Phalloidin ifluor 647

Embryos where dechorionated in bleach and fixed in a 1:1 mixture of heptane and 40% formaldehyde for 9 min. Embryos were then stuck to tape, manually devetillinized in PBS and transferred to eppendorf tubes. Staining to quantify maternal KD and for SIM were carried out in the same way: embryos were blocked in 1% BSA for 1 h, incubated with primary antibodies overnight at 4C, washed in PBS-Triton-X 0.1%, incubated with secondary antibodies for 2 h at room temperature, washed in PBS-Triton-X and mounted in Vectashield mounting medium. Primary antibodies were: 1:100 rat anti-DCAT-1 [Developmental Studies Hybridoma Bank (DHSB)], 1:10 mouse anti-NECD (C458-2H, DSHB) and 1:10 rat anti-DCAD2 (DCAD2, DSHB). Secondary antibodies were: 1:200 anti-Rat-FITC (712-095-153, Jackson ImmunoResearch) for α-Cat KD quantification; 1:200 anti-Mouse-Alexa488 (AB_2338189, Invitrogen) and 1:200 anti-Rat-Alexa568 (A-11077, ThermoFisher) for SIM. Embryos were also stained with 1:1500 Phalloidin-iFluor647 (ab176759, Abcam).

G-actin was immunolabelled as previously described (Lee et al., 2013 (link)). Briefly, cells were fixed with 4% paraformaldehyde for 10 min, followed by 5 min exposure to cold acetone. To immunolabel G-actin, cells were incubated with monoclonal JLA20 anti-actin antibody (Millipore, 1:200) for 1 h at room temperature. F-actin was labelled with Phalloidin-iFluor 647 (Abcam, 1:500).
For quantification of perijunctional G- and F-actin fluorescence intensities cells were co-labelled for E-cadherin, and the E-cadherin channel was used to create a mask for measuring the intensities of the other channels. The mean grey value of the protein of interest was measured in an area 2 µm around the E-cadherin mask in a given field of view. The mean grey value of Tmsb4x-depleted cells was normalized to that of the control cells, and the data are presented as the normalized intensity. To calculate perijunctional G/F-actin ratio the absolute mean grey value of G-actin staining was divided by that of F-actin (Phalloidin) staining in an area 2 µm around the E-cadherin mask.
For cells grown in 50 µM calcium, the fluorescence intensities of F-actin and G-actin were measured using the ImageJ FIJI package (Schindelin et al., 2012 (link)). Cell peripheries were traced manually and a customized ImageJ macro was used to measure the intensity (mean grey value) in a 2-μm-wide band from the cell edge.

MCF7, T47D, MDA-MB-453, SNU216, MKN45, PC3, OE19, HCT116, 427.1.86, and 4 T1 cells were seeded onto chamber slide or 0.1% gelatin coated coverslip, and fixed with 4% paraformaldehyde at room temperature for 10 min. Then the cells were permeabilized with 0.5% PBS-T and nonspecific binding was blocked with 10% goat serum/1% gelatin in 0.1% PBS-T for 30 min. mAb3F3 were used as primary antibodies. FITC-conjugated anti-mouse IgG was used as secondary antibody. Actin was visualized using phalloidin-iFluor647 (Abcam) and nucleus was stained with 0.3 μM DAPI (Molecular Probes) diluted in Mowiol mounting media (Sigma). These stained cells were visualized using confocal microscope LSM510 meta (Zeiss) and processed in Photoshop.

Dulbecco’s Modified Eagle Medium (DMEM) and Fluoroshield histology mounting medium were purchased from Sigma Aldrich (St. Louis, MO, USA); fetal bovine serum (FBS) (Capricorn Scientific), paraformaldehyde (ROTH), glutaraldehyde, 25% solution (Sigma Aldrich, St. Louis, MO, USA), Phalloidin iFluor 647 (Abcam, Cambridge, UK); 4′,6-diamidino-2-phenylindole (DAPI), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), and 2.5% Trypsin-EDTA all were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
The murine macrophage cell line, RAW264.7 (ATTC TIB-71), was purchased from the American Type Cell Culture Collection (ATCC, Manassas, VA, USA). HeLa cells stably expressing histone H2B-GFP were kindly provided by Prof. P.Tchelidze, who had used them in his previous research [12 (link),13 (link),14 (link),15 (link),16 (link)]. Primary mouse skin fibroblasts (pMSFs) had been previously prepared at our laboratory according to the protocol described in [17 (link)], and the early passages of pMSFs were stored at −86 °C. These cells were thawed and passaged for the current experiments.

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.
In vitro, keratinocytes were infected with shScr;puromycin or shAnln;puromycin, selected with 3 μg/ml puromycin (Sigma), and plated on fibronectin-coated coverslips (60,000 cells in a single well of a 24-well plate). When cells created a confluent monolayer, the media were replaced with high-calcium media (1.5mM) or with fresh low-calcium media (50μM). Twenty-four hours later, cells were fixed and stained Phalloidin-iFluor 647 (Abcam, ab176759, 1:500) and DAPI.

The experiment was performed as described in [6 (link)]. Cells seeded in 24-well plates were cultured until 70% confluence was reached and treated with (1) PDT, (2) APE1 inhibitor, or (3) PDT with APE1 inhibitor. Untreated cells were used as a control. Afterwards, cells were washed 3 times with PBS, fixed with 3.7% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS and again washed 3 times with PBS. Subsequently, cells were blocked in SuperBlock (PBS) (Thermo Scientific) with 0.025% Triton X-100, washed 5 times with PBS containing 0.5% BSA, and incubated overnight with primary antibodies (Table 1). The next day, cells were washed 5 times with PBS containing 0.5% BSA and incubated for 1 h at room temperature in the dark with secondary antibodies (Table 1) or phalloidin-iFluor 647 (Abcam) diluted 1:1000 in PBS containing 0.5% BSA.

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.