Mycoplasma elimination reagent

The H22 mouse hepatoma cell line, RAW264.7 mouse macrophage cell line, and attenuated Salmonella typhimurium VNP20009 were preserved in our laboratory. Cells were cultured using Dulbecco’s modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). All strains were cultured using LB solid or liquid medium. The VNP strain constitutively expressing RFP (VNP-RFP strain), green fluorescent protein (GFP) (VNP-GFP strain), bioluminescence (LuxCDABE) (VNP-Lux strain), and the VNP strain controllably expressing RFP with the aid of the sifB promoter (VNP-psifB-RFP strain) were all constructed as described previously.^{33 (link)} All plasmids used in this study were constructed using the ClonExpress Ultra One Step Cloning Kit V2 (Vazyme, C116, Nanjing, China). The constructed plasmids, including pJ23100-RFP, pJ23100-Lux, and psifB-RFP, were electrotransformed into VNP competent cells, with the transferred empty vector plasmids serving as blank control strains and denoted as VNP-NC. DH5α and BL21 competent cells were purchased from Vazyme. Mycoplasma contamination was excluded by a mycoplasma elimination reagent (Yeasen, 40607ES03, Shanghai, China).

BMECs were cultured in DMEM (Gibco, USA) with 100 mg·mL⁻¹ streptomycin sulfate (Life Technologies, USA), 15% fetal bovine serum (FBS; Gibco, USA), and 100 U·mL⁻¹ penicillin. GENE provided the lentivirus used to construct PDGFR-β knockdown or overexpression constructs (Shanghai, China). PDGFR-β overexpression lentivirus (termed oePDGFR-β), a negative control (termed NC), PDGFR knockdown lentivirus (termed shPDGFR-β-7, shPDGFR-β-8, shPDGFR-β-9), or a scramble control (termed shNC) was used to infect BMECs plated in 6-well dishes at 50% confluence. Pools of stable transductions were formed using puromycin (7 μg·mL⁻¹) and mycoplasma elimination reagent (0.1%, Yeasen, Shanghai) for 2 weeks. We carried out transfections employing the GENE transfection kit (GENE, China).

We obtained the human BC cell lines MDA-MB-231, BT549, MCF-7, SKBR3 and normal breast epithelial cell line MCF-10A from Chinese Academy of Sciences (Shanghai, China). All BC cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) with 10% Fetal Bovine Serum (FBS) (Gibco, USA), penicillin (100 units/ml) and streptomycin (100 μg/ml) (Enpromise, China), and mycoplasma elimination reagent (Yeasen, China). MCF-10A were cultured in Mammary Epithelial Basal Medium (MEBM) (Cambrex, USA). All these cells were cultured at 37°C with 5% CO₂. MiR-640 mimics, miR-640 inhibitor and non-specific miR-negative control (miR-640-NC) oligo were purchased from RiboBio (Guangzhou, China). The specific siRNA of Wnt7b (si-Wnt7b) and negative control (si-NC) were purchased from Generay (Shanghai, China). Hieff Trans™ Liposomal Transfection Reagent (Yeasen, China) was used for transfection according to the protocols.