M mlv reverse transcriptase

Wild-type cells were plated at a density of 3 × 10⁵ cells/per well in 35 mm petri dishes. The cells were treated singly with metformin (1 mM), Enz (10 µM), or Abi (5 µM), or in specific combinations for 5 days. Total mRNA was extracted using Trizol (Life Technology, Carlsbad, CA, USA) and isolated with RNA mini-prep columns (Qiagen, Valencia, CA). The mRNA was reverse transcribed to cDNA with M-MLV reverse transcriptase (Roche Diagnostics, Basel, Switzerland). Reverse-transcribed cDNA (10–100 ng) was amplified by real-time PCR using IQ SYBR green mix (Bio-Rad, Hercules, CA, USA) and detected via the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad). Each reaction was performed in duplicate. The sequences of AR, Arv7, MID1, and TBP primers are provided in Table 2. The sequences of Arv7 primers used are in reference [49 (link)].

Total cellular RNA was extracted from cells by using TRIzol, according to the manufacturer’s instructions (Invitrogen). Potential DNA contamination was mitigated by treating samples with RNase-free DNase (Promega). cDNA was prepared using MMLV Reverse Transcriptase (Roche). For amplification, the cDNA was mixed with 1 μL of forward and reverse primers (5 μM each), 5.5 μL of RNase-free water, and 7.5 μL of 2 × PCR SYBR Green Mix buffer in a 15-μL reaction. The PCR protocol comprised 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative quantitation was performed by using an ABI PRISM 7500 sequence-detection system (Applied Biosystems, Foster City, CA, USA) and measuring real-time SYBR green fluorescence, and results were obtained by using the comparative Ct method (2-ΔΔCt) with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. The primers used are listed in Supplementary File 3.

Total RNA for RNA-seq samples was also used for qRT-PCR analysis. First-strand cDNA was synthesized using M-MLV reverse transcriptase (Roche, Switzerland) and oligo (dT) 18. We chose 8 plant hormone-related genes, 3 SL perception-related genes and randomly selected 12 genes for further study and validation of RNA-seq using qRT-PCR. Each transcript primer (Table S1) was designed using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA). OaTublin1 was used as an internal control [89 (link)]. qRT-PCR was conducted using SYBR GreenER™ qPCRSuperMix Universal (Invitrogen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. Thermal cycle conditions for PCR were as follows: 94 °C for 3 min, 40 cycles including 94 °C for 15 s and 60 °C for 30 s. Relative expression levels were calculated using the 2^−ΔΔCt method [90 (link)] and one-way ANOVA (IBM SPSS Statistics 19.0, Armonk, NY, USA).

Total cellular RNAs were extracted from MCF10A and MDA-MB-231 by using TRIzol (Invitrogen). cDNA was prepared using MMLV Reverse Transcriptase (Roche) and amplificated using 2 × PCR SYBR Green Mix buffer in a 15-μL reaction. The PCR process run 40 cycles of 95° C for 15s and 60° C for 1 min in ABI PRISM 7500 sequence-detection system (Applied Biosystems, Foster City, CA, USA). The results were shown by using the comparative Ct method (2-ΔΔCt) with β-actin as an internal control. The primers used were supplied in Supplementary Table 9.

Total RNA was extracted using Trizol® Reagent (Invitrogen). RT-PCR was performed as described previously [7 (link)] using a reaction kit containing M-MLV reverse transcriptase (Roche) and ProTaq® DNA polymerase (Roche).The primers used for c-Maf, CCND2, ITGB7 and GAPDH were described previously [7 (link)]. The thermal cycle conditions used were as follows: 1 cycle at 94 °C for 5 min; 35 cycles at 94 °C for 30 s; 55 °C for 30 s; 72 °C for 30 s; and 1 cycle at 72 °C for 10 min.