Fig. 4: BMP signaling prevents cell differentiation and promotes senescence in the presence of ATRA. | Nature Communications

Fig. 4: BMP signaling prevents cell differentiation and promotes senescence in the presence of ATRA.

From: Bone morphogenetic protein (BMP) signaling determines neuroblastoma cell fate and sensitivity to retinoic acid

Fig. 4

A Immunofluorescent staining for TGW cells treated for 5 days (0.2 µM ATRA, 0.5 µM K02288). MAP2 and NEFH expression and neurite structures are shown in red. Nuclei were stained with DAPI (blue). Representative results of 3 biologically independent replicates are shown. Scale bar = 50 µm. B MAP2 expression in TGW and SK-N-SH cells treated for 6 days (0.02 µM ATRA and 0.5 µM K02288 for TGW, 5 µM ATRA and 0.5 µM K02288 for SK-N-SH). Four isoforms of MAP2 are shown. C Viability of TGW cells treated with ATRA for 6 days in the presence of DMSO or K02288. Cell viability was measured with MTS. Data represent the mean ± SD of 4 independent replicates. At the arrow indicated point, P = 7.51 × 10−5 for 0.05 µM K02288, P = 3.99 × 10−6 for 0.2 µM K02288. D Senescent cells form blue crystals in TGW and SK-N-SH cells treated with 0.2 µM and 2 µM ATRA, respectively. Scale bar = 50 µm. E Senescence levels quantified by intensity of fluorescence. TGW and SK-N-SH cells were treated for 0 (DMSO), 3, 6, and 9 days with 0.2 µM and 2 µM ATRA, respectively. Cell lysates containing 10, 20, and 40 µg protein from each time point were collected for β-gal activity analysis. P values for the arrow indicated samples are 2.11 × 10−5, 1.18 × 10−4, 4.38 × 10−4, 6.03 × 10−5 (from left to right). F Senescence in TGW and SK-N-SH treated for 6 days (0.2 µM ATRA and 0.5 µM K02288 for TGW, 5 µM ATRA and 0.5 µM K02288 for SK-N-SH). Scale bar = 100 µm. G Senescence levels quantified by intensity of fluorescence in TGW and SK-N-SH cell lines treated the same as in (F). P values for ATRA vs ATRA + K02288 are 1.32 × 10−4 for TGW, 6.08 × 10−5 for SK-N-SH. H MAP2 expression in TGW with SMAD9 CRISPR knockout (SMAD9 KO). Cells were treated with DMSO or 0.2 µM ATRA for 6 days. Control, negative control gRNA with no target in the human genome; All, a mixture of 3 independent gRNAs targeting SMAD9; 2972, a single gRNA targeting SMAD9. I Senescence in TGW with SMAD9 KO. Cells were treated with 0.2 µM ATRA for 6 days. Scale bar = 50 µm. J Senescence levels quantified by intensity of fluorescence in TGW with SMAD9 KO. Cells were treated with 0.2 µM ATRA for 6 days. 20 µg protein from each sample was used. P values are 1.46 × 10−3 for KO-All, 4.75 × 10−3 for KO-2972. K Senescence in TGW and SK-N-SH treated for 6 days (0.05 µM ATRA and 1 µM FK506 for TGW, 0.5 µM ATRA and 1 µM FK506 for SK-N-SH). Scale bar = 100 µm. L Senescence levels quantified by intensity of fluorescence in TGW and SK-N-SH cell lines treated the same as in (K). P values for ATRA vs ATRA + K02288 are 0.019 for TGW, 2.24 × 10−3 for SK-N-SH. For panel B and H, β-Actin and GAPDH were used as loading control. Representative results of 3 independent replicates are shown. For panels D, F, I, and K, the representative results of 3 independent replicates are shown. For panels E, G, J, and L, data represent the mean ± SD of 3 independent replicates. P-values were calculated with two-tailed t-tests. Source data are provided as a Source Data file.

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