Bnip3

The siRNA target sequence for mouse GSK-3β, Atg7 or BNIP3 was purchased from Santa Cruz Biotechnology. siRNA transfection was performed as previously described (Lee et al., 2015 (link)). Raw264.7 cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen, United States) according to the manufacturer’s instructions. siRNA was used at a final concentration of 60 pM.

The proteins were subjected to 10-12% gel electrophoresis as described previously 66 (link). The membranes were incubated with the following primary antibodies (1:1000 dilution) at 4 °C overnight: TFRC (Cell Signal Technology, 46222, MA, USA), HO1 (Cell Signal Technology, 43966, MA, USA), GPX4 (Abcam, ab125066, Cambridge, UK), BNIP3 (Santa Cruz Biotechnology, sc-56167, TX, USA), PINK1 (Novas Biologicals, BC100-494, CO, USA), PARK2 (Cell Signal Technology, 2132, MA, USA), VDAC (Abcam, ab14734, Cambridge, UK), COX IV (Cell Signal Technology, 4844, MA, USA), LC3B (Sigma‒Aldrich, L7543, MO, USA), KIM1 (R&D, AF1817, MN, USA) and TUBA (Beyotime, AF0001, Shanghai, China).

Total proteins were extracted from model mouse lung tissue and cells using a prepared SDS lysis buffer for Western blotting analysis. Then, 60 ug of protein was isolated using 12% SDS-polyacrylamide gel and imprinted onto polyvinylidene fluoride membrane (PVDF, Merck Millipore, Germany). Then, the membrane was sealed in 5% skim milk for 1.5 h. β-actin (Abcam, # ab8226), Collagen I (Arigo Biolaboratories, #ARG21965), HIF-1a (Proteintech, #20960-1-AP), LC3A/B (Cell signaling technology, #12741), P62 (Cell signaling technology, #16177S), Phospho-SQSTM1/p62(Ser349) (CST, #E7M1A), Bcl-2 (Abcam, #ab182858), BNIP3 (Santa Cruz Biotechnology, #sc-56167), BNIP3L (Proteintech, #12986-1-AP), BECN1 (Boster, #PB0014), and SOD2 (Boster, #BA4566) were used. The PVDF membranes were subjected to incubation with antibodies above at 4 °C overnight, then HRP-labeled secondary antibody (ZSGB-BIO, Beijing, China) was added and incubated for 1 h, and the immune reaction was detected by the ECL luminescence system.

Total protein was extracted from mouse lung tissues and cells using a prepared SDS lysis buffer for protein blotting analysis. The proteins were separated on SDS–polyacrylamide gels and transferred to a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Germany). The membrane was then blocked with 5% skimmed milk for 2 h. Antibodies against β-actin (Abcam, #ab8226), Collagen I (Arigo Biolaboratories, #ARG21965), TGF-β1 (ImmunoWay, #YT4632), P-NFKB (Wanleibio, #WL02169), NFKB (Bioss, #bsm-33117M), TNF-α (Proteintech, #60291-1-Ig), IL-1β (Wanleibio, #WLH3903), HIF-1a (Proteintech, #20960-1-AP), SOD1 (Wanleibio, #WL01846), SOD2 (Boster, #BA4566), LC3-II (Cell Signaling Technology, #12741), P62 (Cell Signaling Technology, #16177S), LAMP2 (Proteintech, #66301-1-Ig), P-mTOR (Abmart, #T56571), mTOR (Abmart, #T55306), P-ULK1 (ImmunoWay, #YP1544), ULK1 (Abmart, #T56902), Beclin1 (Boster, #PB0014), Atg5 (Bioss, #bs-4005r), BNIP3 (Santa Cruz Biotechnology, #sc-56167), P-AKT (Abmart, #T40067), AKT (Abmart, #BM4400), and TMEM175 (Proteintech, #19925-1-AP) were used. The PVDF membrane was incubated with these antibodies at 4 °C overnight. Subsequently, an HRP-labeled secondary antibody (ZSGB-BIO, Beijing, China) was added and incubated for 1 h, followed by detection using the ECL luminescence system.

Genetic knockdown studies for human ULK1, ATG7, TAX1BP1, BNIP3, PPT1, and MFSD12 were performed in A375P cell lines. Genetic inhibition studies for mouse Ppt1 and Calreticulin were performed in MC38 cells. Nontarget siRNA (sc-37007), human ULK1 (sc-44182), ATG7 (sc-41447), TAX1BP1/T6BP (sc-106831), BNIP3 (sc-37451), PPT1/CLN1 (sc-105216), MFSD12 (sc-97888), mouse Ppt1/Cln1 (sc-142398) and Calreticulin/Calregulin (sc-29895) siRNAs were purchased from Santa Cruz Biotechnology. These siRNAs consist of pools of 3–5 target-specific siRNAs.

The cells were lysed by RIPA (Solarbio, Beijing, China) with 1 mM PMSF (Solarbio, Beijing, China). Protein-extract supernatants were quantified using a BCA protein assay kit. A total of 20 μg protein was subjected to electrophoresis using SDS-PAGE and transferred to PVDF (Bio-Rad, Hercules, CA, USA). After blocking in 5% non-fat milk for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against Col2a1 (ABclonal, Wuhan, China, 1:1000), Mmp13 (Proteintech Group, Rosemont, IL, USA, 1:1000), p16 (Abcam, Cambridge, UK, 1:1000), p21 (ABclonal, Wuhan, China, 1:1000), p62 (ABclonal, Wuhan, China, 1:1000), Lc3 (Cell Signaling Technology, Danvers, MA, USA, 1:1000), Klf10 (Santa Cruz Biotechnology, Paso Robles, CA, USA, 1:1000), Bnip3 (Santa Cruz Biotechnology, Paso Robles, CA, USA, 1:1000), and β-actin (Proteintech Group, Rosemont, IL, USA, 1:5000), followed by incubation of the blots in HRP-conjugated secondary antibodies. The representative images were acquired by ECL reagents (Solarbio, Beijing, China) using an iBright 1500 imaging system (Thermo Fisher, Waltham, MA, USA).