Calyculin a

The primary fibroblasts were treated with TGFβ, FGF, PDGF (7666-MB, 3139-FB, 1447-PC, R&D Systems), or PP1 inhibitor Calyculin A (141784, Abcam) for 15 or 30 min, respectively. After 2 times of PBS washing, the total protein was extracted by lysate with protease inhibitor and phosphatase inhibitor. The total protein concentration was detected by BCA kit, and the protein was separated by 10% SDS-PAGE and then transferred to PVDF membrane. The 5% skimmed milk was used to block the protein at room temperature for 2 h, and then the primary antibodies were added and reacted at 4 °C overnight. On the second day, the corresponding second antibody was added and sealed at room temperature for 1 h, followed by the final step of ECL addition for exposure. The antibodies contain anti-p-Smad2/3 antibody (8828, Cell Signaling Technology, 1:1 K), anti-p-P38 antibody (9211, Cell Signaling Technology, 1:1 K), anti-p-ERK1/2 antibody (4370, Cell Signaling Technology, 1:2 K), anti-p-AKT antibody (4060, Cell Signaling Technology, 1:2 K), p-JNK antibody (4668, Cell Signaling Technology, 1:1 K), anti-GAPDH antibody (30201ES, Yeasen, China, 1:1 W), anti-rabbit IgG secondary antibody (305-035-003, Jackson ImmunoResearch, 1:1 W) and anti-mouse IgG secondary antibody (115-035-003, Jackson ImmunoResearch, 1:5 K).

For in vivo jasplakinolide treatments, wild-type embryos were collected on E15.5 and incubated with 3μM jasplakinolide (Sigma-Aldrich), 30 nM Calyculin A (Cell Signaling Technology), or DMSO in serum-free DMEM (Biological Industries) at 37°C for 2 h before embedding in OCT or processed for whole-mount preparation and immunofluorescence microscopy as described above. For in vitro treatments, keratinocytes were infected with shScr;puromycin or shAnln;puromycin, selected with 3 μg/ml puromycin (Sigma) and plated on fibronectin-coated coverslips (40,000 cells in a single well of a 24-well plate). Twenty-four hours later, the medium was switched to high calcium (1.5mM Ca²⁺) and cells were treated with 100nM jasplakinolide (Sigma-Aldrich) or 2nM Calyculin A for 5 min, and then with 8μM nocodazole for 6 h. Cells were then fixed and labeled with Phalloidin-iFluor 647 (Abcam, ab176759) and pERM (Cell Signaling Technology, 1:200) overnight at 4°C. After washing, sections were incubated with a secondary antibody (1: 500 dilution) at room temperature for 1 h. Data was collected using a Nikon C2+ 60×/1.4 objective that generates optical sections of 0.49 μm at the middle of the cell.

Doxycycline (Sigma-Aldrich) was used at 2 µg/ml. The following doses of inhibitors were used: 4 µM DCB (Sigma-Aldrich), 10 µM p38 inhibitor (SB203580; New England Biolabs), 50 µM blebbistatin (Sigma-Aldrich), 5 µM R0-3306 (CDK1i; Sigma-Aldrich), 1 µM calyculin A (Abcam), 15 µM DDR1-IN-1 (Tocris), 10 µM Y-27632 (ROCKi; Tocris), 10 µM MG132 (Tocris), and 5 nM nocodazole (Sigma-Aldrich).