e, h The proline levels were analyzed using the absorbance method (right, n = 4). A representative set of samples were shown in the left. Scale bar, 1 cm. f The proline levels were analyzed by mass spectrometry (n = 5). g H1299 cells were infected with Sh-PINCH-1 (P1) or Sh-con lentivirus and analyzed by Western blotting. PYCR1 levels were quantified and compared to that in H1299 cells (right, n = 6). i The NADPH/NADP + ratios were analyzed. j, k ROS levels were analyzed with DHE (j). Scale bar, 75 μm. The mean fluorescence intensities (MFI) were calculated (k, n = 3). l ATP levels were analyzed (n = 4). m Mitochondrial respiration was analyzed using Seahorse. OCRs in wild type (green) and PINCH-1 KO (red) A549 cells before and after injection of oligomycin (1 μM), FCCP (1 μM), rotenone (Rot, 0.5 μM) and antimycin A (AA, 0.5 μM) are shown (A549 n = 4, KO n = 5). Data in a–m represent mean ± SEM. Statistical significance in a–l was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis. Statistical significance in m was determined using unpaired two-tail t-test. **P < 0.01; ***P < 0.001; NS no significance. The samples in a, d, and g were from same experiment and blots were processed in parallel. Source data are provided in Source Data file.
