Fig 1: MKL1 accumulates in the nucleus in resistant mouse and human BCCsImmunofluorescence staining using antibodies against MKL1 and cytokeratin-14 (K14) in mouse (a) and human patient (b) BCC tumor sections. c-d and f-g, Compartmental quantification of tumor immunostaining. Positional staining intensity measured using the ImageJ multi-plot particle measure tool. K14 and DAPI used as markers for cytoplasmic and nuclear compartments respectively. n = 16 for resistant and n = 14 for sensitive mouse tumors. n = 24 for resistant and n = 11 for sensitive human tumors. Scale bars = 50µm and 10µm in respective low and high magnification fields. Pearson coefficient (r) used to determine MKL1 staining correlation with K14 (cytoplasmic) staining (***P < 0.001). e, ASZ001 cellular fractionation followed by immunoblotting for SRF and MKL1 highlights nuclear localization.
Fig 2: Expression and subcellular localization of MKL1 and SRF protein in ARMS cells. a Confocal images of MKL1 and SRF expression and subcellular localization in RH30 cells with or without overexpressed PAX3-FOXO1 protein. Protein expression of MKL1 (green) and SRF (red) and the colocalization or association (yellow) between them were attenuated with ectopic PAX3-FOXO1 expression, but almost not changed for their subcellular distribution by immunofluorescence staining. b Analysis of total MKL1 and SRF expression in RH4 cells with increasing amount of PAX3-FOXO1 protein expression. Left: MKL1 and SRF levels determined by western blot. Right: the relative expression of MKL1 and SRF protein shown on the left. c Analysis of total MKL1 and SRF expression in RH4 cells transfected by siRNAs against PAX3-FOXO1 gene. Left: Expression of MKL1 and SRF determined by western blot. Right: the relative expression of MKL1 and SRF protein shown on the left. d Distribution of SRF and MKL1 proteins in cytoplasmic fraction of RH30 cells transfected with increasing amount of PAX3-FOXO1 plasmid. Left: Expression of MKL1, SRF and PAX3-FOXO1 protein determined by western blot analysis. Right: the relative quantification analysis of MKL1, SRF and PAX3-FOXO1 protein shown on the left. a-Tubulin served as a loading control. e Distribution of SRF and MKL1 expression in nuclear fraction of RH30 cells transfected with increasing amount of PAX3-FOXO1 plasmid. Left: Expression of MKL1, SRF and PAX3-FOXO1 protein determined by western blot. Right: the relative quantification analysis of MKL1, SRF and PAX3-FOXO1 protein shown on the left. TBP served as a loading control for nuclear fraction. f Analysis of total RhoA expression in RH4 cells with a different dose of PAX3-FOXO1 plasmid transfected. Left: RhoA level determined by western blot. Right: the relative expression of RhoA protein shown on the left. g Analysis of total RhoA expression in RH4 cells transfected by siRNAs against PAX3-FOXO1 gene. Left: Expression of RhoA determined by western blot. Right: the relative expression of RhoA protein shown on the left
Fig 3: Proposed model of ACTA1 inhibition by PAX3-FOXO1 in ARMS cells. ACTA1 is inhibited by PAX3-FOXO1 through RhoA-MKL1-SRF signaling pathway. In this pathway, Cyto D and CCG-1423 can regulate ACTA1 expression by controlling nuclear accumulation of MKL1 in distinct ways. PAX3-FOXO1 can cooperate with these molecules in ACTA1 regulation. SRF activates ACTA1 expression by binding to the promoter region of ACTA1 gene. Overexpressed ACTA1 protein can inhibit ARMS cell proliferation, migration and tumor growth. Therefore, decreased ACTA1 expression by PAX3-FOXO1 may help to promote cell proliferation, migration and finally tumor growth.
Fig 4: SRF/MKL1 are necessary for resistant BCC growth and potentiate hedgehog pathway activitya, MTS assay carried out in ASZ001 with stable expression of antisense shRNAs against SRF. b, qPCR for GLI1 in ASZ001 cells with transient SRF knockdown. c, Schematic representation of known SRF activating pathways and associated inhibitors. d, MTS assay using the MKL inhibitor CCG-1423 uncovers MKL1 as a necessary SRF activator in ASZ001 cells. Data points represent mean MTS absorbance for biological triplicates ±SD. Red line indicates previously reported IC50 for CCG-1423. e, Relative expression of GLI1 mRNA in response to CCG-1423 treatment. f, MTS growth assay carried out in human resistant BCC cells (UW-BCC1) treated with indicated concentration of MKL inhibitor. g, mRNA expression of GLI1 in NIH-3T3 cells following Smoothened agonist (SAG) treatment in cells expressing full length MKL1 (MKL1-FL) and constitutively-active MKL1 (MKL1-N*). h, RNA-seq used to determine differential expression of genes regulated by CCG-1423 (1µM, left panel) and vismodegib (150nM, right panel) in ASZ001 cells. i, RNA-seq used to identify overlapped genes downregulated by CCG-1423 and vismodegib. j, Chromatin immunoprecipitation followed by sequencing (ChIP-seq) utilized to identify SRF and GLI1 genome-wide binding profiles and overlap within respective genomic peak intervals. k, ChIP-seq peak enrichment for common GLI1/SRF bound loci across 6kb genomic regions centered on SRF peaks. l, Positional ChIP-seq peak enrichment for SRF relative to GLI1 in ASZ001 cells. m, Representation snapshot of local ChIP-seq peak enrichment at the GLI1 genomic locus. n, Chromatin immunoprecipitation (ChIP) followed by qPCR using oligos against the 5’ untranslated region of GLI1, GLI2, CCND2, ACTB, and FOXF1. SRF occupancy is abolished after MKL1 inhibition (CCG-1423) or GLI1 inhibition (PSI) at all tested loci except for FoxF1. o-s, RPKM values from RNA-seq data for genes containing differential ChIP occupancy in panel n. Data represent mean qPCR fold enrichment over IgG control ±SD. Students t-test (two-tailed) used to determine significance for pairwise observations, *P < 0.05, **P < 0.001, ns = not significant. All data points represent the mean of triplicates ± SEM. For all ChIP experiments, vismodegib (Vismo), CCG-1423, and PSI were treated at 150nM, 1µM, and 5µM respectively.
Fig 5: Positive Feedback Loop Makes a Bi-stable Switch(A) The core regulatory network of the SRF-mediated double loop, comprising KLF4/5, MAL-SRF complex, focal adhesion proteins, and G-actin.(B) Bifurcation diagram of the KLF4/5 expression level as a function of the cell-matrix adhesion, for a MAL expression level approximately corresponding to that of the AST cells. Solid black lines represent stable cell states, with the SRF-repressed state in the upper branch and the SRF-activated state in the lower branch. For cell-matrix adhesion between the two limiting points LP1 and LP2, the cell can be in either of the two stable states. With changes of cell-matrix adhesion, the cell can switch between the two states reversibly. The region of negative substrate adhesion (gray bar) is unreachable. Blue line indicates the transition of AST cells.(C) AIT and AST cell adhesion curves on Matrigel-coated substrates with gradient of coating concentrations (0.1–40 µg/cm2) (mean ± SD, n = 4 independent biological replicates, *p < 0.05, **p < 0.01).(D) Relative expression of KLF4, KLF5, and NANOG in AIT and AST cells on Matrigel-coated substrates with three different coating concentrations (0.1, 1, and 10 µg/cm2) (mean ± SD, n = 3 independent experiments, *p < 0.05, NS, not significant).(E) Bifurcation diagrams of KLF4/5 expression level as a function of cell-matrix adhesion. The three diagrams correspond to the three values of MAL expression level indicated on the right. For a MAL expression level approximately that of an AIT cell (1.59 unit), the cell cannot switch to the upper branch by just changing the cell-matrix adhesion. With reduced total MAL expression, the upper branch can become reachable. Below a critical value (0.72 unit) of MAL expression, there is no bi-stability and the properties of the cell change smoothly. Green line indicates the irreversible transition of AIT cells. Violet line indicates the reversible transition of the small interfering RNA (siRNA)-treated AIT cells.(F) The relative change of the position of the two limiting points LP1 and LP2, when each of the 20 parameters in the model is increased or decreased by 15%.(G) Western blot result indicated the interference efficiency of MAL protein in AIT and AST cells.(H) siRNAs were transfected with lipofection. Suspended AIT cells were treated with siRNA for 2 hr, and then seeded on the GNF substrate. These cells formed a dome-like morphology after MAL siRNA transfection. Samples with only addition of liposome were used as a control.(I) Relative expression of KLF4, KLF5, and NANOG in AIT cells on low-adhesion GNF substrate with or without siRNA treatment (mean ± SD, n = 3 independent biological replicates, *p < 0.05, **p < 0.01). To avoid the interference of ROCK inhibitor on cell adhesion, data in this figure were obtained more than 48 hr after withdrawal of the ROCK inhibitor from cell cultures.See also Figure S7.
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