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. 2019 Feb 19;10:845. doi: 10.1038/s41467-019-08772-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Kindlin-2 regulates PYCR1 and proline levels. a Kindlin-2 KO cells were infected with lentiviral vectors encoding 3xFLAG-tagged kindlin-2 (3fl-K2), 3xFLAG-tagged kindlin-2 F1, and F3 deletion mutant (3fl-K2∆F1∆F3) or 3xFLAG vector lacking kindlin-2 sequence (3fl). Three days later, the cells were analyzed by western blotting. b Protein levels of PYCR1 relative to that of GAPDH were quantified by densitometric analyses. The levels of PYCR1 in KO, 3fl, 3fl-K2, or 3fl-K2∆F1∆F3 cells were compared with that of A549 cells (normalized to 1) (n = 3). c The cytosolic (lane 3 and 5), mitochondrial (lane 4 and 6), and total (lane 1 and 2) lysates from A549 or kindlin-2 KO cells were analyzed by western blotting. d The cells were immunofluorescently stained with DAPI and anti-PYCR1 antibodies. Scale bar = 10 μm. e The mRNA levels of PYCR1 in kindlin-2 KO cells were analyzed by RT-PCR (n = 4) and compared with those in A549 cells (normalized to 1). f, g The proline levels were analyzed. The results were visualized (f) and quantified (g, n = 4). Scale bar = 1 cm. h–j A549 cells infected with kindlin-2 shRNA (Sh-K2) or control (Sh-NC) lentivirus were analyzed by western blotting (h). The proline levels were analyzed. The results were visualized (i) and quantified (j, n = 3). Scale bar = 1cm. k–m NCI-H358 cells were infected with kindlin-2 shRNA (Sh-K2) or control (Sh-NC) lentivirus for 5 days and then analyzed by western blotting (k). l, m The proline levels were analyzed. The results were visualized (l) and quantified (m, n = 3). Scale bar = 1 cm. n, o The effect of kindlin-2 on PYCR1 enzyme activity. The enzyme activity of purified PYCR1 (PY1) with or without GST-kindlin-2 (GST-K2) was analyzed. n The absorbance at 340 nm during the first 5 min of the reaction. o The relative activities of PYCR1 with or without GST-Kindlin-2 were calculated from n (n = 3). Data in b, e, g, j, m, and o represent means ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis in b, j, and m or using two-tailed unpaired Student’s t-test in e, j, and o, **P < 0.01; ***p < 0.001. NS, no significance