Pink1

Complementary DNA (cDNA) was synthesized by a Verso cDNA RT kit (Cat# AB1453B, Thermo Scientific) per the manufacturer's protocol. Real-time PCR for target mRNAs was performed using TaqMan gene expression assays for Lipocalin 2 (Lcn2, Mm01324470_m1), Ccl2 (Mm00441242_m1), Ccl7 (Mm00443113_m1), Gbp2 (Mm00494576_g1), IL-1β (Mm0133-6189_m1), Myc (Mm00487804_m1), IL-6 (Mm00446190_m1), suppressor of cytokine signalling 3 (SOCS3, Mm00545913_s1), C5ar1 (Mm00500292_s1), Trem2 (Mm04209424_g1), Ptgs2 (Mm00478374_m1), Nlrp3 (Mm00840904_m1), Mb21d1 (Mm01147496_m1), Tmem173 (Mm00727224_s), Tnfrsf1 (Mm00441883_g1), Fos (Mm00487425_m1), Tgm2 (Mm00436979_m1), Lamp1 (Mm01217070_m1), Pink1 (Mm00550827_m1), Apoe (Mm01307192_m1), Blnk (Mm01197846_m1), and GAPDH (Mm99999915_g1) (Applied Biosystems, Carlsbad, CA] on an QuantStudio™ 5 Real-Time PCR System (Applied Biosystems). Gene expression was normalized by GAPDH and compared to the control sample to determine relative expression values by the 2-ΔΔCt method 31 (link), 41 (link).

Rapamycin (Sigma) was used in a final concentration of 0.5 µM and LY294002 (Jena Bioscience) in a final concentration of 25 µM. RNA was isolated with the RNeasy mini kit (Qiagen) and then treated with DNase I. cDNA was synthesized with SuperScript III reverse transcriptase using oligo(dT)₂₀ and random primers (Invitrogen). cDNA from 30 ng RNA were utilized in a 20 µl reaction volume using the StepOnePlus Real-Time PCR System and the appropriate TaqMan gene expression assays (Applied Biosystems): PINK1 (Hs00260868_m1), ATG5 (Hs00355494_m1), ATG6 (Hs00186838_m1), ATG7 (Hs00197348_m1), ATG8E (Hs01076567_g1), p62/SQSTM1 (Hs00177654_m1), PARK2 (Hs01038318_m1) LAMP-1 (Hs00931464_m1), LAMP-2 (Hs00903587_m1), Mfn1 (Hs00566851_m1S), Mfn2 (Hs00208382_m1), Opa1 (Hs01047019_m1), Drp1 (Hs00247147_m1), Fis1 (Hs00211420_m1) March5 (Hs00215155_m1), ATP13A2 (Hs00223032_m1) SOD2 (Hs00167309_m1), HIF1A (Hs00936368_m1) NFKBIA (Hs00153283_m1), FOXO3A (Hs00921424_m1), SGK1 (Hs00178612_m1), ACTB (Hs99999903_m1), GAPDH (Hs99999905_m1). mRNA expression was normalized to the TATA box binding protein (TBP: Hs99999910_m1). Relative expression changes were calculated with the 2^-ΔΔCt method [48] (link).

Cells were lysed in lysis buffer containing 20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 1 mM Na₃VO₄, and 5 mM NaF. Proteins were extracted by centrifugation, and equal amounts of proteins were loaded onto a gel, resolved by SDS-PAGE, and transferred onto a PVDF membrane (Millipore, MA, USA). The membranes were blocked for 1 h with 5% skim milk in 1 × TBS-T (20 mM Tris–HCl, 137 mM NaCl, pH 7.5, and 0.1% Tween-20). The membranes were probed overnight with a primary antibody at 4 °C. The following primary antibodies were used: p53, p21 (ABclonal Technology, MA, USA), LC3B (Cell Signaling Technology), PINK1, Parkin, BNIP3 (Thermo Fisher Scientific), β-actin (Santa Cruz, CA, USA). The primary antibody was detected for 2 h with anti-mouse in sheep and anti-rabbit in donkey (Jackson ImmunoResearch, PA, USA). Protein bands were visualized using a western blot detection system (Millipore) and developed using an image analyzer (Vilber, Collégien, France). Protein levels were quantitated with Image J (NIH, MD, USA).