Journal: bioRxiv

Article Title: HDAC Inhibitors recapitulate Human Disease-Associated Microglia Signatures in vitro

doi: 10.1101/2024.10.11.617544

Figure Lengend Snippet: Bulk RNAseq data from the HMC3 DAM model. A. Heatmaps showing the expression of Cluster 11 (left), Microglia 13 (middle) and DAM1/DAM2 (right) marker gene sets in bulk RNAseq data generated 24hrs following exposure to DMSO (control), Entinostat (red; 10µM) or Vorinostat (green; 1µM). Each column represents a single sample, each row a single gene represented in the respective marker set. Pairwise differential testing between DMSO control and each of the treatment conditions (Entinostat, 10µM; Vorinostat, 1µM) was conducted using a Wald test with the Benjamini-Hochberg correction (FDR alpha < 0.05). The legend represents Z scores, with lower scores indicated in red and higher scores indicated in blue. Data represents n=3 independent experiments for each treatment group with each n for all compounds being performed at the same time. B. Volcano Plots depicting the distribution of differentially expressed genes from different signatures (DAM1, DAM2 , Cluster 11 , Microglia 13 ) for each treatment condition (Entinostat or Vorinostat) in comparison to DMSO control. HMC3 microglia were treated for 24hrs with DMSO as control, Entinostat (10µM) or Vorinostat (1µM) followed by bulk RNA-Seq. Volcano plots depict all genes present in each marker set (DAM1: 10 genes; DAM2: 20 genes; Cluster 11: 89 genes, Microglia 13: 127 genes) plotted based on log2FC (fold change expression) and -log10(p value) with the ones significantly upregulated marked in red and labelled with the gene name. Plots are organized from Cluster 11 (top left), to DAM1 (bottom left), to DAM2 (top right) to Microglia 13 (bottom right). C. PCA plot of bulk RNAseq results from HMC3 microglia treated with DMSO, Vorinostat or Entinostat . Principal component analysis (PCA) was calculated on log-normalized bulk RNA-Seq data derived from compound-treated HMC3 microglia following 24hrs of exposure to DMSO (control; blue), Entinostat (10µM; red) or Vorinostat (1µM; green). Data represents n=3 independent experiments for each of the treatment group with each n for all compounds being performed at the same time. D. Pie chart depicting the number of significantly upregulated genes by Entinostat or Vorinostat for each of the queried marker signatures DAM1, DAM2, Cluster 11 , Microglia 13 . The number of significantly upregulated genes across all three replicates for each treatment group (Entinostat or Vorinostat) in comparison to DMSO control was identified and converted to a percentage of marker genes upregulated/ marker set. Data for DAM1 are depicted in purple, for DAM2 depicted in red, for Cluster 11 depicted in violet and for Microglia 13 depicted in teal. E. Signature-specific markers induced by Vorinostat and Entinostat. Markers significantly induced by Vorinostat and Entinostat for each signatured are depicted for DAM1 (purple), DAM2 (red), Cluster 11 (violet), Microglia 13 (teal).

Article Snippet: The next day, microglia were treated with the respective concentrations of Vorinostat (1µM; Ambeed; Cat #: A234507), Cholic acid (10µM; Cayman chemical; Cat #: 20250), Flavokavain B (10µM; ChromaDex; Cat #: ASB-00006058- 005), Wiskostatin (1µM; Cayman chemical; Cat #: 15047), Trimipramine (10µM; Cayman chemical; Cat #: 15921), Naftopidil (10µM; APExBIO; Cat #: 57149-07-2), Ramipril (10µM; APExBIO; Cat #: B2208), Valporic acid (90µg/ml Sigma; Cat #: PHR1061), Geranylgeraniol (10µM; Sigma; Cat #: 24034-73-9), Entinostat (10µM; Ambeed; Cat #: A122285), Amiodarone hydrochloride (10µM; Sigma; Cat #: A8423), Temozolomide (100µM; APExBIO; Cat #: B1399) or DMSO (Sigma-Aldrich, Cat #:472301) as control and incubated for 6hrs and 24hrs before harvest for RNA extraction.

Techniques: Expressing, Marker, Generated, Control, Comparison, RNA Sequencing Assay, Derivative Assay

Journal: bioRxiv

Article Title: HDAC Inhibitors recapitulate Human Disease-Associated Microglia Signatures in vitro

doi: 10.1101/2024.10.11.617544

Figure Lengend Snippet: Bulk RNA-Seq of the human iPSC-derived microglia (iMG) DAM model. A. Volcano Plots depicting the distribution of differentially expressed genes from different signatures (Cluster 11 , Microglia 13 , iMG Cluster 2+8 ) for Vorinostat treatment in comparison to DMSO control. iPSC-derived microglia at Day 28-29 of differentiation were treated for 24hrs with DMSO as control or Vorinostat (0.1µM) followed by bulk RNAseq. Volcano plots depict all genes present in each marker set (Cluster 11: 89 genes, Microglia 13: 127 genes, iMG Cluster 2+8: 134 genes) plotted based on log2FC (fold change expression) and -log10(p value) with the ones significantly upregulated marked in red and of the most significantly changed genes, a selection of nine genes was labeled with the gene name. Plots are organized from Cluster 11 (left), to Microglia 13 (middle), to iMG Cluster 2+8 (right). B. Heatmaps showing the expression of Cluster 11 (left), Microglia 13 (middle) and iMG Cluster 2+8 (right) marker sets in bulk RNAseq data generated 24hrs following compound treatment with DMSO (control) or Vorinostat (green; 0.1µM). Each column represents a single sample, each row a single gene represented in the respective marker set. Pairwise differential testing between DMSO control and each of the treatment conditions (Entinostat, 10µM; Vorinostat, 1µM) was conducted using a Wald test with the Benjamini-Hochberg correction (FDR alpha < 0.05). The legend represents Z scores, with lower scores indicated in red and higher scores indicated in blue. Data represents n=5 independent experiments per treatment group from one batch of iPSC-derived human microglia. For data replication in a second batch see Supple. . C. Venn diagram depicting significantly induced markers across the signatures for Cluster 11 , Microglia 13 and iMG Cluster 2+8 in Vorinostat-treated iMGs. Each circle shows significantly induced markers from each marker set - Cluster 11 (violet), Microglia 13 (green), Dolan et al. (red). Overlays of circles depict induced marker genes shared across different combinations of marker sets. Percentage indicates ratio of each marker set in relation to the total number of significantly induced markers across all three signatures. D. MITF expression in HMC3 and iMG DAM models. Violin plots depict the expression of the transcription factor MITF in transcripts per million (TPM) across treatment conditions in HMC3 microglia (top; DMSO (blue), Vorinostat (1µM; green), Entinostat (10µM; red); n=3/group) and iMG (bottom; DMSO (blue), Vorinostat (0.1µM; green); n=6 per group, one iMG batch; for data replication see Suppl. ). For statistical analysis of HMC3 data, one-ay ANOVA followed by Dunnett’s multiple comparisons test was performed. For iMG data, unpaired t-test was performed. Each dot represents a replicate, central interrupted line represents the median and fine dotted lines represent the interquartile range. *p.adj ≤ 0.05, **p.adj ≤ 0.01, ***p.adj ≤ 0.001 test

Article Snippet: The next day, microglia were treated with the respective concentrations of Vorinostat (1µM; Ambeed; Cat #: A234507), Cholic acid (10µM; Cayman chemical; Cat #: 20250), Flavokavain B (10µM; ChromaDex; Cat #: ASB-00006058- 005), Wiskostatin (1µM; Cayman chemical; Cat #: 15047), Trimipramine (10µM; Cayman chemical; Cat #: 15921), Naftopidil (10µM; APExBIO; Cat #: 57149-07-2), Ramipril (10µM; APExBIO; Cat #: B2208), Valporic acid (90µg/ml Sigma; Cat #: PHR1061), Geranylgeraniol (10µM; Sigma; Cat #: 24034-73-9), Entinostat (10µM; Ambeed; Cat #: A122285), Amiodarone hydrochloride (10µM; Sigma; Cat #: A8423), Temozolomide (100µM; APExBIO; Cat #: B1399) or DMSO (Sigma-Aldrich, Cat #:472301) as control and incubated for 6hrs and 24hrs before harvest for RNA extraction.

Techniques: RNA Sequencing Assay, Derivative Assay, Comparison, Control, Marker, Expressing, Selection, Labeling, Generated