Fig. 6: SNAI2 anti-differentiation effects are mediated by the blockade of MYOG, MEF2A/C/D and CDKN1A. | Nature Communications

Fig. 6: SNAI2 anti-differentiation effects are mediated by the blockade of MYOG, MEF2A/C/D and CDKN1A.

From: Interaction between SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in Fusion Negative Rhabdomyosarcoma

Fig. 6

a Schematic representation of role of SNAI2 in RAS-mutated RMS and induction of differentiation after SNAI2 knock down. b, c, e, f, h, i Representative images of RD shScr and shSNAI2 cells transfected with siRNA stained for Myosin Heavy Chain 1 (MyHC, green), MEF2C (red) and DAPI for nuclei (blue). Green numbers bottom of each image represents the average number of percentage of positive cells for three images. Scale Bar in b = 100 μM. d, g Western blot of RD shScr and shSNAI2.1 cells transfected with siRNAs against MEF2A, and MYOG along with Control siRNA (scramble) probed for MEF2A and MYOG. Representative blot, n = 3 biologically independent experiments. j Box plot depicting SNAI2 myogenic direct target gene expressions (RNA-seq) in SMS-CTR cells transduced with shRNAs or treated with trametinib. TPM, Transcripts per Million. Box plots show quartiles, black bar shows the median, and whiskers show the 1.5 × interquartile range. The exact p values are reported in the figure (n = 3 biologically independent experiments). k Scatter plot of log2 fold change (L2FC) of MYOG-activated SE gene expression (RNA-seq)20 (top) and shSNAI2/shScr transduced vs trametinib/DMSO treated cells for RAS-dependent SE gene expression (RNA-seq)20 (bottom) in SMS-CTR cells. l Representative ChIP-seq tracks for SNAI2 (blue), MYOD (green), H3K27ac (yellow) (top) and gene expression (RNA-seq) (bottom) in SMS-CTR. Δ, trametinib minus DMSO; TPM, Transcripts per Million. m Representative ChIP-seq tracks for SNAI2 (blue), MYOD (green), H3K27ac (yellow) (top) and gene expression (RNA-seq) (bottom) in SMS-CTR. Δ, shSNAI2.1 minus shScr; TPM, Transcripts per Million. n Western blot of RD, JR1, and SMS-CTR cells treated with 10 nM trametinib compared to vehicle control (DMSO), probed for p-ERK (phosphorylated), ERK, SNAI2, MEF2C, and actin (loading control). Representative blot, n = 3 biologically independent experiments. o Schematic model: SNAI2 expression in FN-RMS is regulated by MYOD and SNAI2 binding is able to dampen MYOD binding at myogenic transcription factor genes, which contributes to maintenance of a myogenic differentiation block (left). MEK inhibition with trametinib and/or SNAI2 knockdown in FN-RMS releases SNAI2, allowing MYOD to activate genes important for myogenic differentiation (MYOG, MEFs, TNNTs) thus inducing muscle differentiation (middle). Silencing of myogenic SNAI2 target genes (like MYOG or MEF2A) blocks differentiation downstream of SNAI2. Bubbles depict transcriptional co-activators in inactive (gray) or active (yellow) status.

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