Anti f actin

RAW264.7 cells were seeded onto six-well culture plates (2 × 10⁶ cells/well) and cultured for 24 h. RAW264.7 cells were fixed with 4% PFA in PBS for 10 min. The cells were permeabilized with 0.1% Triton-X 100 in PBS for 5 min and incubated with rhodamine-conjugated phalloidin (Biotium, Fremont, CA, USA) to visualize F-actin. All the experiments were carried out three times. Anti-F-actin that marked with FITC was purchased from Abcam and diluted to 1:600.

Formalin-fixed paraffin-embedded BLCA samples were cut into 5-μm-thick sections. Antigen retrieval and immunostaining of sections were performed as described previously^{37 (link)}. Primary antibody (1:100) anti-ANLN was purchased from Abcam (ab154337). Staining intensity was classified in a blinded fashion by pathologists. The immunohistochemical stain was scored on the percentage of positively tumor cell nucleus (negative, score 0; <1/3, score 1; 1/3–2/3, score 2; >2/3, score 3) and the color intensity of cytoplasm (negative, score 0; stramineous, score 1; buffy, score 2; dark brown, score 3). The two scores were combined, scores of 0–3 were defined as low expression, and 4–6 were defined as high expression. Immunofluorescence was performed using a standard protocol as described previously^{23 (link)}. Primary antibodies were as follows: anti-ANLN (1:100), anti-F-actin (1:100, Abcam, ab130935). Images of immunofluorescence were taken by confocal microscope (Leica).

Dimethyl sulfoxide (DMSO), bicinchoninic acid (BCA), trichloroacetic acid (TCA), sulforhodamine B (SRB), and catalase were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), trypsin-EDTA solution (1X), antibiotic-antimycotic solution (100X) and phosphate-buffered saline (PBS) (1X) were purchased from HyClone Laboratories, Inc. (South Logan, UT, USA). Anti-p-AMPKα, anti-AMPKα, anti-p-ACC, anti-ACC, anti-p-mTOR, anti-mTOR, anti-p-4EBP1, anti-4EBP1, anti-p-eIF4E, anti-eIF4E, anti-p-P70S6K1, anti-P70S6K1, anti-p-RPS6, anti-RPS6, anti-rictor, anti-p-Akt, anti-Akt, and anti-E-cadherin were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-PKC-α, anti-p-Rac1, anti-Rac1, anti-PCNA, anti-β-actin, and all secondary antibodies were purchased from Santa Cruz Biotechnology. (Santa Cruz, CA, USA). Anti-p-rictor was purchased from Millipore (Temecula, CA, USA). Anti-p-PKC-α, anti-F-actin, and anti-Ki-67 were purchased from Abcam (Cambridge, MA, USA). Gefitinib was purchased from Selleckchem (Houston, TX, USA) and rapamycin was purchased from Tocris Bioscience (Bristol, UK).

Tissues or cells were collected and lysed in radioimmunoprecipitation assay (RIPA, Solarbio, R0020) buffer and phenylmethylsulfonyl fluoride (PMSF, Solarbio, P0100) (RIPA buffer: PMSF = 100:1). The protein concentration was measured by bicinchoninic acid protein assay kit (Coolaber, SK1070). After separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was transferred to polyvinylidenefluoride membranes. The protein was sealed for 2 h with 5% skim milk and incubated overnight at 4°C with antibodies: anti-collagen I (Affinity, AF7001), anti-collagen III (Affinity, AF0136), anti-vimentin (Affinity, AF7013), anti-α-SMA (Affinity, AF1032), anti-FAP1 (Cell Signaling Technology, 66562S), anti-S100A4 (Cell Signaling Technology, 13018S), anti-Myo1c (Abcam, ab194828), anti-YAP1 (Cell Signaling Technology, 14074S), anti-phospho-YAP1 (Cell Signaling Technology, 53749S), anti-F-actin (Abcam, ab130935), anti-GAPDH (Affinity, AF7021). 1×Tris buffered saline Tween was used to wash the membranes for three times. Then membranes were incubated for 1 h with secondary antibodies at room temperature. The expression of proteins was detected by enhanced chemiluminescence reagent kit (SparkJade, ED0015-B).

Transforming growth factor-β1 (cat #: T1654), UA (cat #: 03240595), and protein A agarose (cat #: P1406) were purchased from Sigma–Aldrich (St. Louis, MO, United States). Matrigel (cat #: 354263) was purchased from Corning Incorporated (New York, NY, United States). Puromycin (cat #: REVG1001) was purchased from GENECHEM (Shanghai, China). The anti-NOX4 (cat #: ab109225), anti-RhoA (cat #: ab187027), anti-ROCK1 (cat #: ab205829), anti-α-SMA (cat #: ab32575), anti-Collagen-I (cat #: ab6308), anti-p67^phox (cat #: ab109366), anti-Rac1 (cat #: ab33186), anti-TIMP1 (cat #: ab61224), anti-MMP1 (cat #: ab137332), anti-MMP2 (cat #: ab37150), anti-MMP9 (cat #: 119906), and anti-F-actin (cat #: ab205) antibodies were purchased from Abcam (Cambridge, MA, United States). The anti-GAPDH antibody was purchased from OriGene (cat #: TA802519) (Rockville, MD, United States). The full-length human recombinant NOX4 (cat #: H00050507-G01) and RhoA (cat #: H00000387-P01) proteins were purchased from Abnova (Walnut, CA, United States).