Chemidoc system

Cells were seeded at a density of 3 × 10⁵ cells per well in a 6-well plate with 2mL of complete supplemented Keratinocyte SFM and allowed to rest overnight. The next day, culture media was replaced with 2mL of media without FBS supplement and serum starved prior to 24-hour groups receiving treatment. This process was repeated 18 h later for the 6-hour group cells and then again 5 hours later for the 1-hour group cells. Following treatment, media was removed, wells were washed with warm PBS, and then 200µL of warm buffer containing 100mM NEM (Thermo Scientific, 23030) was added to the well and allowed to incubate at room temperature for 10 min. Cells were lysed by the addition of 1% w/v SDS, scrapped on ice, and lysate was collected. Protein concentration was determined via BCA and 20 µg was loaded onto a non-reducing, denaturing 12% polyacrylamide gel. Gels were run at 120v for 90 min before being transferred to a nitrocellulose membrane by TransBlot Turbo (BIO-RAD) using the mixed molecular weight setting. A Ponceau stain was used to confirm transfer. Membranes were blocked with 5% NFDM (BIO-RAD, #1706404) at room temperature for 1 h and then incubated with either PRDX1 (proteintech, 15816-1-AP) or PRDX3 (proteintech, 10664-1-AP) at a 1:2000 dilution overnight at 4 °C. The next day, membranes were washed, incubated with anti-rabbit HRP linked secondary antibody (Invitrogen, # 31460) at a 1:10,000 dilution for 1 h at room temperature. Membranes were developed via enhanced chemiluminescence (Thermo Scientific, 32106), imaged on a ChemiDoc system (BIO-RAD), and band intensity was quantified via ImageJ.

The sequences of the specific primers and the product sizes are listed in Table 1. The presence of Mt was assessed via the use of 10 µL of Crystal Taq Master Mix (2x), 0.8 µL of oligos (0.3 µM), 2 µL of cDNA (150 ng), and 7.2 µL of RNase-free water. The reaction was performed in the MiniPCR thermocycler under the following conditions: 95°C for 120 s, 95°C for 15 s, 60°C (or the optimal temperature for each primer) for 30 s, and 72°C for 60 s; the last three steps were repeated for 30 cycles, followed by a final cycle at 72°C for 5 min. The products were loaded onto a 2 % agarose gel along with the products of the constitutive gene. The relative expression intensities of the genes were obtained via the Bio-Rad ChemiDoc™ system.Table 1

Primer characteristics according to Primer-BLAST.

Table 1

Gen		Sequence (5′→3′)	Variant	Product	Tm (°C)
β–Actin	FR	TTCTACAATGAGCTGCGTGTGGGGGTGTTGAAGGTCTCAAA	α1, 2, 9, 1, 11, 5, 10, 3, 8, 4, 6, like 2, X1.	122 pb	60
Mt−1	FR	TCTCCTTGCCTCGAAATGGACGGGCACACTTGGCACAGC	Mt−1X, Mt−1E, Mt−1E “like”, Mt−1E V2, Mt−1A, Mt-1M, Mt-1F, Mt-1F V1Mt−1E V1	151 pb499 pb	59
OGG1(BER)	FR	ACTCCCACTTCCAAGAGGTGGGATGAGCCGAGGTCCAAAAG	X8, X7, X6, X5, X4, X3, X2, X1, 2e, 2c, 2 f, 1b, 1a, 2 h, 2d, 1c, 1e, 1d, 2b, 2a, 2 g.	165 pb	58
APE1(BER)	FR	CAATACTGGTCAGCTCCTTCGTGCCGTAAGAAACTTTGAGTGG	2, 4, 3, 1.	88 pb	53
XPD(NER)	FR	TCTGCCTCTGCCCTATGATCGATTCCCTCGGACACTTT	X2, 1.CNOT3 (X16, X54, X52, X29, X28, X24, X16, X21)WIPF1	363 pb1991 pb3893 pb	54
XPB(NER)	FR	CCAGGAAGCGGCACTATGAGGGGTCGTCCTTCAGCGGCATTT	X2, X1, 1, 2, 3.	171 pb	53
Rad50(HR)	FR	CTTATACAGGACCAGCAGGAACCCTTTCTGTCGCCCTAATGC	Rad50	686 pb	58
MRE11(HR)	FR	CCAGAGAGCCCTTGTACGTTCCACCTCTTCGACCTCTTC	X10, X9, X7, X5, X4, X1, X10, X9, X7, X5, X4, X1, 1, 3, 2.ZBTB4.- X11, X12, X10, X9, X8, X2, X1, X7, X6, X5, X4, X1, X3, X2, 1, 2.FAM120A.- X9, X8, X7, X6, X5, X4, X3, X2, X1.SALL2.- X2, X1, 1, 6,SETD1A.- X6, X5, X4, X2, X3, X1.LRRC41	667 pb1675 pb2737 pb1543 pb949 pb208 pb	57
Ku70(NHEJ)	FR	CCGAGATACAGGCATCTTCCTAGCTTTAACCTGCTGAGTGCT	X1, 4, 3, 2, 1.PHYKPL.- X7	204 pb96 pb	54
Ku80(NHEJ)	FR	AGCATAGACTGCATCCGAGCTCCCCATACATCCACGACCT	1, X1.	315 pb	59

F, Forward; R, Reverse; pb, base pairs; Tm, Melting temperature (°C). The genes were purchased from Alpha DNA, PROBIOTEK.

Cell lysates were separated on 10–15% SDS–polyacrylamide gels with Laemmli buffer and Tricine buffer system. The proteins were then transferred onto the nitrocellulose membrane or PVDF membrane at 250 mA for 2 h or 100 mA for 8–12 h at 4 °C. The membrane was incubated with blocking buffer (5% BSA or milk in TBST) for 1 h at room temperature followed by incubation with respective antibodies (antibodies are listed in Supplementary Table 4) at 4 °C overnight. After washing with TBST three times, the membrane was incubated with IRDye goat anti-rabbit 800CW and goat anti-mouse 680RD secondary antibodies (LI-COR Biosciences) or HRP-conjugated anti-rabbit or mouse antibody. Imaging was performed by Odyssey Clx infrared imaging system (LI-COR) or Chemidoc system (BIO-RAD) or iBright FL1500 and bands were quantified and normalized using ImageJ.

Activated/inhibited/transfected BMDMs or BMDCs were collected, lysed in RIPA buffer (containing Tris-HCl, EDTA, NaCl, Triton X-100, PMSF, protease, and phosphatase inhibitors), and centrifuged at 13,475 × g for 20 min at 4 °C. Protein concentrations were measured with the Bradford assay. Equal protein amounts were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 0.1% TBS/T with 5% non-fat milk, incubated with primary antibodies overnight at 4 °C, followed by HRP-conjugated secondary antibodies. Target proteins were detected using chemiluminescent substrates and imaged with a BioRad ChemiDoc system.

Cell lysates were resolved on 10% SDS/PAGE gels for electrophoresis and transferred to PVDF membranes (Millipore) by a Trans-blot Turbo transfer system (Bio-rad). After blocking with 5% BSA at 37 °C for 1 h, probed with primary antibody overnight at 4 °C, the membrane was incubated with goat anti-mouse IgG HPR secondary antibody for 1 h at room temperature. Clarity Western ECL Substrate Kit (Bio-rad) and Chemi-doc system (Bio-rad) were used to detect protein bands.