Additional file 2 of Investigation of the impact of bromodomain inhibition on cytoskeleton stability and contraction
Creators: name: Bigger-Allen, Alexander
name: Gheinani, Ali Hashemi
name: Adam, Rosalyn M.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Cancer
subject: Hematology

description: Additional file 2: Supplementary Figure 1. PCA plots of PDGF and TGFB stimulated SMC and Fibroblasts datasets.4 microarray datasets were identified from NCBI GEO database in which fibroblasts or smooth muscle cells were stimulated with PDGF or TGFB. PCA and scree plots were generated using the raw expression data to determine the extent of variation between conditions and replicates. The first plot in each pair of plots contains the first two principle components (PC1 and PC2) on the x and y axis respectively along with the percentage of the total variation they account for in each dataset. The second plot in each of the panels represent all principle components and the percentage of total variation with the dataset that each principle component accounts for ranked from greatest to least variation. A) The dataset, GSE52488, is previously published by us and contains primary Human bladder smooth muscle cells (pHBSMC) stimulated with vehicle control (Ctrl) or PDGF for 24hrs.B) GSE63883 comprises Human airway smooth muscle cells stimulated with vehicle control (Ctrl) or TGFB for 8hrs. C) GSE14256 comprises human fibroblasts stimulated with vehicle control (Ctrl) or PDGF for 24hrs. D) GSE61128 comprises human fibroblasts stimulated with vehicle control (Ctrl) or TGFB for 6hrs. Supplemental Figure 2. Secondary TF enrichment analysis using UCSC TFBS. Volcano plots of top 3000 differentially expressed genes in which the log2 fold (log2FC) changes and –log10 transformation of the p-values were plotted on the x and y axis respectively. Genes that met the log2FC cutoffs of less than -0.25 or greater than 0.25 as well as an adjusted p-value cut off of &lt; 0.05 were colored in red. In green are genes that only meet the log2FC cutoffs, in blue are the genes that only meet the p-value cutoffs and the genes in grey meet none of the cutoffs. A) Differentially expressed genes from GSE63383, comprising Human Airway smooth muscle cells stimulated with TGFB vs control (Ctrl). B) Differentially expressed genes from GSE14256, comprising Human foreskin fibroblasts stimulated with PDGF vs control (Ctrl). C) Differentially expressed genes from GSE61128, comprising Human arterial fibroblasts stimulated with TGFB vs control (Ctrl). Genes that met both cutoffs, identified in read in the volcano plots, were compared with differentially expressed genes from our previously published dataset of primary human bladder smooth muscle cells stimulated with PDGF or vehichle control. Roughly 800 differentially expressed genes (DEG) were common between our dataset and each of the three other datasets. These lists of genes were subjected to transcription factor (TF) enrichment analysis using the UCSC transcription factor binding sequence database. Nearly 170 TF were identified for each list of shared DEGs. D) The top shared TFs (y axis corresponding to each dot) were rank ordered by the percentage of genes in the list of DEGs that were calculated to be regulated by that TF (indicated by the size of each dot). The –log10 transformed adjusted p-values were reported as the color of each dot and the fold enrichment of each TF were plotted on the x axis. Supplemental Figure 3. No Enrichment of cytoskeleton and chromosome related terms using traditional GSEA approach for GSE63383. A) Standard Gene Set Enrichment Analysis (GSEA) was performed on genes shared between our previously published dataset and those from GSE63383 comprising Human airway smooth muscle cells stimulated with PDGF and compared to control. GO terms for molecular function (MF), biological process (BP) and cellular compartment were determined using a running sum statistic on the shared DEG list rank ordered by log2 fold change (log2FC). Enriched terms were than rank ordered based on the ratio of genes in the DEG list that match the total number of genes associated with each term. Each term is colored based in its adjusted p-value.P-values were adjusted using Bonferroni method. The size of the dot, corresponding to each term indicate the number of genes from each DEG list that map to each term. GSEA curves where plotted for three or four B) MF terms, C) BP terms and D)CC terms that were statistically most significant and or have the greatest absolute value of normalized enrichment score (NES). Supplemental Figure 4. Enrichment of Cytoskeleton and chromosome related terms using traditional GSEA approach for GSE14256.A) Standard Gene Set Enrichment Analysis (GSEA) was performed on genes shared between our previously published dataset and those from GSE14256 comprising Human foreskin fibroblasts stimulated with PDGF and compared to control. GO terms for molecular function (MF), biological process (BP) and cellular compartment were determined using a running sum statistic on the shared DEG list rank ordered by log2 fold change (log2FC). Enriched terms were than rank ordered based on the ratio of genes in the DEG list that match the total number of genes associated with each term. Each term is colored based in its adjusted p-value.P-values are adjusted using Bonferroni method. The size of the dot, corresponding to each term indicate the number of genes from each DEG list that map to each term. GSEA curves where plotted for three or four B) MF terms, C) BP terms and D) CC terms that were statistically most significant and or have the greatest absolute value of normalized enrichment score (NES). Supplemental Figure 5. No Enrichment of cytoskeleton and chromosome related terms using traditional GSEA approach for GSE61128. A) Standard Gene Set Enrichment Analysis (GSEA) was performed on genes shared between our previously published dataset and those from GSE61128 comprising Human aortic fibroblasts stimulated with TGFB and compared to control. GO terms for molecular function (MF), biological process (BP) and cellular compartment were determined using a running sum statistic on the shared DEG list rank ordered by log2 fold change (log2FC). Enriched terms were than rank ordered based on the ratio of genes in the DEG list that match the total number of genes associated with each term. Each term is colored based in its adjusted p-value. P-values are adjusted using Bonferroni method. The size of the dot, corresponding to each term indicate the number of genes from each DEG list that map to each term. GSEA curves where plotted for three or four B) MF terms, C) BP terms and D) CC terms that were statistically most significant and or have the greatest absolute value of normalized enrichment score (NES). Supplemental Figure 6. Enrichment analysis using GO-CC terms; changes in chromatin remodeling and cytoskeleton. Shared DEGs were identified between our PDGF stimulated pHBSMC dataset and each of the three other datasets. These DEG lists, along with the log2 fold change and p-values for each gene were subjected to active subnetwork searches against the BIOGRID protein-protein interaction network followed by gene ontology enrichment analysis for terms related to cellular compartment. Similar terms were then clustered. These analyses were performed using functions from the pathfindR package. In all comparisons of our dataset, either with A)GSE63383 in which smooth muscle cells were stimulated with TGFb, B) GSE14256 in which fibroblasts were stimulated with PDGF, or C) GSE61128 in which fibroblasts were stimulated TGFb, there was an enrichment of terms related to chromatin (clusters highlighted with orange boxes) and for changes in the cytoskeleton (clusters highlighted with green boxes). Supplemental Figure 7. Identification of shared genes between all 4 datasets, TF regulator analysis and enrichment analysis. Differentially expressed genes (DEGs) form each of the four microarray datasets were compared via A) a 4 way Venn-diagram to identify the number of genes that were common to all four datasets. B) MYC and MAX were among the top TF predicted to regulate these shared genes based on -log Fisher's exact test (FET) p-value. Enrichment analysis of the shared genes was performed using enrichGo function from the clusterprofiler R package without the use of Log2 fold changes or p-values searching for C) GO-MF terms, D)GO-BP terms and E) GO-CC terms. Supplemental Figure 8. Quality assessment of Data sets using PCA plots to determine homogeneity among treatment groups. Quality assessment of publicly available data in which mechanically active cells are stimulated with A)PDGF (GSE111714), B)TGFb (GSE127229) or C) Growth media (GSE138323) with or without JQ1 using PCA plots. More than 80% of the variation for each dataset was between the conditions and not within the replicates as captured in the first two principle components. Supplemental Figure 9. Enrichment analysis of JQ1 sensitive genes using GO-MF terms. JQ1 sensitive genes for each dataset were identified with the DEGreport package on normalized count data. The JQ1 sensitive genes, regardless of the direction of change in expression were then subject to active subnetwork search against the BIOGRID database of protein-protein interaction networks followed by enrichment analysis and clustering using functions from the pathfindR package. Clusters of enriched GO terms related to molecular function were rank ordered by adjusted p-value of the first term in each cluster and plotted as dot plots. A-C)Clusters of terms for chromatin related changes are highlighted in orange and cytoskeleton related changes are highlighted in green. Supplemental Figure 10. Pathway and expression of JQ1 sensitive cytoskeleton associated genes from PDGF and TGFB datasets. A) JQ1 sensitive genes from the PDGF and TGFB datasets were filtered for genes in the KEGG pathway, “Regulation of the actin cytoskeleton”. Green genes were perturbed by TGFB and returned near to baseline with JQ1. Red genes were perturbed by PDGF and returned near baseline with JQ1. Red and Green colored genes were perturbed in both datasets and returned to near baseline expression by JQ1. B)The same 39 unique genes were plotted based on their baseline mean expression and log2 fold change. The color of each gene reflects the-log10 transformation of the adjusted p-value. Triangles indicate JQ1 sensitive genes from the TGFB dataset. Circles indicate JQ1 sensitive genes from the PDGF dataset. Genes with a mean expression count of greater than 3000 and an absolute log2FC of greater than 0.15 or 20% were labeled and considered for in vitro validation.Supplemental Figure 11. RNAseq dataset of fibroblasts stimulated with JQ1 or shBRD4 under proliferative or quiescent conditions. RNA-seq dataset comprising human fibroblasts under conditions of growth or quiescence and treated with JQ1, DMSO, shCtrl, or shBRD4 (GSE189585). A) Principle component analysis identified 75% of the total variation in the dataset in the first two principle components was attributed to the difference among the conditions.B) Skree plot of all principle components plotted on the x axis and the percentage of the total variation they account for within the dataset. Supplemental Figure 12. Pattern Analysis identifies groups of genes attenuated by JQ1 and/or BRD4. Three conditions were assesed to identify the JQ1 sensitive and shBRD sensitive genes. These conditions included DMSO or control shRNA treated quiescent cells (Ctrl_Qsnt), DMSO or control shRNA treated proliferating cells and JQ1 or shBRD4 treated proliferative (Expt_Pro) cells indicated in pink and light blue respectively. JQ1 and BRD4 sensitive genes were calculated with the DEGreport package and grouped into three categories; genes sensitive to JQ1 and not shBRD4 (indicated with a red boarder), genes sensitive to shBRD4 and not JQ1 (indicated with a light-blue boarder) and genes sensitive to both that change in the same direction (indicated with a purple boarder). Supplemental Figure 13. Enrichment analysis using CC and MF terms of BRD4 and JQ1 attenuated DEGs induced to near baseline. JQ1 and shBRD4 sensitive genes (JQ1_attenuated and BRD4_attenuated respectively) were separated based on the change in expression comparing the JQ1 or shBRD4 treated proliferative cells to the DMSO or control shRNA proliferative cells. The upregulated genes are subjected to active subnetwork searches using the BIOGRID database of protein-protein interactions followed by enrichment analysis of these networks of genes using A)GO terms for cellular compartment and B)Molecular function. Enriched terms are plotted on the y axis and their fold enrichments are plotted on the x axis. Terms are plotted as dots whose sizes correspond to the number of genes from the DEG lists that are associated with each term. The color of each dot corresponds to the –log10 transformed p-values. Supplemental Figure 14. Representative genes of JQ1 on and off target attenuation of cytoskeleton associated genes. A)Comparative analyses via an upset plot of 5 groups of gene A) 569 unique genes in the GO-CC library of terms for stress fiber, focal adhesion, and actin cytoskeleton, which were identified as enriched from genes attenuated by both the JQ1 and shBRD treatments. B) 1312 shared genes attenuated by both JQ1 and shBRD C) 1920 JQ1 sensitive genes attenuated only by JQ1. D) 1574 genes predicted to be JQ1 sensitive perturbed by PDGF in dataset GSE111714 and E) the 1108 JQ1 sensitive genes predicted from the TGFb dataset, GSE127229. 4 groups of shared genes were identified. Group 1 (indicated in dark blue) comprises 1 gene (ITGB1) which is shared between gene groups A, B, D and E and represents a BRD4 and JQ1 sensitive (or on-target) cytoskeleton associated gene that is perturbed by both PDGF and TGFB. Group 2 (indicated in light blue) comprises 12 genes and represents JQ1 sensitive, cytoskeleton-associated genes not identified as JQ1 off-targets (exclusive to JQ1 sensitive genes in GSE189585) or as JQ1 on-targets (genes sensitive to both JQ1 and shBRD4). Group 3 and Group (indicated in red and yellow respectively) represent 8 and 9 JQ1 off-target cytoskeleton-associated genes stimulated in the PDGF and TGFB datasets respectively. An additional 3 genes encoding kinases were assessed do to their relevance for initiating, propagating and facilitating changes to the cytoskeleton. These include PDGFRA, SRC, LIMK1. These 33 genes were visualized in two KEGG pathways that represent the majority of these genes, B)regulation of the actin cytoskeleton, and C) focal adhesion. Genes are color coded based on the group to which they were identified. Supplemental Figure 15. HBSMC and RBMC comparison and JQ1 dose and time effect on cellular viability. A)A heatmap of Ct values from qPCR data comparing RBMC and HBMC gene expression included ECM, smooth muscle and fibroblast specific gene expression. Higher expression is color coded in blue and lower expression is color coded in red. B)Dose and time response of RBMC stimulated with JQ1. Supplemental Figure 16. Validation of JQ1 sensitive cytoskeletal genes in pHBSMC. A heatmap of Log2FC values from qPCR data assessing a time course of Veh, PDGF or TGFb stimulation with or without JQ1. Data are representative of 3 independent trials. Supplemental Figure 17. JQ1 robustly reduces transcription of contractile genes human and rat bladder contractile cells. A)Smooth muscle markers were assessed in both the RBMC and pHBSMC via qPCR and B)immunoblots. MYC was also assessed via immunoblot to confirm JQ1 reduced MYC protein expression. Supplemental Figure 18. JQ1 inhibits contraction of RBMCs on collagen gels after 16hrs with low cytotoxicity. A) A dose and time response of JQ1 mediated inhibition of RBMC contraction of collagen gels and B)assessment of cell viability using Alamar blue. Bar charts represent the means three independent replicates. Error bars reflect the standard deviation. p-values were calculated with student ttest. * p-value &lt; 0.05, ** p-value &lt; 0.01, *** p-value &lt; 0.001. Data are representative of 3 independent trials. Supplemental figure 19. Uncropped immunoblots of RBMC time course with PBS, PDGF, TGFB, JQ1, and/or DMSO. Representative blots of lysates from RBMC stimulated with vehicle control (V), PDGF (P), or TGFB (T) concurrently with DMSO or JQ1 for 2, 4, 8, and 16 hrs. Blots were stained for ITGA5, ITGB1, PDGFRB, PDGFRA, phosphorylated and total CFL1, SRC, LIMK1. Supplemental figure 20. Uncropped immunoblots of pHBSMC and RBMC with PBS, PDGF, TGFB, JQ1, and/or DMSO. Representative blots of lysates from RBMC and pHBSMC stimulated with vehicle control (V), PDGF (P), or TGFB (T) concurrently with DMSO or JQ1 for 16 hrs. Membranes were stained with antibodies against, MYC, CNN1, SM22a, BACT, and Vim