Bs 1448r

Proteins were extracted using RIPA lysis buffer (Biosharp, Shanghai, China) supplemented with protease inhibitors. The proteins were separated in 10% SDS-PAGE (Beyotime, Shanghai, China) and electrophoretically transferred to PVDF membrane (Milipore, USA). The membranes were incubated with primary antibodies: Actin(1:3000, #bs0061R, Bioss, Beijing, China), Musashi (1:1000, Bioss, Beijing, China), Snail (1:1000, #bs1371R, Bioss, Beijing, China), Notch1 (1:100, #AF5307, Affinity Biosciences, OH, USA), Hes1 (1:200, #BM4488, Boster Biological Technology Co. Ltd., Wuhan, China), and JAG1 (1:500, #bs-1448R, Bioss, Beijing, China) at 4 °C overnight. HRP-conjugated secondary antibodies (1:5000, #bs0295M, Bioss, Beijing, China)were used for 1 h at 37 °C. The protein bands were observed using ECL reagents (Milipore, USA). Protein immunoreactivity derived from vehicle groups (LV-NC) was normalized to 1.0. Values of LV-miR-153 groups were normalized according to vehicle groups (LV-NC).

Cells were cultured on coverslips, washed in cold PBS, fixed in 95% ethanol for 30 min, and permeabilized in 0.3% Triton X-100 (Solarbio, Beijing, China) for 15 min. Fixed cells were blocked in PBS containing 5% goat serum (ZSGB, Beijing, China) at 37 °C for 1 h. Cells were incubated with the primary antibody CD133 (1:1000, #66666-1-lg, Proteintech, Wuhan, China) and Jagged1 (1:400, #bs-1448R, Bioss, Beijing, China) at 4 °C overnight. The secondary antibody goat anti-rabbit IgG (Cy3, #GB303, Servicebio, Wuhan, China) was added on cells which were washed three times at 37 °C in the dark for 1 h. DAPI (Solarbio, Beijing, China) was added on cells for 5 min. The fluorescent signal was captured using a TS100 inverted fluorescence microscope (Nikon, Japan).

For immunohistochemistry, tissue samples were incubated with antibody against proliferating cell nuclear antigen (1:400, #bs-2007R, Bioss, Beijing, China) or Jagged1 (1:400, #bs-1448R, Bioss, Beijing, China) at 4 °C overnight. Sections were subsequently incubated with HRP for 60 min at 37 °C. Coloration was conducted with 3,3-diaminobenzidin (DAB), then kept at room temperature without light for 10 min. Staining score was determined as 0: 0–5% of staining cells, 1: 6–25% of staining cells, 2: 26–50% of staining cells, 3: 51–75% of staining cells, and 4: > 75% of staining cells. Staining intensity was scored as 0–1: negative, 2–3: weak positive, 4–5: intermediate, and 6–7: strong positive. The sum of both extent and intensity score was defined as the staining final score [24 (link)].