Cfx instrument

The samples were prepared as previously described [45 (link)]. Briefly, total RNA was isolated from each cell line homogenized in Trizol reagent (Thermofisher) using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instruction. RNA was reverse-transcribed to synthesize cDNA using iScript cDNA Synthesis kit (Bio-rad). Real-time quantitative PCR was performed on a CFX instrument (Bio-rad) with SYBR Green reagent (Bio-rad) and specific primers as followed: KRT18-forward, 5′-CAGAGACTGGAGCCATTACTTC-3′; KRT18-reverse, 5′-GCCAGCTCTGTCTCATACTTG-3′; CDH1-forward, 5′-CTGCCAATCCCGATGAAATTG-3′; CDH1-reverse, 5′-TCCTTCATAGTCAAACACGAGC-3′; CDH2-forward, 5′-CCCAAGACAAAGAGACCCAG-3′; CDH2-reverse, 5′-GCCACTGTGCTTACTGAATTG-3′; VIM-forward, 5′-ACCCTGCAATCTTTCAGACAG-3′; VIM-reverse, 5′-GATTCCACTTTGCGTTCAAGG-3′; FN1-forward, 5′-GTGGCAGAAGGAATATCTCGG-3′; FN1-reverse, 5′-GAGAATACTGGTTGTAGGACTGG-3′; CTGF-forward, 5′-CGACTGGAAGACACGTTTGG-3′; CTGF-reverse, 5′-AGGCTTGGAGATTTTGGGAG-3′; CYR61-forward, 5′-GAGTGGGTCTGTGACGAGGAT-3′; CYR61-reverse, 5′-GGTTGTATAGGATGCGAGGCT-3′; AXL-forward, 5′-TTTATGACTATCTGCGCCAGG-3′; AXL-reverse 5′-TGTGTTCTCCAAATCTTCCCG-3′; AMOTL2-forward 5′-GACTTCAACCGGGATCTTAGAG-3′; AMOTL2-reverse 5′-CCAGCTTCTCTTGCTCCTG-3′; 18 s RNA-forward, 5′-GTAACCCGTTGAACCCCATT-3′; 18 s RNA-reverse, 5′-CCATCCAATCGGTAGTAGCG-3′.

The pMV261 empty and casR-overexpressing strains were grown to OD₆₀₀ of 1.0 in 100 mL of 7H9 medium. The extraction of mRNA from the cultures and qRT-PCR analysis was performed as described previously [23 (link)]. The cDNA was obtained using the HiScript II Q RT kit (Vazyme, Nanjing, China). The qRT-PCR system consisted of 20 μL of solution containing 10 μL of 2 × SYBR Green qPCR mix, 400 nM specific primers, and 1 μL of cDNA. The reactions were performed in a Bio-Rad CFX instrument under the following program: 95 °C for 1 min and 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s. The expression level of each gene was normalized with sigA as an internal reference. Gene relative expression was determined according to the 2^−ΔΔCt method [28 (link)].

PCR assays for transcripts that are correctly spliced around the IAP insertion and for reference genes were designed relative to exon junctions using Primer3 [49 (link)]. Assays were selected for use if they produced a single product by melt profile, single band by gel imaging, and low coefficient of variation among replicate samples. Specific primers used are provided in S4 Table. RNA was prepared from tissues homogenized in Trizol, converted to cDNA by reverse transcription, and assayed by quantitative PCR using SYBR green fluorescence in a Bio-Rad CFX instrument as previously described [19 (link)]. Normalization was performed by geometric averaging among the indicated reference genes, as described [50 (link)], and implemented in software supplied by the manufacturer. Four reference gene assays, Gapdh, Ppia, Sdha, and Pitpna were selected based on high cycle efficiencies, accurate dose titration, and low quantitative variation across tested conditions (only the first three were used for comparisons including vibrator samples, which express ~18% normal level of Pitpna).

Quantitative RT-PCR (qRT-PCR) was conducted as already depicted^{3 (link)}. Total RNA was isolated from CSC221 cells using RNAiso Plus (TaKaRa, Otsu, Japan). 1 mg of RNA was converted to cDNA using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). qPCR was performed using SYBR Green (Enzynomics, Seoul, Korea)^{99 (link)}. Primers for real-time PCR are listed in Supplementary Table 2. qRT-PCR reactions and analyses were performed on a CFX instrument (Bio-Rad, Hercules, CA, USA)¹⁰⁰ .

Total RNA was extracted from CRC cells using RNAiso Plus (TaKaRa, Otsu, Japan). cDNA was synthesized from one microgram of RNA by M-MLV (Invitrogen, Carlsbad, CA, USA). SYBR Green reagents (Enzynomics, Seoul, Korea) were used to determine relative expression of candidate genes. The list of primers used in this research is reported in Additional file 1: Table S1. CFX instrument (Bio-Rad, Hercules, CA, USA) was used to perform the analysis.