Fig. 2

APLF downregulation influence cellular machinery and impede invasive, tumorigenic and metastatic potential of metastatic MDAMB-231 cells. a MDAMB-231 cells were transduced with lentiviral particles expressing shRNA against APLF or empty pLKO.1 vector (empty vector). Lentiviral vectors containing shRNA targeting human APLF was cloned in the pLKO.1 (Addgene) vector [1]. Extent of knockdown was measured at the protein level by western blot. b Viability of the control and APLF-kd MDAMB-231 cells were determined by MTT assay. c Cell cycle analysis for both control and APLF-kd cells were performed. Representative plot indicate the percentage of cells present in a given phase for control and APLF-kd cells. d G1/S-phase specific marker, CYCLIN D1 level was determined in control and APLF-kd cells by western blot analysis. e Control and APLF-kd cells were exposed to DNA DSB inducing agent etoposide (10 μM) for 4 h followed by recovery in absence of etoposide. γH2AX-positive foci cells were determined by immunofluorescence analysis to demonstrate the defect in DNA repairs after 0 h and 24 h of recovery period. Bar graph representing the fraction of γH2AX–positive foci in control and APLF-kd cells. Nuclei with ≥5 foci were counted as positive. f Same set of samples was analyzed for the expression of cleaved Caspase 3 by western blot as a measure of apoptosis in response to APLF-knockdown. g Invasion assay was performed in invasion chamber from Corning (Corning® BioCoat™ Matrigel® Invasion Chamber; 354,480). The graph represent the percentage of cells invaded and expressed in terms number of cells invaded to total number of cells added to the upper chamber at the start of the experiment. h Same set of cells were investigated for their migration or wound healing potential. Bar graph represents percentage of wound recovery expressed in terms of [1-(Width of the wound at a given time/width of the wound at t = 0)] for control and APLF-kd MDAMB-231 cells. i Control and APLF-kd MDAMB-231 cells were subcutaneously injected in female NOD/SCID mice (n = 3 for each group; age = 6-8 weeks). After 5 weeks, mice injected with control cells developed tumors of significantly bigger size than in mice injected with APLF-kd cells. Representative picture has been included and the experiment was repeated independently 3 times. j, k To determine the effect of APLF on in vivo metastatic potential, both control and APLF-kd cells were injected into the lateral tail vein of female NOD/SCID mice (n = 3 for each group; age = 6-8 weeks). Prior to this, control and APLF-kd MDAMB were transfected with pEGFPC1 (Clonetech; 6084-1). After 6 weeks of injection, lungs were dissected and examined for the presence of metastatic nodules (black arrows). Representative lung and H&E staining of metastatic tumor are shown. l Expression of APLF in lungs was determined by RT-PCR. Human APLF and ACTIN confirmed the presence of MDAMB-231 cells in the lungs section. Mouse Gapdh was used as the negative control. m Control and APLF-kd MDAMB-231 cells were investigated for the expression of DNA repair genes associated with breast cancer metastasis. mRNA was extracted and analyzed for the expression of genes by qRT-PCR. Error bar = S.E.M for three independent experiments. Statistical analyses were performed using Student t-Test function, *p < 0.05, **p < 0.01