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. 2019 Sep 18;48:353–363. doi: 10.1016/j.ebiom.2019.09.017

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© 2019 The Authors. Published by Elsevier B.V.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Fig. 6 — MFF regulation of tumour cell proliferation. (a) PC3 cells were transfected with siCtrl or two independent MFF-directed siRNA (siMFF #A and siMFF #C) and analysed for cell proliferation by direct cell counting during a 3-day interval. Each tracing corresponds to an individual experiment (n = 3). ***, p = .0002–0.0003 (by two-sided unpaired t-test). (b) DU145 (left) or PC3 (right) cells stably transduced with pLKO or MFF-directed shRNA (shMFF) were analysed for cell proliferation by direct cell counting at the indicated time intervals. Each tracing corresponds to an individual experiment (n = 3). ***, p < .0001 (by two-sided unpaired t-test). (c and d) PC3 cells transfected with siCtrl or siMFF were analysed for DNA content by PI staining and flow cytometry (c, representative experiment; numbers correspond to the percentage of cells in each peak) and the percentage of cells in the indicated cell cycle phase was quantified (d). Mean ± SD (n = 2). (e and f) PC3 cells transduced with pLKO or shMFF were reconstituted with MFF2 cDNA and analysed by Western blotting (e) or cell proliferation by direct cell counting after 72 h (f). The position of endogenous or Flag-MFF2 is indicated. Mean ± SD (n = 8). ***, p < .0001; ns, not significant (by two-sided unpaired t-test). (g and h) PC3 or DU145 cells stably transduced with two independent MFF-directed shRNA (clones #1 and #2) or pLKO were analysed for colony formation after 14 d by crystal violet staining (g, representative experiment) and quantified (h). Mean ± SD (n = 4). ***, p ≤.0001–0.003 (by two-sided unpaired t-test). (i) PC3 cells stably transduced with pLKO (top) or shMFF (bottom) were injected s.c. on the flanks of immunocompromised athymic nude mice and tumour growth was measured at the indicated time intervals with a calliper. Each symbol corresponds to an individual tumour. Tumour measurements (mm³) at day 30 are as follows: pLKO, 292.3 ± 40.1 (n = 9); MFF shRNA, 124.4 ± 35.4 (n = 9). **, p = .006 (by two-sided unpaired t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)