Journal: bioRxiv

Article Title: Actomyosin contractility modulates Wnt signaling through adherens junction stability

doi: 10.1101/220178

Figure Lengend Snippet: (A,B) Wnt-responsive luciferase TOPFLASH assay measuring TCF/LEF reporter activity in (A) MCF7 and (B) RKO cells following Wnt3A transfection and siPP1β and siMYPT3 transfection. Data are presented as mean ± SEM with letters above representing significant difference from corresponding column, (P<0.01). (C) Western blot analysis of total cellular levels of E-cad, β-cat, MYPT3, Wnt3A and PP1β. β-tub was used as a loading control. (D) Western blot analysis of β-cat in cytoplasmic (Cyto), membranous (Mem), chromatin-bound (CB), and whole cell extract (WCE) fractions in MCF7 cells. GAPDH, Na + /K + ATPase, and Histone 3 were used as loading controls for corresponding fractions. Complete fraction, see . (E) Quantification of relative levels of β-cat from each fraction taken from (D), normalized to Control + siScramble conditions. Data shown as mean ± SEM (n = 4 biological replicates), ns = not significant, *P<0.05.

Article Snippet: Proteins were transferred onto nitrocellulose membranes, and probed against the following primary antibodies: rabbit anti-β-catenin (1:1000, Cell Signaling), rabbit anti-E-cadherin (1:1000, Cell Signaling), mouse anti-PPP1R16A (MYPT-3) (1:500, abcam), mouse anti-Protein Phosphatase 1 beta (PP1β) (1:1000, abcam), rabbit anti-Wnt3A (1:1000, Cell Signaling), mouse anti-β-tubulin (1:1000, ABM), rabbit anti-GAPDH (1:3000, Cell Signaling), mouse anti-Na+/K+ ATPase (1:50 DSHB), rabbit anti-Histone H3 (1:1000 Cell Signaling).

Techniques: Luciferase, TOPFlash assay, Activity Assay, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of endogenous EZH2 (A) or SUZ12 (B) immunostaining in MDA-MB-231 and BoM-1833 cells. Insets highlight exemplary nuclear bodies of EZH2 or SUZ12 accumulation (arrows) in the BoM-1833 cells. Scale bar: 10 µm. Images were acquired and are displayed with identical settings. ( C ) Violin plot quantifying PRC2 body diameter in BoM-1833 cells. Each dot represents a single PRC2 body; data from 3 biological replicates (N = 16–32 cells). ( D ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in A-B. Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.0102. Error bars indicate mean ±SEM. ( E ) Representative confocal fluorescence microscopy image of BoM-833 cells stained for endogenous PRC2 (SUZ12, green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PRC2 body. Scale bar: 5 µm. ( F ) Schematic representation of the 3D photo-biotinylation approach used to map the proteome of endogenous PRC2 bodies. Total EZH2 (green) is spatially distributed within the cell and selectively photo-biotinylated at defined regions of interest (magenta) upon light activation. Following cell lysis, biotinylated proteins are captured using avidin-based immunoprecipitation and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The figure was created using Biorender. ( G ) Volcano plot illustrating the proteomic content of PRC2 bodies in BoM-1833 cells. Analysis was performed on the 1384 proteins identified as enriched in the labeled versus control condition in all 4 biological repeats, with unique peptides ≥ 2, fold change ≥ 1.5; and t-test significance ≤ 0.05. The x-axis represents the log 2 enrichment ratio (2P/CTL), and the y-axis represents the -log 10 p-value, indicating statistical significance. The dotted horizontal line corresponds to the p-value threshold (p < 0.05). Members of the core PRC2 complex are labeled in green. ( H ) Representative confocal fluorescence microscopy images of endogenous PHF19 immunostaining in MDA-MB-231 and BoM-1833 cells. The arrow highlights exemplary accumulations of PHF19 within nuclear bodies in BoM-1833 cells. Scale bar: 20 µm. The images were acquired and are displayed with identical settings. ( I ) Violin plot showing the quantification of endogenous PHF19 body diameter in BoM-1833 cells based on the images representatively shown in (H). Data represent measurements from N = 14–17 cells across n = 3 biological replicates, with each dot representing the diameter of a single PHF19 body. Biological repeats are color coded. ( J ) Quantification of percentage of cell nuclei with PHF19 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in (I). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.003. Error bars indicate mean ±SEM. ( K ) Representative confocal fluorescence microscopy image of endogenous PHF19 (green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PHF19 body. Scale bar: 5 µm. ( L ) Representative confocal fluorescence microscopy images of BoM-1833 cells, 24 h post transfection with a GFP-PHF19 (green) expression plasmid and immunostained for endogenous core PRC2 subunits (SUZ12, purple). The arrow indicates an exemplary area of co-localization. Scale bar: 10 µm.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Immunostaining, Staining, Activation Assay, Lysis, Avidin-Biotin Assay, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Labeling, Control, Transfection, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of BoM-1833 cells transfected with the indicated siRNAs. Cells were fixed 96 hours post-transfection and immunostained for endogenous EZH2 (A) or SUZ12 (B). Regions of interest (ROIs) are highlighted, with inset images showing magnified views of the immunostained cells. Scale bar: 10 µm. Images that are to be directly compared where imaged and are displayed with identical settings. ( C ) Quantification of the percentage of nuclei exhibiting PRC2 bodies in BoM-1833 cells treated as in (A-B) and immunostained for PRC2 core subunits. Data represent measurements from N = 50–60 cells across n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, *** = 0.0003, ns= not significant. Error bars indicate mean ±SD. ( D ) BoM-1833 cells were transfected with the indicated siRNAs and lysed 96 hours later for Western blot analysis using the specified antibodies. GAPDH was used as loading control. ( E-I ) Densitometric analysis of PHF19 (E), EZH2 (F), SUZ12 (G), PHF1 (H) and MTF2 (I) protein levels in cell lysates obtained from BoM-1833 cells treated as described in (D). GAPDH was used for relative normalization of the chemiluminescence signal obtained for the different PRC2 subunits. Data represent measurements from n = 3 biological replicates, whereby the values for siPHF19 are reported relative to the mean value of the control (siNT) within each biological replicate. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, **** < 0.0001, ns = not significant. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A ) PHF19 gene expression analysis across a TCGA BRCA cohort sorted by molecular subtype subtype. Box plots display the expression levels of PHF19 in normal (grey) and tumor (green) tissue for the indicated breast cancer subtypes. Data are derived from TCGA/GTEx datasets and visualized using GEPIA2. Statistical significance between tumor and normal samples was determined by unpaired t-test (*p < 0.05). n= 291 (Normal), 194 (Luminal B), 415 (Luminal A), 66 (HER2), 135 (Basal-like). ( B-C ) Representative confocal microscopy images of EZH2 (B) and SUZ12 (C) immunostaining in the indicated cell lines. Scale bar: 20 µm. Images that are to be directly compared were recorded and are displayed using identical settings. ( D ) Quantification of the percentage of cell nuclei with PRC2 bodies in the indicated cell lines based on confocal microscopy images as shown in (B-C). Data represent measurements from N = 35– 55 cells across n = 3 biological replicates. Biological repeats are color coded. ( E ) Representative immunoblot analysis of full cell lysates prepared from the indicated cell lines and using the annotated antibodies. GAPDH was used as the loading control. ( F-G ) Densitometric quantification of EZH2, SUZ12 (F) and PCL family (G) subunit protein expression in the TNBC cell line panel used in this work. GAPDH was used for normalization of the chemiluminescence signal of the PRC2 subunits across cell lines. The data for siPHF19 are reported relative to the mean values for the siNT control. Data represent measurements from n = 3 biological replicates, error bars are mean ±SD. Measurements stemming from cell lines forming detectable PRC2 bodies by Airyscan microscopy were highlighted in red. ( H-I ) Representative confocal fluorescence microscopy images showing co-immunostaining of H3K27me3 with the endogenous PRC2 core subunit SUZ12 (H) and PHF19 (I) in MDA-MB-436 cells. Arrows indicate exemplary regions of colocalization. Scale bar: 10 µm (H), 5 µm (I). ( J ) Violin plot showing the quantification of PRC2 core and PHF19 protein body diameter as based on the images representatively shown in (F-G). Data represent measurements from N = 14–29 (core PRC2 subunits) and N= 19-22 (PHF19) cells across n = 3 biological replicates, with each dot representing the diameter of a single protein body. Biological repeats are color coded. ( K ) Representative confocal fluorescence microscopy images of MDA-MB-436 cells, 24 h post transfection with GFP-PHF19 (green) and immunostained for endogenous SUZ12 (purple). The arrow indicates an exemplary area of co-localization. Scale bar: 5 µm. ( L-M ) MDA-MB-436 cells were transfected with the indicated siRNAs followed by fixation 96 h later and immunostaining for endogenous EZH2 (L) or SUZ12 (M). The bottom row shows magnified views of the cropped fields of view. Images that are to be directly compared were acquired and are displayed using identical settings. Scale bar: 10 µm ( N ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-436 cells transfected with the indicated siRNAs and imaged as representatively shown in (L-M). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA, ****= 0.001, ns= not significant. Error bars indicate mean ±SD. ( O ) MDA-MB-436 were treated as described in (L-M), followed by cell lysis. The material was analyzed by Western blot using the indicated antibodies. See also Figure S4. ( P , S ) Representative confocal microscopy images and ( R , T ) quantification of HS578T (P, R) and BT549 (S, T) fixed 24 h after transfection with a plasmid encoding for GFP-PHF19 (magenta) and immunostained for endogenous SUZ12 (PRC2 core). ROIs (Regions of Interest) are highlighted and magnified, showing the endogenous localization of SUZ12 in cells transfected with GFP-PHF19 (ROI 1) versus un-transfected cells (ROI 2). Scale bar: 20 µm. The bar diagrams show the endogenous SUZ12 localization phenotype in relation to the GFP-PHF19 expression status. Data represent measurements from N = 7–30 cells from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, * = 0.0123, **= 0.0038. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Gene Expression, Expressing, Derivative Assay, Confocal Microscopy, Immunostaining, Western Blot, Control, Microscopy, Fluorescence, Transfection, Lysis, Plasmid Preparation

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of generating D50 MCT8-COs and obtaining single-cell suspension for sc-RNA seq analysis. B . UMAP plot showing the cell clusters identified by principal component analysis in the indicated COs C . UMAP plots showing the distribution of markers for NPCs and neurons. D . Dot plot showing the relative expression levels of the gene markers used to identify each cell population. E . UMAP plot showing the cell types identified by manual curation in control COs. F . Dot plot showing the relative expression levels of SLC16A2, THRB, and THRA in each cell population of the dorsal projection trajectory. G . Interpretation of sequential gene expression changes during dorsal projection trajectory progression. H . UMAP plot showing the cell types identified by manual curation in MCT8-COs. I . Histogram of the relative number of cells in control and MCT8-COs. J . Volcano plots showing the distribution of differentially expressed genes in MCT8-deficient vs. control COs. iPSCs, induced pluripotent stem cells; COs, cortical organoids; NPCs, neural precursor cells; IPCs, intermediate precursor cells; DPT, dorsal projection trajectory; UMAP, Uniform Manifold Approximation and Projection. The X 2 test for multiple comparisons and pairwise cell proportions. **P < 0.01; ***P < 0.0001. Differentially expressed genes thresholds: p-value < 0.05, and Average Log 2 Fold-Change of 0.26.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Suspension, RNA Sequencing, Expressing, Control, Gene Expression

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of obtaining spatial transcriptomic data. B . Image showing the cell segmentation on the indicated COs, based on transcript localization data. Scale bar: 100 µm. C . Heatmap showing the relative expression levels of the genes used to identify the indicated neural cell types. D . Heatmap of the differentially expressed genes in the indicated neural cells between control vs. MCT8-COs. E . Same as in B , except the cells are classified into the indicated types. F . Distribution of distances between the indicated cells and groups. G . Histogram of the distribution of cell densities. Considering a radius of 100 µm, if only one cell was in contact with another cell, it was considered as sparse density. If one cell was in contact with five or more cells, it was considered a dense density. H . Violin plots show the expression levels of the indicated genes, considering the indicated cellular densities. I. Ligand-receptor interaction between the indicated neural cells in Control and MCT8-deficient COs. Differentially expressed genes threshold: p-value < 0.05.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of generating NPCs from iPSCs and the subsequent differentiation of the NPCs into neurons. B . Brightfield and confocal fluorescence images showing iPSCs-derived NPCs stained for SOX2 (magenta), NESTIN (yellow), MCT8 (magenta), and Dapi (blue; nuclear). C . Relative mRNA levels of the indicated genes in Control and MCT8 NPCs after 1, 6, and 12 days of neurodifferentiation. D . Brightfield images showing NPCs after six days of neurodifferentiation. E . Principal component plot illustrating differences between control and MCT8-deficient NPCs. F . Volcano plots showing the distribution of differentially expressed genes in control vs. MCT8-NPCs; each point represents the average of 5 control and 5 MCT8-deficient samples of pooled NPCs for each transcript. G . Heatmap depicting the top 20 differentially expressed genes related to neurogenesis between control vs. MCT8-NPCs identified by bulk-RNA seq. H . Venn comparison of differentially expressed genes belonging to the gene set related to neurogenesis between control vs. MCT8-NPCs identified by bulk-RNA seq. (blue) and sc-RNA seq analysis (green) and between control vs. MCT8-IPCs identified by sc-RNA seq analysis (purple); common differentially expressed genes were identified (grey box). I . Brightfield images of control and MCT8-NPCs after twelve days of neurodifferentiation; TUJ1 staining in green and Dapi (blue; nuclear). J . The upper two panels are MCT8 staining in red, TUJ1 in green, and Dapi (blue); the lower panels are RBFOX3 staining in green, NEUROD1 in red, and Dapi in blue. K . MCT8-NPCs after being treated with 60nM T3 during the twelve days of neurodifferentiation. TUJ1 staining is green, and SOX2 is red. L . SOX2 staining in red and Dapi in blue on the indicated cells and treatments. Scale bars: B: 50 µm; D, I, J: 100 µm. Differentially expressed genes thresholds: p-value < 0.05, and Average Log 2 Fold-Change of 1.5 in the Partek Flow platform. Expression values are mean ± SD of n = 3–6 RNA samples, each of them consisting of 2 pooled 6-well plates of NPCs from either control or MCT8-NPCs; Two-tailed Student’s test for comparing D2 deiodination and relative mRNA expression between D1, D6 and D12 of neurodifferentiation; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Fluorescence, Derivative Assay, Staining, Control, RNA Sequencing, Comparison, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of treating iPSCs-derived NPCs with radioactive T4 I125 to measure T3 I125 production. B . Representative chromatograms of the medium after control and MCT8-NPCs were incubated with T4 I125 for 24 hours. C . Quantitation of the DIO2 deiodination in control and MCT8 NPCs; n = 5 DIO2 assays. D . Volcano plots showing the distribution of differentially expressed genes in control + 1nM T4 vs. control NPCs; each point represents the average of five control + 1nM T4 and five control samples of pooled NPCs for each transcript. E . Interpretation of the findings in A-D . F . Schematic of the generation and timing of COs generation, starting with iPSCs to a culture of embryoid bodies, followed by neural induction, neuroepithelial bud expansion, and maturation. G . Quantitation of DIO2 deiodination in control COs during their first 20 days in culture. n = 4 DIO2 assays per timepoint, each consisting of 4 pooled COs from control COs. H . Relative SOX2 mRNA levels in control COs during their first 20 days in culture. Expression values are mean ± SD of n = 3–6 RNA samples, each of them consisting of 4 pooled COs from control COs; I . Schematic representation of the experiment: COs are treated with 1nM T4 from D7 to D50 and then dissociated into a single-cell suspension for sc-RNA seq. J . UMAP plot showing the cell types identified. K . Histogram of the relative number of cells in T4-COs and control COs. L . Histograms of the relative number of cells in clusters of NPCs. The identification number of each cell cluster is indicated at the bottom right corner of each rectangle. M . Volcano plots showing the distribution of differentially expressed genes in T4-CO vs. control COs. H . Gene set enrichment analysis reveals gene ontology terms enriched in T4 TX COs. O . Histograms of the relative number of cells undergoing the indicated cell cycle phase in clusters one and nine of control and NPCs. Two-tailed Student’s test for comparing DIO2 deiodination in iPSC-derived NPCs; ***P < 0.001. Differentially expressed genes thresholds: p-value < 0.05, and Average Log 2 Fold-Change of 0.26.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Derivative Assay, Control, Incubation, Quantitation Assay, Expressing, Suspension, RNA Sequencing, Two Tailed Test

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of the protocol to isolate mNPCs, propagate them in culture, and differentiate into neurons. B, C. Brightfield images showing representative neurospheres containing mNPCs ( B ) and the mNPCs cultured in collagen-coated plasticware ( C ). D-F . Confocal images showing mNPCs expressing Nestin (green) and Sox2 (red) ( D ), and Mct8 (red) ( E ). The insets in E depict two dividing mNPCs that exhibited higher intensity of Mct8 immunofluorescence. F . Quantitation of the DIO2 deiodination in mNPCs; n = 6 DIO2 assays. G. Brightfield images showing representative mNPCs after two days of neurodifferentiation. H - J . Relative mRNA levels of the indicated genes in mNPCs under the indicated conditions; n = 4. K . Brightfield images showing representative mNPCs after four days of neurodifferentiation. Note the neuronal process extension. L . Tuj1+ cells under the indicated conditions and the quantitation of the percentage of Tuj1+ cells. Values are the mean ± SD of 5 replicates. Scale bars: B: 300 µm, C-L: 25 µm. Two-tailed Student’s test for comparing DIO2 deiodination in iPSC-derived NPCs; **P < 0.01; ***P < 0.001.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Cell Culture, Expressing, Immunofluorescence, Quantitation Assay, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS

doi: 10.1101/2023.12.13.571549

Figure Lengend Snippet: Effects of HDAC inhibitors, arimoclomol and combined treatments on HSPA1A in motor neurons expressing TDP-43 G348C . A Phase contrast image of spinal cord – DRG culture. Arrows point to motor neurons. B Small but significant increase in the percentage of motor neurons with HSPA1A immunoreactivity three days following microinjection of plasmid encoding TDP-43 G348C , compared to absence of labeling in neurons injected with mCherry (control). C-F Percentage of HSPA1A immunopositive motor neurons after three days of treatment with vehicle, the HDAC inhibitors C SAHA, D RGFP109, E RGFP963 or F Tubastatin A, or with arimoclomol alone or in combination with the respective HDAC inhibitor. HDAC inhibitors with class I activity ( C-E ) significantly increased the percentage of neurons expressing HSPA1A. Arimoclomol and the HDAC6 inhibitor Tubastatin A were ineffective. Data presented as mean ± S.D., n = 9 cultures. Statistical significance was evaluated by one-way ANOVA followed by Bonferroni post hoc analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar = 20μm.

Article Snippet: Primary antibodies were: rabbit anti-TDP-43 (EPR18554 1:500 Abcam, Cambridge, UK), rabbit anti-FUS (11570-1-AP 1:300, Proteintech, Rosemont, IL, USA); mouse antibody specific for human SOD1 (SD-G6 1:100, Millipore Sigma), Mouse anti-human HSP70 specific for stress-inducible HSPA1A (SMC-100B 1:100, Stressmarq Biosciences Inc., Victoria, BC, Canada), mouse anti-HSPA8/HSC71/Hsc70 (13D3) antibody (NB120-2788 1:300, Novus Biologicals, Colorado, USA), mouse anti-FLAG M2 (F1804 1:400, Millipore Sigma), rabbit anti-acetylated H3K9/K14 (9677 1:400, Cell Signaling, Danvers, MA, USA), rabbit anti-Brg1 (21634-1-AP 1:600, Proteintech), rabbit anti-MAP2 (AB5622 1:500, Millipore Sigma), chicken anti-GFP (GFP1010 1:500 AVES, CA, USA).

Techniques: Expressing, Microinjection, Plasmid Preparation, Labeling, Injection, Control, Activity Assay

Journal: bioRxiv

Article Title: Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS

doi: 10.1101/2023.12.13.571549

Figure Lengend Snippet: Hspa1a mRNA in ALS culture models using single molecule fluorescence in situ hybridization (smFISH). Cultures containing motor neurons expressing TDP-43 G348C , FUS R521G or SOD1 G93A were treated with vehicle (DMSO), 4 µM arimoclomol, 1µM RGFP963 or the combination of arimoclomol and RGFP963 (Combo). smFISH was conducted on day three. A The highly expressed Hspa8 mRNA is presented as a reference control for comparison to the minimal labeling of Hspa1a mRNA in B,C,D . B No significant impact of TDP-43 G348C or drug treatments on the number of Hspa1a mRNA spots. C Scarcity of Hspa1a mRNA spots in neurons expressing FUS R521G ; no significant effect of drug treatments. D Low numbers of Hspa1a mRNA spots in neurons expressing SOD1 G93A ; a small but significant increase in a subset of neurons by arimoclomol and RGFP963 treatments. Data are presented as mean ± S.D. n = 9-24 neurons per group. E-G Number of transcription sites by smFISH. E Example of H spa1a transcription site labeling. F,G Effect of ALS variants and combination drug treatment on the number of Hspa1a transcription sites on F day one and G day three following microinjection of expression vectors. n = 6-30 neurons per group. Statistical significance was evaluated through one-way ANOVA followed by Bonferroni post hoc analysis. *p<0.05. Scale bar = 15μm.

Article Snippet: Primary antibodies were: rabbit anti-TDP-43 (EPR18554 1:500 Abcam, Cambridge, UK), rabbit anti-FUS (11570-1-AP 1:300, Proteintech, Rosemont, IL, USA); mouse antibody specific for human SOD1 (SD-G6 1:100, Millipore Sigma), Mouse anti-human HSP70 specific for stress-inducible HSPA1A (SMC-100B 1:100, Stressmarq Biosciences Inc., Victoria, BC, Canada), mouse anti-HSPA8/HSC71/Hsc70 (13D3) antibody (NB120-2788 1:300, Novus Biologicals, Colorado, USA), mouse anti-FLAG M2 (F1804 1:400, Millipore Sigma), rabbit anti-acetylated H3K9/K14 (9677 1:400, Cell Signaling, Danvers, MA, USA), rabbit anti-Brg1 (21634-1-AP 1:600, Proteintech), rabbit anti-MAP2 (AB5622 1:500, Millipore Sigma), chicken anti-GFP (GFP1010 1:500 AVES, CA, USA).

Techniques: Fluorescence, In Situ Hybridization, Expressing, Control, Comparison, Labeling, Microinjection