Journal: Nature Communications

Article Title: The single-cell transcriptomic atlas and RORA-mediated 3D epigenomic remodeling in driving corneal epithelial differentiation

doi: 10.1038/s41467-023-44471-w

Figure Lengend Snippet: a Smoothed expression trends in pseudotime of TFs whose expression correlates best with terminal fate probabilities calculated by CellRank. b Immunofluorescence staining of the indicated genes in the induced CECs that differentiated for 7 days. Scale bar, 100 μm. n = 3 biologically independent experiments. c Heatmap showing the differentially expressed genes between cultured LSCs and induced CECs. TPM: transcripts per kilobase million. d Overlapping of CellRank TFs ( a ) and CEC-specific TFs ( c ). e UMAP plots of RORA and PITX1 density. f Immunofluorescence staining of RORA, PITX1 and KRT3 in PCW18, PCW21, PWC25 and adult limbal–corneal tissues. Scale bar, 100 μm. n = 3 biologically independent experiments. g Ranked SCENIC TFs in adult corneal suprabasal and superficial layers. h UMAP plots showing SCENIC regulon activities of RORA and PITX1. i UMAP plots showing the scores for moments of spliced RNAs ( Ms ) of RORA and PITX1 . j kNN-DREMI analysis (DREVI plots and DREMI values) indicates the relationship between the indicated genes after imputation with MAGIC (Markov affinity-based graph imputation of cells) .

Article Snippet: The antibodies used for immunofluorescence are as follows: anti-KRT3 (Abcam, #ab68260, 1:200), anti-KRT12 (Abcam, #ab124975, 1:200), anti-LGALS3 (Biolegend, #125401, 1:200), anti-S100A2 (Abcam, #ab109494, 1:200), anti-KRT15 (NeoMarkers, #MS-1068-P0, 1:200), anti-IFTIM3 (Proteintech, #11714-1-AP, 1:200), anti-PAX6 (Sigma, #AMAB91372, 1:200), anti-ALDH3A1 (GeneTex, #GTX30042, 1:200), anti-CLU (Proteintech, #12289-1-AP, 1:200), anti-KRT19 (Biolegend, #628502, 1:200), anti-RORA (Immunoway, #YT4166, 1:100), anti-PITX1 (Sigma, #HPA008743, 1:100), Anti-KRT14 (Thermo, #MA511599, 1:200), Anti-KI67 (Cell Signaling Technology, #9129 S, 1:200), Anti-TP63 (Cell Signaling Technology, 67825 S, 1:200), anti-rabbit IgG (Alexa Fluor 488 Conjugate, CST, 4412 S,1:1000), anti-mouse IgG (Alexa Fluor 488 Conjugate, CST, 4408 S,1:1000), anti-rabbit IgG (Alexa Fluor 594 Conjugate, CST, 8889 S,1:1000), and anti-mouse IgG (Alexa Fluor 594 Conjugate, CST, 8890 S,1:1000).

Techniques: Expressing, Immunofluorescence, Staining, Cell Culture

Journal: Nature Communications

Article Title: The single-cell transcriptomic atlas and RORA-mediated 3D epigenomic remodeling in driving corneal epithelial differentiation

doi: 10.1038/s41467-023-44471-w

Figure Lengend Snippet: a CellOracle RORA and PITX1 knockout simulation vectors with perturbation scores and normal development vector. b Dynamo in silico double perturbation and double activation simulation vectors of RORA and PITX1 . c Dynamo response heatmaps showing Jacobian regulation from RORA to the selected genes versus RORA Ms . White dashed lines indicate the zero value. Positive value represents activation from RORA to the indicated genes and negative represents repression from RORA to the indicated genes. d 5-Ethynyl-20-deoxyuridine (EdU) staining for LSCs treated with control, RORA - and PITX1 -overexpression. Bar plots showing statistics on the percentage of EdU-positive cells for each group. Scale bar, 100 μm. Data are represented as means ± SEM ( n = 6 independent experiments, *** P < 0.001). SEM: Standard Error of the Mean. P -values were calculated using two-sided unpaired Student’s t tests. Source data are provided as a file. e Heatmap of bulk RNA-seq data showing the selected differentially expressed genes upon RORA and PITX1 overexpression in LSCs. f , g GSEA (Gene Set Enrichment Analysis) for the indicated gene sets using bulk RNA-seq data of RORA - and PITX1 -overexpression versus control. h Single cell kNN-DREMI analysis (DREVI plots and DREMI values) indicates the relationship between RORA and PITX1 after imputation with MAGIC. i Linear regression test for gene fold changes induced by RORA and PITX1 overexpression. j Dynamo Jacobian from RORA to RORA and PITX1 in the UMAP space. Positive value (red) indicates positive regulation. k Real-time quantitative PCR (qRT-PCR) analysis for RORA upon PITX1 overexpression and PITX1 upon RORA overexpression. Data are represented as means ± SEM ( n = 3 independent experiments, *** P < 0.001). n.s., nonsignificant. P -values were calculated using two-sided unpaired Student’s t tests. Source data are provided as a file. l qRT-PCR analysis for KRT3 , KRT12 , KRT24 and CLU in CECs treated with the indicated groups for one week upon differentiation from LSCs. Data are represented as means ± SEM ( n = 3 independent experiments, * P < 0.05, ** P < 0.01, *** P < 0.001). P -values were calculated using two-sided unpaired Student’s t tests. Source data are provided as a file.

Article Snippet: The antibodies used for immunofluorescence are as follows: anti-KRT3 (Abcam, #ab68260, 1:200), anti-KRT12 (Abcam, #ab124975, 1:200), anti-LGALS3 (Biolegend, #125401, 1:200), anti-S100A2 (Abcam, #ab109494, 1:200), anti-KRT15 (NeoMarkers, #MS-1068-P0, 1:200), anti-IFTIM3 (Proteintech, #11714-1-AP, 1:200), anti-PAX6 (Sigma, #AMAB91372, 1:200), anti-ALDH3A1 (GeneTex, #GTX30042, 1:200), anti-CLU (Proteintech, #12289-1-AP, 1:200), anti-KRT19 (Biolegend, #628502, 1:200), anti-RORA (Immunoway, #YT4166, 1:100), anti-PITX1 (Sigma, #HPA008743, 1:100), Anti-KRT14 (Thermo, #MA511599, 1:200), Anti-KI67 (Cell Signaling Technology, #9129 S, 1:200), Anti-TP63 (Cell Signaling Technology, 67825 S, 1:200), anti-rabbit IgG (Alexa Fluor 488 Conjugate, CST, 4412 S,1:1000), anti-mouse IgG (Alexa Fluor 488 Conjugate, CST, 4408 S,1:1000), anti-rabbit IgG (Alexa Fluor 594 Conjugate, CST, 8889 S,1:1000), and anti-mouse IgG (Alexa Fluor 594 Conjugate, CST, 8890 S,1:1000).

Techniques: Knock-Out, Plasmid Preparation, In Silico, Activation Assay, Staining, Over Expression, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Nature Communications

Article Title: The single-cell transcriptomic atlas and RORA-mediated 3D epigenomic remodeling in driving corneal epithelial differentiation

doi: 10.1038/s41467-023-44471-w

Figure Lengend Snippet: a Selected tracks for promoter-anchored chromatin interactions in control and RORA -overexpressed LSCs. b Bar plots showing the percentages of P-P and P-NPR loops respectively in RORA -overexpression- and control-specific interactions. c RORA ChIP-seq signal at promoter and NPR anchors of RORA -overexpression-specific interactions. d GO terms enriched in RORA -overexpression-specific promoter anchors. GO enrichment analysis were performed and P -values were calculated in Metascape . e Scatterplots showing the differential H3K27ac, H3K4me3 and H3K27me3 peaks upon RORA overexpression. f Heatmaps showing the indicated ChIP-seq signals across the peaks with H3K27ac reduction and increase upon RORA overexpression. g Heatmaps showing the indicated ChIP-seq signals across the peaks with H3K27ac and H3K27me3 shifts upon RORA overexpression. h , i Genome browser tracks for promoter-anchored chromatin interactions and the indicated ChIP-seq and RNA-seq signals across the RORA , PITX1 , KRT12 , KLF4 , LGALS3 and EMP3 loci in control and RORA -overexpressed LSCs.

Article Snippet: The antibodies used for immunofluorescence are as follows: anti-KRT3 (Abcam, #ab68260, 1:200), anti-KRT12 (Abcam, #ab124975, 1:200), anti-LGALS3 (Biolegend, #125401, 1:200), anti-S100A2 (Abcam, #ab109494, 1:200), anti-KRT15 (NeoMarkers, #MS-1068-P0, 1:200), anti-IFTIM3 (Proteintech, #11714-1-AP, 1:200), anti-PAX6 (Sigma, #AMAB91372, 1:200), anti-ALDH3A1 (GeneTex, #GTX30042, 1:200), anti-CLU (Proteintech, #12289-1-AP, 1:200), anti-KRT19 (Biolegend, #628502, 1:200), anti-RORA (Immunoway, #YT4166, 1:100), anti-PITX1 (Sigma, #HPA008743, 1:100), Anti-KRT14 (Thermo, #MA511599, 1:200), Anti-KI67 (Cell Signaling Technology, #9129 S, 1:200), Anti-TP63 (Cell Signaling Technology, 67825 S, 1:200), anti-rabbit IgG (Alexa Fluor 488 Conjugate, CST, 4412 S,1:1000), anti-mouse IgG (Alexa Fluor 488 Conjugate, CST, 4408 S,1:1000), anti-rabbit IgG (Alexa Fluor 594 Conjugate, CST, 8889 S,1:1000), and anti-mouse IgG (Alexa Fluor 594 Conjugate, CST, 8890 S,1:1000).

Techniques: Over Expression, ChIP-sequencing, RNA Sequencing Assay

Journal: Brain

Article Title: Autophagy induction reduces mutant ataxin-3 levels and toxicity in a mouse model of spinocerebellar ataxia type 3

doi: 10.1093/brain/awp292

Figure Lengend Snippet: Temsirolimus inhibits the mTOR pathway in vivo , as rapamycin does in cultured primary neurons. ( A ) Sections from mouse brains treated with either temsirolimus (CCI-779) or placebo were immunostained for phosphorylated S6 protein (Phospho-S6, top panels) or total S6 protein (bottom panels), nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Total S6 immunoreactivity was seen in the perinuclear region of all cells. In placebo treated animals some cells stained positive for phosphorylated S6, however, this staining was not seen in sections from animals treated with temsirolimus. Scale bar represents 5 µm and is valid for all panels. In cultured primary neurons, LC3-II levels were assessed by western blot ( B ). Two different exposures are shown to allow comparison of weaker bands in untreated lanes (−Baf A1) and stronger bands in Bafilomycin A1 (+Baf A1) lanes without saturation. Densitometric quantification of LC3-II levels relative to actin in triplicate experiments is shown in ( C ). ** P < 0.01 and * P < 0.05 by t -test. Effect of rapamycin treatment of phosphorylation of downstream mTOR targets was investigated by western blotting, ( D ) phosphorylated p70 S6 kinase levels and phosphorylated S6 ribosomal protein and ( E ) phosphorylated eukaryotic initiation factor 4E-binding-protein (EBP)-1.

Article Snippet: Primary antibodies used were mouse anti-ataxin-3 (Millipore, Watford, Hertfordshire), rabbit anti-light-chain-3 (LC3: Novus Biologicals), rabbit anti-phospho and total p70S6Kinase, rabbit anti-phospho and total S6 ribosomal protein, rabbit anti-phospho and total eukaryotic initiation factor 4E-binding-protein 1 (Cell Signaling Technology), goat anti-ubiquitin specific peptidase (Usp)-15, rabbit anti-Grik2, mouse anti-splicing factor proline/glutamine-rich and rabbit anti-RORa (all Abcam, Cambridge) (see for expansions).

Techniques: In Vivo, Cell Culture, Staining, Western Blot, Binding Assay