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. 2020 Feb 19;6(8):eaaw9960. doi: 10.1126/sciadv.aaw9960

Fig. 5. MSN-NONO interaction activated CREB signaling to promote breast cancer progression by facilitating the nuclear localization of pPKC.

Fig. 5

(A) CREB phosphorylation level was measured by Western blot after overexpressing NONO in MDA-MB-231 cells by Western blot. (B) CREB phosphorylation level was determined in MSN-overexpressing MDA-MB-231 cells by Western blot. (C) CREB phosphorylation level was determined in MSN knockdown MDA-MB-231 cells by Western blot. (D) Western blot was used to measure CREB phosphorylation level after overexpressing MSN with different mutation status in MDA-MB-231 cells. (E) The downstream genes of CREB signaling from the results of RNA-seq of MDA-MB-231–shCTRL and MDA-MB-231–shMSN cells were shown as heatmap according to relative expression level. (F) The expression of CREB downstream genes ALS2 and CCNA1 was measured by qRT-PCR in MSN knockdown MDA-MB-231 cells. (G) The expression of CREB downstream genes ALS2 and CCNA1 was measured by qRT-PCR in MSN-overexpressing MDA-MB-231 or T47D cells. (H) Phosphorylation level and total proteins of CREB were determined in MSN-overexpressing and NONO-knocking down MDA-MB-231 cells by Western blot. (I) The expression of CREB downstream genes ALS2 and CCNA1 was measured in MSN-overexpressing and NONO-knocking down MDA-MB-231 cells by qRT-PCR. (J) The expression relationship between MSN and CREB downstream genes ALS2 or CCNA1 was analyzed in breast cancer cell lines from the dataset (Heiser 2012) contained in the UCSC Xena database. (K) CREB phosphorylation level was detected in MDA-MB-231 cells overexpressing NONO in different truncated forms by Western blot. (L) The immunoprecipitated samples obtained with anti-FLAG M2 Magnetic Beads of CTRL or FLAG-MSN–overexpressing MDA-MB-231 cells were conducted with Western blot. (M) Nuclear protein level of pPKCζ was observed after MSN knockdown by Western blot. (N) Nuclear protein level of pPKCζ was observed after NONO knockdown by Western blot. (O) pPKCζ level in cytoplasm and nucleus was determined by Western blot after overexpressing MSN with different mutagenesis. (P) We examined the effect of PKC inhibitor on the phosphorylation of CREB when overexpressing MSN by Western blot. *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired t test of triplicates. Error bars, means ± SEM.