Cdkn1a

Total RNA was extracted from HCT116 and HT29 cells using a RNeasy Plus Mini Kit (Qiagen, Venlo, Netherlands). Reverse transcription was performed using the SuperScript VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific). Polymerase chain reaction amplification was performed using the following primers: CDKN1A forward primer (5′-CCGGCTGATCTT CTCCAAGAG-3′) and reverse primer (5′-AAGATGTAGAGCGGGCCTTTG-3′), TP53 forward primer (5′-ATCCTCACCATCATCACACTGG-3′) and reverse primer (5′-ACAAACACGCACCTCAAAGC-3′), and TBP forward primer (5′-TGCTGCGGT AATCATGAGGATA-3′) and reverse primer (5′-TGAAGTCCAAGAACTTAGCTG GAA-3′) (Thermo Fisher Scientific). Real-time PCR was performed using a Light Cycler LC480 (Roche Diagnostics, Rotkreuz, Switzerland) with SYBR Green I dye (Roche) and TB Green Premix Ex Taq II (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. A constant amount of RNA was reverse transcribed to complementary DNA, and the complementary DNA was then amplified using the following conditions: 1 cycle of denaturation at 95 °C for 5 min, and 48 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 5 s, and extension at 72 °C for 3 s. The expression of each gene was normalized to TBP mRNA level and is represented as fold changes compared to the control group.

Real-time quantitative RT-PCR (qRT-PCR) was performed for selected genes using Taqman chemistry and the ABI 7900 Real Time PCR System. The Globin cleared purified RNA from 5 control and irradiated animals was used for cDNA synthesis using the High-Capacity cDNA Archive Kit (Life Technologies). Gene expression assays (primer/probe sets) were purchased from Thermo Fisher for the following genes: Ifit3 (Mm00366278_m1), Ifit1 (Mm00365614_m1), Cdkn1a (Mm04205640_g1), Aen (Mm00471554_m1), Gstt1 (Mm00492506_m1), Il18r1 (Mm00515178_m1), Ccng1 (Mm00438084_m1), Alas1 (Mm001235914_m1), Acsbg1 (Mm00547366_m1), Cyp2b6 (Mn00456591_m1), Actb (Mm00607939_s1). The ΔΔCT method was used to calculate expression relative to controls, using normalization to Actb expression. Each reaction was run in triplicate with 5 control and 5 irradiated samples, and means were compared using an unpaired t-test.

Total RNA was purified using the RNeasy Plus Mini Kit (Qiagen, Copenhagen, Denmark). The RNA concentration was measured by the Qubit RNA HS kit and the Qubit 4 Fluorometer (ThermoFisher Scientific) and stored at −80 °C until use. Two μg total RNA was reverse transcribed using the RevertAid Minus First strand cDNA synthesis kit (ThermoFisher Scientific) using oligo(dT) primers, following the manufacturer’s instructions. qPCR was performed with TaqMan Fast Advanced Master Mix TaqMan assays for SOX2 (Hs01053049_s1), NANOG (Hs04260366_g1), POU5F1 (Hs0099632_g1), CD44 (Hs01075864_m1), PROM1 (Hs01009250_m1), BAD (Hs00188930_m1), BAK1 (Hs00832876_g1), BAX (Hs00180269_m1), BCL2 (Hs00608023_m1), TP53 (Hs01034249_m1), CDKN1A (Hs00355782_m1), HPRT1 (Hs02800695_m1), and GAPDH (Hs99999905_m1), all from ThermoFisher Scientific. Ten ng cDNA was used per reaction, as recommended by the manufacturer’s protocols. The qPCR was performed in a QuantStudio 3 (ThermoFisher Scientific) for 2 min at 50 °C, 2 min at 95 °C, followed by 40 cycles at 95 °C for 1 sec and 60 °C for 20 s. Expression levels were normalized to HPRT1 and GAPDH, and relative expression levels were calculated using the Pfaffl-method [54 (link)].

Total RNA was isolated using the Trizol reagent according to manufacturer’s protocol (MilliporeSigma). Altogether, 1 μg of total RNA was used to synthesize cDNA using High Capacity RNA-to-cDNA (4387406, Thermo Fisher Scientific) kit per manufacturer’s protocol. Primers for HPV 16 and -18 native and vector sequences were synthesized by Integrated DNA Technologies, from reference sequences (8 (link), 43 (link)–45 (link)) or as de novo sequences (Supplemental Table 1). Taqman probes were used for the p53 target genes: CDKN1A, NOXA, PUMA, BAX, GADD45A, and MDM2 (Thermo Fisher Scientific). Probes for the DREAM target genes — BIRC5, CDC25c, KIF23, MYBL2, and PLK4 — were obtained from Thermo Fisher Scientific. GAPDH and ACTB were used as reference genes. The qPCR was run at 50°C for 2 minutes, 95°C for 10 minutes, and 60°C for 1 minute using the 7900HT Fast Real-Time PCR System (Applied Biosystems). The specificity of the reaction was verified by melt curve analysis. Data processing was performed using SDS 2.4 software (Applied Biosystems), and the relative quantitation of each mRNA was performed using the comparative Ct method as described earlier (46 (link)).