Vectashield mountant with dapi

IF staining was performed as previously described8 (link). DSB foci were detected by immunostaining with a monoclonal antibody to γH2AX (EMD Millipore; 05-636, 1:400), a polyclonal antibody to 53BP1 (Novus Biologicals; NB100-904, 1:100) or FANCD2 (Abcam; ab108928, 1:50). Localization studies were performed using antibody to PTEN (Cell Signalling; 9188, 1:100) or CK2β (Santa Cruz; sc-12739, 1:50) followed by incubation with a polyclonal goat anti-mouse or rabbit IgG Alexa Fluor 488/568 (Invitrogen) at 1:400 for 1 h. Cover glasses were mounted in Vectashield mountant with DAPI (Vector Laboratories) as nuclear stain. Images were captured using oil immersion 63 × objectives Zeiss Axio Imager A1/Axio Cam MRC and Axiovision LE software (Carl Zeiss, Oberkochen, Germany).

Immunofluorescence staining was performed as previous described^{38 (link)}. Localization studies were performed using antibody to CXCR4 (Invitrogen; PA3-305, 1:100), CXCR7 (Novus; NBP2-58162, 1:100), integrin αvβ3 (Millipore; MAB1976, 1:100), HIF1α (Cell Signaling Technology; #3434, 1:50), CD31 (Abcam; ab28364, 1:100), and CD34 (Abcam; ab8563, 1:100), and further incubated with anti-rabbit IgG fluorescence (Invitrogen) or anti-mouse IgG fluorescence (Invitrogen). Cover glasses were mounted in Vectashield mountant with DAPI (Vector Laboratories) as nuclear stain. Images were captured using oil immersion 63 × objectives Zeiss 510 microscopy (Carl Zeiss MicroImaging, Röttingen, Germany) and processed using Zen software (ZEISS), and particularly images for 3-dimensional endothelial sprouting stained with Alexa Fluor 488 conjugated monoclonal antibody against CD31 (1:200) (Invitrogen) and Hoechst 33342 (1:1000) (Thermo Fisher Scientific) were captured using a confocal microscope (Olympus FV1000, Zeiss LSM 880). Z-projection of the 3D stacks of microvascular network were obtained with ZEISS ZEN lite, and further analyzed with ImageJ (National Institutes of Health, Bethesda, MD) to obtain binary images and calculate the proportion of the fluorescent pixels within the ROI of each image, deriving the angiogenic sprout area coverage in the vessel channel.

Immunofluorescence (IF) staining was performed as previous described [12 (link)]. HaCaT cells were plated on matrigel-coated cover glasses (1:8 dilution; growth factor-reduced, Corning, USA) in a 24-well plate and grown for 24 h prior to IF staining. Localization studies were performed using antibody to PFN1 (Abcam; ab118984, 1:500), Phalloidin (Abcam; ab176753, 1:1000), Vinculin (Merck; FAK100-Part No.90227, 1:100), E-cadherin (Abcam; ab1416-500, 1:100), EpCAM (Abcam, 187270, 1:100), γH2AX (Abcam; ab11175, 1:300), cleaved caspase 3 (Cell Signaling; 9661s, 1:100), and further incubated with anti-rabbit or anti-mouse IgG fluorescence (Invitrogen). Cover glasses were mounted in Vectashield mountant with DAPI (Vector Laboratories) as nuclear stain. Images were captured using oil immersion 63 × objectives Zeiss 510 microscopy (Carl Zeiss MicroImaging, Röttingen, Germany) and processed using Zen software (ZEISS) and further analyzed with ImageJ (National Institutes of Health, Bethesda, MD) to measure and analyze the intensity of IF staining.