Loading buffer

Total RNA was isolated using RNAiso plus (TaKaRa, Japan) according to the manufacturer’s instructions. For RNase R treatment, 2 mg of total RNA of THP-1 was incubated 20 min at 37 °C with or without 3 U/μg RNase R (Epicentre Technologies, USA). Reverse transcription of mRNA and miRNA was conducted using random primers system (TaKaRa, Japan) and stem-loop primers in the GeneCopoeia system (MD, USA), respectively. gDNA was extracted by QuickGene DNA whole blood kit S (KURBO, Japan). PCR was performed using PrimerSTAR HS DNA Polymerase (TaKaRa, Japan) system. After PCR amplification, 10 μl amplified product was mixed with loading buffer (TaKaRa) and ran on 1% agarose gel with gel red. The amplified product was visualized under UV. Quantitative RT-PCR was performed using a 2× Taq PCR mix (TaKaRa). The All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia) was used for miRNA RT-PCR. The primers are listed in Additional file 1: Table S2.

RNA samples (50 μg) were denatured for 10 min at 65°C in loading buffer (TAKARA, China). The treated RNA was separated on 10% urea-polyacrylamide gels for 1.5 h at 120 V after a pre-run for 0.5 h at 200 V and transferred to Hybond-N nylon membranes (Amersham, Germany) by electroblotting for 1 h at 300 mA. Gene-specific oligonucleotides were labeled with [γ-³²P]ATP (PerkinElmer, USA) by the exchange reaction of T4 polynucleotide kinase (NEB, USA) using 10 U of enzyme, 10 pmol oligonucleotide, and 40 μCi [γ-³²P]ATP in reaction buffer for 1 h at 37°C. The membranes were UV-crosslinked at 1,200 J and the blots were prehybridized at 45°C for 1 h in Hybridization Solution (#HYB-101, TOYOBO, Japan). Hybridization with specific [γ-³²P]ATP end-labeled oligonucleotides was then performed overnight at 45°C. The membranes were washed in 0.1% SDS in 5X SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) at 45°C, followed by 0.1% SDS in 1X SSC for 30 min per wash. Signals were detected and analyzed using a Cyclone Plus Phosphor Imager (#C431200, PerkinElmer, USA). All DNA oligonucleotides used for RNA blot analysis are listed in Supplementary Table 1.

The PCR reaction is affected by many factors. Therefore, the parameters of the m-PCR assay were optimized by varying concentration of deoxyribonucleotide triphosphate (dNTPs; 0.1–0.4 mM), Mg²⁺ (0.2–0.5 mM), Taq DNA polymerase (1.0, 1.5, 2.0, and 2.5 U), and betaine (0.05–0.4 mM) in a 25-µL reaction volume.
A mixture of the genomic DNA, which contained same amount of genomic DNA of the six types of bacteria, was used as a template to amplify the corresponding target genes. The total volume of each reaction system (recommended system) was 25 μL, which included 1 μL of template DNA (about 150 ng of genomic DNA).
PCR cycles were as follows: pre-denaturation at 94 °C for 4 min, denaturation at 94 °C for 40 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, for 25–35 cycles, extension at 72 °C for 10 min, and preservation at 16 °C. After the reaction, 5 μL of the reaction solution was mixed with 1 μL of loading buffer (6×; TaKaRa Biotechnology) for 1.5% agarose gel electrophoresis.

Cell proteins were extracted with RIPA lysis buffer (KeyGen, Nanjing, China), and measured with the BCA kit (Thermo Scientific, Waltham, MA, USA) to determine their concentration. The protein mixture (30 μg) with the loading buffer (Takara, Kusatsu, Japan) was loaded onto SDS–polyacrylamide gel (10%; Invitrogen, Carlsbad, CA, USA), electrophoresed for 2h, transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) and then blocked with WB Blocking Buffer (KeyGen, Nanjing, China) for 1h.The membranes were then incubated with the 1:1000 diluted primary antibody anti-light CDK19 (SAB, Cat No. 37480), MMP-7 (CST, Cat No. 3801S, CDH1 (CST, Cat No. 14472S), CDH2 (CST, Cat No. 13116S), VIMENTIN (CST, Cat No. 5741S), MMP-7 (CST, Cat No. 3801S), PCNA (CST, Cat No. D3H8P), CCND1 (CST, Cat No. E3P5S), BCL-2 (CST, Cat No. 5071S), and internal reference anti-β-actin (CST, Cat No. 4970), respectively, for 4 h, followed by 1:4000 diluted HRP-anti-rabbit IgG (CST, Cat No. 7074) for 1 h at 25 °C. Finally, the immune-conjugated signals were examined with Immobilon ECL Ultra Western HRP Matrix (Millipore, Cat No. WBULS0500), and the density of the bands was analyzed with the image station 4000 mm pro (Carestream, YTO, Toronto, ON, Canada).