P19arf

Bacterially produced full-length mouse p16^Ink4a was used to immunize rabbits, and antibodies were affinity purified. Frozen lung sections (8 μm in thickness) were immunostained using p19^Arf (1:100 dilution, sc-32748, Santa Cruz Biotechnology) and p16^Ink4a antibodies. Sections were visualized using Alexa Fluor 647-conjugated anti-rat IgG (1:1000 dilution, 712-606-150, Jackson ImmunoResearch) and Alexa Fluor 488-conjugated anti-rabbit IgG (1:1000 dilution, 711-545-152, Jackson ImmunoResearch). Sections were counterstained with DAPI. SA β-gal staining was performed using a Cellular Senescence Detection Kit—SPiDER βGal (SG03, Dojindo).

Commercially available antibodies to HRP-conjugated p53, cyclin D1, CDK2, TIM23, LC3, p62, phospho-p53 (Ser392 and Ser46), p19-ARF, cMyc, Caspase 8, DRAM1, and LAMP2 were purchased from Santa Cruz Biotechnology, CA. Phospho-MDM2 (Ser166 and Ser186), total MDM2, cleaved PARP, total PARP, pro-caspase 9, Caspase 3, phospho-Drp1 (Ser616), Beclin1, phospho-Ulk1 (Ser757), phospho-AMPKα (Thr172), phospho-Beclin1 (Ser15), and phospho-mTOR (Ser2481) were procured from Cell Signaling Technology, MA. HRP-conjugated antibody to actin, Mdivi1, Z-VAD-fmk, chloroquine, and FH535 were purchased from Sigma-Aldrich, MO. LAPTM4B was purchased from Novus Biologicals, CO. Commercially available AZD5363, rapamycin, and mTOR inhibitor (AZD8055) were procured from Cayman Chemicals, MI.

Tissues were fixed in 10% buffered formalin, embedded in paraffin wax and sectioned at 5 mm. For histological examination sections were stained with hematoxylin and eosin, according to standard procedures as previously described [62 ]. CC3 Cleaved Caspase 3 Asp175 (Cell Signaling Technology 9661), CC10 (Santa Cruz Biotechnology sc-9772), CD4 (Cell Signaling Technology 25229, prosurfactant protein C (millipore AB3786), p21 (291 H/B5, homemade), γH2AX Ser 139 (Millipore 05-636), PPERK Thr202/Tyr204 (Cell Signaling Tehcnology 9378), Ki67 (Cell Signaling 12202), MPO (Dako A0398), F4-80 (ABD Serotec MCA497), p16 (33B, homemade), p19 ARF (sc-32748 Santa Cruz), pRb (ser807/811, #9308, Cell Signaling), pSMAD3 (ser423/425 ab52903, Abcam), pSTAT3 (tyr705, #9145 Cell Signaling), p53 (POE316A, homemade), cyclin D1 (M3635, Dako), c-MYC (ab32072, Abcam), PPARγ (Cell Signaling, #2435), HIF1α (Cell Signaling, #36169), 8-hydroxy-2’-deoxyguanosine (Abcam, ab48508), 4-hydroxy-2-nonenal (Alpha Diagnostic, HNE11-S) antibodies were used for immunohistochemistry in tissue sections. Pictures were taken using Olympus AX70 microscope. The percentage of positive cells was identified by eye and the areas were calculated by ImageJ and Zen 3.1 (Zeiss) softwares.