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. Author manuscript; available in PMC: 2016 Aug 15.
Published in final edited form as: Oncogene. 2016 Feb 15;35(32):4235–4243. doi: 10.1038/onc.2015.488

Figure 2. Insulin receptor knockdown reduced insulin-regulated signaling and growth in TamR cells, but not in parental cells.

Figure 2

MCF-7L and T47D and their counter TamR cells were InsR knock-downed with lentiviral shRNA (shIR#2, shIR#6) or plasmid control (shSrcb or pLKO.1). InsR protein and mRNA expression levels were respectively determined using Western blot analyses as shown in lentiviral transduced (A) MCF-7L and T47D cells and qRT-PCR as shown in (B) MCF-7L and T47D cells. Cells were plated, serum started for 24 hours and treated with or without 10nM insulin for 15 minutes. Whole cell lysates were collected, separated by SDS-PAGE and subjected for indicated immunoblotting analyses. For qRT-PCR analysis, total RNA was collected from cells in full media. Data was normalized to housekeeping gene, GAPDH. Results represent mean ± SD of triplicates from three independent experiments. (C) Cell monolayer growth was determined using MTT assay. Transduced MCF-7L and TamR cells were serum starved for 24 hours and treated with or without insulin for 5 days. (D) Anchorage-independent growth of lentiviral transduced MCF-7L and T47D was measured after 19 days and 25 days, respectively. Values were normalized to untreated group and were represented in fold change (mean ± SD, n=3). Two-way ANOVA with Bonferroni comparison was performed to identify significance among untreated versus treated groups and shIR versus control groups. *, p<0.05; **, p<0.01.