Anti cav1

Cav-1 human shRNA Plasmid Kit (ORIGENE-TR314183) was used to obtain the knockdown of cav-1 in STO cells. We transfected 1 × 10⁵ cells with the pRS-HuSH plasmid containing either a scramble (SCR) or the shcav-1 sequence and a puromycin resistance cassette using the Fugene HD Transfection Reagent (Promega). Then, 40 h after transfection, cells were split and cultured in the presence of 2 μg/mL of puromycin into the medium. We kept cells under puromycin selection for 1 week and selected the growing clonal cells. After 10 passages, cells were solubilized in RIPA buffer with added protease inhibitors. Then, 30 µg of total proteins were loaded and run on pre-casted 8–12% gel (NuPage Thermo Fisher) and transferred onto PVDF membranes. The PVDF membrane was incubated overnight at 4 °C with anti-cav-1 (Abcam, Cambridge, UK; 1:1000 in TBS-tween) and anti-tubulin (1:2500 in TBS-Tween) antibodies. After a few washes in TBS, membranes were incubated with HRP-conjugated anti-rabbit IgG (for cav-1) and anti-mouse IgG (for tubulin) antibodies. The SuperSignal™ West Femto Maximum Sensitivity Substrate (Life Technologies, Carlsbad, CA, USA) was used for the chemiluminescence detection.

AFCs were fixed in 4% paraformaldehyde for 15 min and then permeabilized with 0.3% Triton X-100 in PBS for 10 min. Non-specific binding was blocked by 4% bovine serum albumin (BSA) at room temperature for 2 h. After incubation with anti-Cav1 (1:200, Abcam, Cambridge, UK ), anti-integrin β1 (1:500, Novus Biologicals, CO, USA) or anti-p65 (1:200, Abcam, Cambridge, UK) antibodies at 4 °C overnight, appropriate Alexa Fluor 488 second antibodies (1:1000, Abcam Cambridge, UK) were used for fluorescent labeling. Nuclei were stained with DAPI. Images were acquired using a fluorescence microscope (Carl Zeiss Microscopy, Thornwood, NY).

Cells seeded on gelatin coated glass coverslips were fixed 0, 2, 4, 6, 12, and 24 hours after treatment using 4% formalin. After washing, the cells were permeabilized with 0.01% triton X-100 and blocked with 1% BSA. F-actin was stained overnight with Rhodamine-Phalloidine (Life Technologies, Eugene, OR, USA, 1:40) and Cav-1 with anti-Cav-1 (abcam, Cambridge, UK, 1:2000). Nuclei were stained with Hoechst and Cav-1 was detected using second antibody goat anti-rabbit DyLight 488 (abcam). The coverslips were mounted with Vecta Shield (Vector Laboratories, CA, USA) and screened using a Leica DMRA fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

Cells were seeded and grown on 25 × 25-mm cover slips in 6-well tissue culture plates. Next day, cells were transfected with p195 or antimiR-195 and 24 h post-transfection, the cells were fixed in 4% formaldehyde solution in PBS for 15 min. Cells were permeabilized for 5 min in 0.1% Triton X-100 followed by 3 washes with 1× PBS. Cells were incubated in blocking buffer (1% BSA and 0.1% Triton X-100 in 1× PBS) for 1 h, primary antibody anti-CAV1 (Abcam, Cambridge, UK) for 2 h followed by Alexa Fluor 488-conjugated secondary antibody (Invitrogen) for 1 h. The fluorescence images were captured using inverted microscope Nikon ECLIPSE Ti (Nikon Corporation, Tokyo, Japan at 40× magnification under 488/519 nm excitation and emission wavelengths.

Cells were lysed in Radio Immunoprecipitation Assay lysis buffer (RIPA, Beyotime, China). The protein was prepared and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). The same amounts of protein were extracted by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The blocked protein with 5% skim milk powder was incubated with primary antibody anti-CAV1 (1:10000, abcam, MA, USA), anti-β-catenin (1:1000, Cell Signaling Technology, MA, USA), anti-cyclin D1 (1:1000, Cell Signaling Technology, MA, USA), anti-E-cadherin (1:1000, Cell Signaling Technology, MA, USA) and anti-GAPDH (1:5000, Cell Signaling Technology, MA, USA) at 4 °C overnight. Then the prepared membranes were incubated with secondary antibody (1:5000, Cell Signaling Technology, MA, USA) for 2 h. Finally, the blots were visualized by ECL chemiluminescent reagent (Millipore, Germany) and related data was analysed by Image Lab Softwave.

The 786-O cells were lysed on ice in 20 mM Tris-HCl (pH 7.4), 1% Triton X-100, 0.025% SDS, 100 mM NaCl, 1 mM Na₃VO₄, 10 mM NaF, and 1% protease inhibitor cocktail (Sigma, United States). Soluble extracts were incubated for 2 h at 4°C with relevant antibodies: anti-CAV-1 (Abcam, United States), anti-LOX-1 (Proteintech, United States) and a negative isotype control mouse immunoglobulin (IgG) (Santa Cruz Biotechnology, United States). Immune complexes were precipitated with protein A/G agarose (Santa Cruz Biotechnology, United States). Western blotting was performed as described before.