Alexa555 phalloidin

Mouse anti–YAP (cat. #sc-101199; Santa Cruz Biotech) was used at 1:200 (IF) and 1:500 (WB). Rabbit anti-p38 (cat. #sc-535; Santa Cruz Biotechnology) was used at 1:2000 for WB. Rabbit anti-α-actinin (cat. #3134; Cell Signaling Technology) was used at 1:500 for WB. Rabbit anti-β-actin (cat. #8457; Cell Signaling Technology) was used at 1:1000 for WB. Phalloidin-Alexa555 (A34055; Thermo Fisher Scientific) was used at 1:100 and DAPI (cat. # D9542; Sigma-Aldrich) was used at 5 µg/ml for IF. Mouse anti-β1 integrin (cat. #ab24693, Abcam) was used at 1:200 for IHC. Mouse anti-β1 integrin (cat. #sc-374429, Santa Cruz Biotechnology) was used at 1:1000 for WB. Rat anti–β4 integrin (cat. # 14-1049-82; Thermo Fisher Scientific) was used at 1:500 (IF) and 1:1000 (WB).
For IF, Alexa 488-, 555- or 647-conjugated secondary antibodies (Thermo Fisher Scientific) were used at 1:500. For WB, IRDye 680 or 800-conjugated secondary antibodies (LI-COR Biotechnology) were used at 1:10,000.

Transfected CHO cells or infected THP-1 cells were fixed and permeabilized for immunofluorescence microscopy as described previously, using Triton X 0.1% for cell permeabilization. For labelings, we used mouse anti-FLAG (Sigma; 1:200), mouse anti-myc (Calbiochem; 1:200), rat anti-HA (Sigma; 1:200), followed by appropriate fluorophore-conjugated anti-mouse antibodies (Jackson ImmunoResearch; 1:200). 4′,6-Diamidino-2-phenylindole (DAPI; 1:30.000) was used to label DNA, and actin staining was carried out by incubating CHO cells with Phalloidin-Alexa555 (Thermo Fisher Scientific, 1:200) during 30 min. Images were acquired on an Axio Imager D2 (Zeiss) and processed with ZEN or Fiji software. For each enhanced GFP (EGFP) fusion protein, the proportion of protein in the nucleus was determined by calculating the ratio between the average GFP fluorescence in the nucleus and the average GFP fluorescence in the cytosol. Quantification of these values was made in Fiji, using the DAPI stain to delineate the nucleus and the F-actin stain for the cell outline. For each protein construct, fluorescence was quantified for at least 60 cells from 3 independent experiments. Statistical significance was assessed with Student’s T-test.

Immunostaining of the pupal wings was performed according to [56 ]. The following primary antibodies were used: rabbit and mouse anti-pH3 1/500 (Merck Millipore #06–570 and Cell signal technology #9796), rabbit anti-cleaved Dcp1 1:200 (Cell Signalling Technology #95785), and anti-ß Galactosidase 1:500 (MP Biomedics #559761 and Promega #Z378A, 1:1000). To stain cellular F-actin we used Phalloidin-TRITC (Sigma-Aldrich Cat#P1951) (1:200), Phalloidin-Alexa 488 (ThermoFisher A-12379) and Phalloidin-Alexa 555 (ThermoFisher A-34055) (1:200). To stain nuclei we used DAPI Merck (Ref268298).
Secondary antibodies (ThermoFisher) were used at dilutions of 1:200.
Pupal wings were mounted in Vectashield mounting fluorescent medium (Vector Laboratories, Inc. REF H-1000).

MDA-MB-468 and MCF-7 breast cancer cells were subjected to control precursor or miR-142-3p precursor transfection and analyzed by confocal immunofluorescence microscopy on a Leica TCS microscope (Leica, Wetzlar, Germany), or a Zeiss LSM 780 (Carl Zeiss, Jena, Germany) (Z-stacks) using Plan-Apochromat 63x/1.40 Oil DIC objectives. Human skin fibroblasts [23 (link)] were used as a control. Samples were prepared as previously described [10 (link)] using ALEXA555-phalloidin (Invitrogen, Eugene, OR, USA, 1:1000) for staining of actin filaments, as well rabbit-anti-Integrin-αV (Cell Signaling, 1:100), mouse mAb anti–human integrin αvβ3 (Millipore, 1:100), mouse Ab anti-human vinculin (Sigma, 1:300) and appropriate ALEXA488 or ALEXA594-labeled secondary antibodies (Invitrogen, 1:600). Cell nuclei were visualized by 4’,6’-diamidino-2-phenylindole (DAPI) staining (Sigma, 1:5,000).

The following antibodies were used: DCAD2 (rat-anti-DE-Cad) used at 1/200 and Mab N27A1 (mouse-anti-Armadillo) used at 1/200 (Developmental Studies Hybridoma Bank); Rabbit-anti-Frazzled: a kind gift from Florence Maschat [4 (link)] used at 1/500; Rabbit-anti-GFP used at 1/500 (Life Technologies). Guinea-pig anti-Sqh1P [36 (link)] a kind gift from Robert E. Ward IV used at 1/100. To label F-actin, tissues were incubated with 50 μM Rhodamine-conjugated phalloidin (or alternately Alexa-488 Phalloidin or Alexa-555 Phalloidin; Invitrogen) in PBS+0.1% Triton X-100 (hereafter PBS-T).
Wing imaginal discs were dissected out of wandering third instar larvae in PBS, and fixed in 3.7% formaldehyde in PBS for 15 minutes. The discs were then washed three times in PBS-T, each wash for 15 minutes. Primary antibodies diluted in PBS-T were incubated with the discs for 3 hours at 25°C or overnight at 4°C. The incubation with the primary antibodies could be combined with phalloidin. Then, discs were washed three times in PBS-T quickly and then four more times, each for 15 minutes, incubated with secondary antibodies in PBS-T for 2 hours at 25°C or overnight at 4°C, washed again in PBS-T three times quickly and then four more times, each for 15 minutes, then cleared in 70% glycerol in PBS and mounted for imaging. The mounted discs were then imaged using confocal microscopy.