Supplementary Figure 5: Knockdown studies confirm that integrin inhibition functions specifically in the tumour cell compartment to induce chemosensitization, without affecting cell survival.
From: Targeting the perivascular niche sensitizes disseminated tumour cells to chemotherapy
a) Representative immunoblots of integrin-β1 and the nuclear envelope proteins Lamin A/C (used as a loading control). ‘K’ depicts the Kaleidoscope molecular weight marker. Lanes 1 and 3 are lysates of independently generated T4-2 cells transduced with non-targeting shRNA (shNT). Lanes 2 and 4 are lysates of independently generated T4-2 cells transduced with shRNA targeting ITGB1 (shITGB1, clone 391001). b) Densitometry from n = 3 independent experiments, normalized to the band intensity of shNT control. *** P < 0.0001 by unpaired, two-tailed t test. shNT and shITGB1 T4-2 cells were seeded on bone marrow MVNs as depicted in Fig. 2a and analyzed 12-days later. c) Representative images of bone marrow MVNs at this time point. Scale bar, 200 μm. d) Number of clusters and e) Tumour cell outgrowth at day 12, to assess the effect of ITGB1 knockdown on tumour cell phenotype in the absence of doxorubicin (n = 5 independent experiments). ** P = 0.0039. P values obtained by unpaired, two-tailed t test. Cultures were treated at days 12 and 15 with 2500 nM doxorubicin, then fixed and stained two days later to assess DTC viability. f) Representative images of TUNEL stained shNT and shITGB1 DTCs on bone marrow MVNs. Scale bar, 20 μm. White arrowhead, TUNEL-positive tumour cell. g) Tumour cell outgrowth over the treatment period (n = 5 independent experiments). * P = 0.050 by unpaired two-tailed t test. The total percentage of h) TUNEL-positive tumour cells, i) TUNEL-positive single DTCs, and j) TUNEL-positive cells within 2–4 cell clusters was analyzed across n = 116 (shNT) and n = 124 (shITGB1) total cells/clusters across three independent experiments. *** P < 0.0001 by unpaired, two-tailed t test. For b, d-e, g-h, j centre line represents the mean, error bars the s.e.m. Statistical tests and P values are indicated. Source data are provided in Supplementary Table 1.
