Investigation of Transcript Variant 6 of TPD52L2 as a Prognostic and Predictive Biomarker in Basal-Like MDA-MB-231 and MDA-MB-453 Cell Lines for Breast Cancer

Figure 5

The effect of TPD52L2 on the tumorigenesis of BLBC cells in vitro and in vivo. (a–e) The MDA-MB-231 cell line was transfected with either shCON or 3 different shRNAs targeting TPD52L2. After puromycin selection and positive colony enrichment, MDA-MB-231 cell lines with stable knockdown were established. (a) Cell viability assays. The samples were assayed in triplicate. Each point represents the mean value from three independent samples. (b) Colony formation assays. (c) Cell migration assays. Representative photographs and bar graphs were from three independent experiments (, , -test, ). (d) Growth curves of xenograft tumors obtained upon subcutaneous implantation of shCON or shTPD52L2 cells. Tumor volumes were monitored every 3 days by measuring tumor diameters (, , ). (e) Images and weights of xenograft tumors. The 5tumors were removed, photographed, and weighed (, , -test, ). (f–i) MDA-MB-453 cells were infected with lentiviruses expressing CAS9 under the control of a doxycycline-inducible promoter and lentiviruses expressing a sgRNA targeting TPD52L2 (sgTPD52L2). Stable inducible Cas9-sgTPD52L2 cell lines were established after two rounds of selection using blasticidin and puromycin. (f) Cell viability assays of inducible Cas9-sgTPD52L2 cell lines. The samples were assayed in triplicate. Each point represents the mean value from three independent samples. (g) The stable cell lines were treated with doxycycline for 48 h and then washed and transfected with plasmids containing the cDNAs of V5 or V6 tagged with HA. Forty-eight hours after transfection, the cells were lysed and immunoblotted as indicated. (h) Colony formation assays. (i) Cell migration assays. Representative photographs and bar graphs are from three independent experiments. The data are presented as the (, -test, ).

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