Skip to main content
. 2021 Jun 29;12:4019. doi: 10.1038/s41467-021-24064-1

Fig. 5. MLK3 links cell cycle to the MAPK stress pathway to activate the HSF1 response.

Fig. 5

WTp53 activation represses MLK3. a The expression of cell cycle progression genes is inhibited by p53 activation. HCT116 cells were transfected with siRNAs for p53 or scrambled control siRNA (scr2) for 48 h, followed by DMSO or 10 µM Nutlin treatment for 24 h. qRT-PCRs for the indicated mRNAs normalized to RPLP0 mRNA. MLK3 mixed lineage kinase 3, PLK4 polo-like kinase 4. b MLK3 silencing suppresses pSer326-HSF1, mimicking the effect of p53 activation. The indicated cells were transfected with an siRNAs pool against MLK3 or scrambled control siRNA (scr2). Forty-eight hours post transfection, cells were treated with DMSO or 10 µM Nutlin for 24 h. Immunoblot analysis. GAPDH, loading control. c MLK3 silencing abrogates HSF1 target gene expression, mimicking the effect of p53 activation. HCT116 cells were transfected and treated as in b. qRT-PCRs for the indicated mRNAs normalized to RPLP0 mRNA. d HSF1 target gene expression is attenuated after MLK3 depletion. Indicated cells were transfected with an siRNAs pool against MLK3 or scrambled control siRNA (scr2). Seventy-two hours post transfection, qRT-PCRs for the indicated mRNAs were performed. Normalized to RPLP0 mRNA. e–g Cell cycle inhibition reduces MLK3 expression and causes MEK1 inactivation. The indicated cells were treated for 24 h with DMSO, 10 µM Nutlin, or 10 µM Palbociclib (CDK4i) (e, g), RG7112 (f), and RO3306 (g) at the indicated concentrations. Immunoblot analysis. GAPDH, loading control. MLK3/GAPDH, MLK3 densitometry normalized to loading control. h Mlk3 mRNA levels of isolated p53Q/fl;vilCreERT2 tumor organoids treated as in Fig. 2c. qRT-PCR normalized to Hprt1 mRNA. Mean ± SD of eight independent experiments from ≥3 organoid cultures/3 different mice. i Mlk3 mRNA levels of heterozygous p53Q/fl;vilCreERT2 organoids treated as indicated for 24 h. Dox Doxorubicin in µM, 5-FU 5-Fluorouracil in µM. qRT-PCR normalized to Hprt mRNA. Mean ± SD from two independent experiments, one included a technical replicate from the same organoid culture (total n = 3). j p53Q/fl;vilCreERT2 organoids treated with MEKi U0126 for 6 h. qRT-PCR of the indicated HSF1 target genes normalized to Hprt mRNA. Mean ± SD of ≥2 different organoid cultures with two technical replicates each. Student’s (a, c, d) mean ± SD of two independent experiments, at least one with a technical replicate. Relative values as ratio (2−ddCT). a, c, d, h, I, j Students t test, two-sided. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns not significant. b, e–g Source data are provided as Source data file.