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. 2022 May 12;13:2642. doi: 10.1038/s41467-022-30375-8

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Three-day proliferation of CDK12-OE and EV MCF10A cells treated with: (i) varying glucose concentrations; (ii) 5 mM 2-deoxyglucose (2DG); (iii) +/− 0.4 mM serine. Data are the mean ± SD (n = 3), expressed as relative to day 0 in each condition (=1, dashed line). Statistical significance was evaluated for each cell line relative to controls (25 mM, −2DG, + Ser) in each condition, and for control-treated CDK12-OE vs. EV cells in the same experimental condition. **P = 0.002; ***P < 0.001, two-sided unpaired t-test. b Three-day proliferation of CDK12-OE and EV MCF10A cells silenced for PSAT1 (PSAT1-KD), MTHFD1 (MTHFD1-KD) and CDK12 (CDK12-KD) or treated with THZ531 (100 nM) and MTX (1μM). Data are the mean ± SD, expressed as relative to day 0 in each condition (=1, dashed line). Statistical significance, two-sided unpaired t-test (n = 3): (i) relative to Ctr-KD in each condition; (ii) relative to CDK12-OE cells silenced for CDK12 (CDK12-KD), for CDK12-OE cells treated with THZ531, silenced for CDK12 and concomitantly treated with THZ531, or treated with MTX. **P = 0.002; ***P < 0.001; n.s., not significant. c RT-qPCR analysis of knockdown (KD) efficiency in the cells described in (b). Data are the mean ± SEM relative to control-silenced cells (Ctr-KD = 1, indicated by horizontal dashed line) for each cell line (n = 1, performed in technical triplicates). d RT-qPCR analysis of the indicated SGOC enzymes in THZ531-treated (100 nM for 48 h) CDK12-OE vs. EV MCF10A cells. Data are the mean ± SEM relative to mRNA levels in control-treated (Veh.) cells (one representative experiment, out of two). e Acini formation in 3D-Matrigel by CDK12-OE vs. EV MCF10A cells treated with THZ531 (100 nM), methotrexate (MTX, 1 μM), or control-treated (Veh.). Left, data are the mean ± SD, expressed as relative to EV in vehicle-treated condition. Right, data are the mean ± SD and are expressed as percentage of disorganized acini (%) vs. total acini number. Statistical significance, two-sided unpaired t-test (n = 3) relative to vehicle in each condition, and relative to EV in the same experimental condition for vehicle-treated CDK12-OE cells. Source data are provided as Source Data file.