P mlc

Cells were grown on a cover slip, serum deprived for 18 h and then exposed to treatments for 16 h. Next, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100, washed 3 times with PBS and incubated at 4 °C overnight with a primary antibody (1:250) against GPER (AB137479) (Abcam, DBA, Milan, Italy) or Phospho-Myosin Light Chain 2 (Ser19) (p-MLC) (Cell Signaling, Euroclone, Milan, Italy). After incubation, the slides were extensively washed with PBS, probed with alexa Fluor 594 goat anti-rabbit IgG (1:300, Thermo Fisher Scientific) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:1000; Sigma-Aldrich). Then, the images were obtained using the Cytation 3 Cell Imaging Multimode reader (BioTek, AHSI, Milan Italy) and analyzed by the Gen5 software (BioTek, AHSI, Milan Italy).

Cells and tissue were collected, and processed for western blotting by solubilizing extracts in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor cocktail (Roche #04693159001) and phosSTOP (Roche, # 04906837001)). Protein lysates were resolved by polyacrylamide gel electrophoresis (PAGE) under reducing conditions (4–12% sodium dodecyl sulfate–PAGE Bis–Tris gels; MOPS buffer system; Invitrogen; NuPAGE-MOPS system). The gels were blotted onto nitrocellulose or PVDF membranes and blocked in either 5% milk or 1% bovine serum albumin and followed by primary antibody incubation for overnight (NUAK2 (Cat#sc-374348, 1:100, Santa Cruz), YAP (Cat#14071, 1:1000, Cell Signaling), GAPDH (Cat#3683, 1:10000, Cell Signaling), actin (Cat#A5316, 1:1000, Sigma), pMLC (Cat#3671, 1:1000, Cell Signaling), MLC (Cat#M4401, 1:1000, Sigma), TAZ (Cat#4883, 1:1000, Cell Signaling), NF2 (Cat#12888, 1:1000, Cell Signaling)). MYPT1 and p-MYPT1 (S445) antibodies were a kind gift from Dr. Dario Alessi^{43 (link)}. After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. Blots were washed with TBST and developed with the enhanced chemiluminescence system. Uncropped Western blot images of data shown in Figures can be found in Supplementary Figure 6.

Cellular samples were collected and lysed, and the protein concentration was determined using the BCA method. Based on the target protein molecular weight, a suitable concentration of protein electrophoresis gel was selected for protein electrophoresis, membrane transfer, and then blocked for 1 h. Primary antibodies against ZO-1 (1:1000, Cat#AF5145, Affinity Biosciences, China), MLCK (1:800, Cat#AF5314, Affinity Biosciences, China), p-MLC (1:1000, Cat#95777, Cell Signaling Technology, USA), MLC (1:1000, Cat#8505, Cell Signaling Technology, USA), and β-actin (1:2000, Cat#4970, Cell Signaling Technology, USA) were incubated overnight at 4° C on a shaker. Secondary antibodies (1:5000, Cat#58802, Cell Signaling Technology, USA) were then incubated for exposure, and the images were recorded using a Bio-Rad Chemidoc XRS+ imaging system. The protein expression levels were calculated using the software ImageJ for grayscale analysis.

Cells were fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS) for 10 min at room temperature, followed by three washes in PBS. Cells were permeabilized in 0.01% Triton/PBS for 1 min, followed by three washes in 0.01% Tween/PBS and then blocked with PBS supplemented with 10% donkey serum for 1 h. Cells were incubated with primary antibody (YAP (Cat#14071, 1:200, Cell signaling), pMLC (Cat#3671, 1:100, Cell signaling), actin (Cat#A5316, 1:200, Sigma) and Flag (Cat#F3165, 1:400, Sigma)) in blocking buffer for overnight. After three washes in 0.1% Tween PBS, cells were incubated in secondary antibody and 1 μg/ml DAPI for 1 h at room temperature. Cells were then washed, mounted and examined with a confocal microscope (Zeiss LSM 700 Laser Scanning Confocal), equipped with a 25× or 40× oil objective lens or epimicroscope (Zeiss Axio Observer Z1).

Tissue fragments and cultured cells were lysed with Laemmli buffer. Lysates were separated by a 10% SDS-PAGE and Western blotting was carried out following standard protocols. For detection, the following primary antibodies were used: MRCKα (#374568), MRCKβ (#374597), GAPDH (#25778; all SantaCruz Biotechnology, Dallas, TX, USA), pMLC (#3674), pCofilin (#3311; all Cell Signaling, Danvers, MA, USA), β actin (#AB6276, Abcam, Cambridge, MA, USA). For detection, appropriate HRP coupled secondary antibodies were used: horse-anti-mouse IgG (#VECTPI2000), goat-anti-rabbit IgG (#VECTPI1000, all Vector Laboratories, Burlingame, CA, USA). LuminataTM Western HRP Chemiluminescence Substrates detection reagent (Milipore, Hellerup, Denmark) was used for chemiluminescence, which was then detected with Medical X-ray film (AGFA, Birkerød, Denmark). The intensity of the bands was quantified using ImageJ software.