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. 2024 Sep 14;10:80. doi: 10.1038/s41523-024-00687-7

Fig. 4. Overactivity of Ck2 toward Stat3 in Py230 shMcam cells drives the alveolar progenitor phenotype.

Fig. 4

a Upregulated Stat3 expression and phosphorylation in Py230-A cells and its inhibition by the kinase inhibitors, CX-4945 10 μM, Ag490 50 μM, Stattic 1 μM for 30 min in WB analysis. b Representative FACS analysis of shCon and shMcam Py230-A cells stained for Epcam, Ncam1, and Sca1 with gating (blue outline) of Epcamhigh Ncam1low cells to examine Sca1 levels. Representative plots of shMcam cells treated with 10 μM CX-4945 for 7 days (right two panels) show restoration of the Epcamhigh Ncam1low Sca1high population. c–e Compiled FACS analysis showing the rescue of Epcamhigh Ncam1low Sca1high subpopulation frequencies in Py230-A and Py230-B cells treated with CX-4945 10 μM for 7 Days (c), with Stattic 1 μM for 7 days (d), and with Ag490 30 μM for 7 days (e). Whiskers mark the 5th and 95th percentile; boxes demarcate 25th, 50th, and 75th percentile. One-way ANOVA (Geisser-Greenhouse correction) with Tukey multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. f Western blot analysis in Py230-A cells showing higher basal levels of pPten (Ser380/Thr382/383) and pAKT in Mcam KD. g Gene expression in Py230-A cells treated with CX-4945 10 μM for 7 Days (scRNA-seq). h Western blot analysis in select cell lines showing differential representation of phosphorylated CK2 substrates in siCon and siMcam transfected cell lines (Py230 and Met1) in culture for 48 h post transfection or with shCon and shMCAM transduction (MCF10A). red circle = increased in MCAM KD, blue triangle = decreased in MCAM KD. i Co-immunoprecipitation western blot analysis of a transiently co-transfected Flag-tagged Mcam variants and HA-Ck2a/Myc-Ck2b expression constructs in HEK293A cells. FL full length Mcam, S629A = point mutant in the CK2 phosphorylation site of Mcam, Δ72 = deletion of the distal 72 amino acids of the Mcam tail, Δ120 = deletion of the membrane proximal 120 amino acids of the Mcam tail, ΔCyto = complete deletion of the cytoplasmic tail of Mcam.