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. 2020 May 19;11:2498. doi: 10.1038/s41467-020-16352-z

Fig. 4. MTOR inhibition induces breast cancer plasticity.

Fig. 4

a NODAL immunoblot from 0 to 24 h INK 20 nM treated T47D cells (n = 3). b Mean sphere counts from 960 T47D cells pre-exposed to DMSO, INK (20 nM), rhNODAL (100 ng/mL), or INK + SB431542 (10 μM) for 24 h (n = 4) with representative images. Micron bars = 250 μm. c Mean fold change in colony number from T47D cells cultured as in b (n = 3). d Mean percentage of CD44high/CD24low T47D cells treated with DMSO, INK, or INK + SB431542 (n = 6) with representative scatterplots defining CD44 and CD24 subpopulations. e Mean log2-fold change relative to DMSO qRT-PCR of NANOG, SOX2, OCT4, TWIST, ZEB1, SNAIL, VIM transcripts from DMSO or INK (20 nM) for 24 or 24 h INK then 24 h media treated T47D cells (n = 3). f Immunoblot analyses of NANOG, OCT4, and SNAIL in 24 h DMSO or INK (20 nM) treated T47D cells. g Mouse lung colonies 8 weeks following tail vein injection of pretreated SUM149 cells (24 h with DMSO or INK (20 nM)) (n = 10) with representative lung colony images (brown). h Mean PDX401 tumor volumes in mice receiving DMSO or INK (30 mg/kg) every second day for two weeks. Arrow indicates treatment cessation (n = 8). i Mean sphere counts from 96,000 PDX401 cells from endpoint tumors from h (DMSO n = 6, INK n = 7) with representative images. Micron bars = 500 μm. j NODAL and 4E-BP1 immunoblot of T47D cells transfected with 4E-BP1 shRNA, shRNA scrambled (SCR) control, 4E-BP1 ORF, or empty vector. k Mean first and second generation sphere counts from 96 T47D cells described in j (n = 3) with representative images. Micron bars = 250 μm. l Kaplan–Meier plot: 4E-BP1 RNA-Seq expression correlates with survival in 1100 breast cancer patient samples (5.4 years, p = 0.0187). 4E-BP1 RNA-Seq transcript abundance in tumor versus healthy tissue (normal, n = 113; tumor, n = 1062; p = 2.2 × 10−16). Data represents independent experiments. Error bars indicate mean ± SD. Box and whisker plot represents median, IQR, whiskers extend to maximum and minimum. Two-sided t-test for paired samples. The asterisks denote p-values < *0.05, **0.01. Multiple comparisons tested by ANOVA. The same letters indicate relationships with a p ≥ 0.05. Different letters indicate statistical differences (p < 0.05).