C2 60 1.4 objective

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.

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, at ~60% confluency, cells were treated with high-calcium (1.5mM Ca²⁺) media, supplemented with 8μM nocodazole (Sigma-Aldrich) for 6 h, and then fixed and labeled with Phalloidin-iFluor 647 (Abcam, ab176759), phospho-ERM (Cell Signaling Technology, 1:200), phospho-MLC (Ser10) (Cell Signaling Technology, 3671,1:1000), or Myosin-IIA (BioLegend, PRB-440P, 1:500) overnight at 4°C. After washing, sections were incubated with the appropriate 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. The cortical intensity was measured with the “freehand line tool” (ImageJ) with a width of 5 pixels. The mean gray value of Anln KD cells was normalized to that of the control cells. The same raw data was used to measure the axial ratio, using the “fit ellipse” tool (ImageJ).

Embryos were injected with lentiviruses encoding shScr;H2B-GFP or shAnln;H2B-GFP on E9 and harvested at E14.5. Whole-mount samples were immunolabeled for E-cadherin antibody (1:500) overnight at 4°C, followed by incubation with a secondary antibody and DAPI at room temperature for 2 h. Data were collected using a Nikon C2+ 60×/1.4 objective that generates optical sections of 0.49 μm at the middle of the basal layer. The mitotic stage of each cell was determined by chromosome organization (DAPI staining). In every microscopic field, the percentage of mitotic cells in each stage was calculated (number of mitotic cells in each stage/total number of mitotic cells). Every embryo was represented by a dorsal skin sample of ~0.5cm². Defects in chromosomal segregation were determined by chromosome organization (H2B-GFP or DAPI staining).

Embryos of pregnant females were injected with lentiviruses encoding shScr or shBcam on E9 and harvested at E16.5. Embryos were frozen in OCT, sectioned (10 μm), fixed, and stained for K6 (1:1,000) and nidogen (1:2,000) overnight at 4°C, followed by secondary antibody at room temperature for 1 h. K6 and nidogen staining was imaged using a Nikon C2+/60×/1.4 objective to generate optical sections of 0.49 μm. Skin thickness was analyzed through the back skin interfollicular epidermis. The distance between the K6+ periderm and the BM was measured using the “freehand lines” tool in ImageJ.