Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: (a) Top 1000 genes ranked by enrichment or depletion. (b-c) Categorization of top 200 genes enriched (b) or depleted (c) in cardiac myocytes compared to undifferentiated hiPSCs by biological groups. (d-e) Gene ontology (GO) analysis of top enriched (d) or depleted (e) hits (see Methods for details). (f-g) Venn diagrams demonstrating the number of CHD (f) or Non-CHD (g) probands with predicted damaging DNVs in hits identified in our screen as enriching hiPSC:CM differentiation or depleting hiPSC:CM differentiation. Arrows depict Venn diagrams representing the number of probands from each cohort with predicted damaging DNVs identified in our screen that have mutations in known dominant CHD genes or where these candidate CHD genes may potentially be causative. (h) Venn diagram demonstrating the number of CHD probands with (purple) or without (brown) damaging DNVs in hits identified in our screen highlighting no enrichment for extracardiac anomalies (p=0.43) or neurodevelopmental delay (NDD; p=0.23) and a slight enrichment for conotruncal CHD (p=0.04). (i) TNNT2+ cells quantified by flow cytometry at day 10 of hiPSC to CM differentiation in WTC11 cells treated with 100nM JQ1 starting at day 6 (n=3 biologically independent samples). (j) MZ3 treatment (500 nM for 0, 3.5, 7, and 16 hours) effectively degrades BRD4 in SV20 hiPSCs as assessed by immunoblot analysis for BRD4 with β-actin expression as a loading control. In all graphs, error bars represent ±1 SEM. * represents p=0.0271 (two-tailed unpaired t test).

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Flow Cytometry, Western Blot, Expressing, Control, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: a-c, UMAP plots of single-cell gene expression of Brd4flox/flox (n = 2) and Mef2c-AHF-Cre; Brd4flox/flox (n = 2) embryos visualized by genotype (a), sample identity (b), or cluster identity (c). d, Gene expression analysis of single-cell data. Heat map shows genes most enriched in each cluster and their inferred identity. e, Violin plots showing gene expression of critical regulators in select clusters. f, Percentage of cells in each cluster in each genotype highlighting the expansion of cluster 0 (MSX1/2+ progenitors) upon BRD4 deletion. g,h, Multiple lineage and pseudotime trajectory inference of cells from control (Brd4flox/flox) (g) and mutant (Mef2c-AHF-Cre; Brd4flox/flox) (h) embryos. i, Proposed model for BRD4 action in SHF CPCs: BRD4 is critical for CM differentiation in a subset of SHF CPCs, with its loss leading to persistence of Wnt-activated, ISL1+, and MSX1/2+ CPCs. A, atrium; pSHF, posterior second heart field.

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Expressing, Control, Mutagenesis

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: (a) Image of Isl1Cre/+; Brd4flox/+ embryo appearing in Figure 2i with region microdissected for bulk RNA-seq highlighted in red. (b) Principal component analysis of RNA-seq from Isl1Cre/+; Brd4flox/+ (blue) and Isl1Cre/+; Brd4flox/flox (red) E9.5 embryos. (c) Volcano plots of E9.5 Isl1Cre/+; Brd4flox/+ vs. Isl1Cre/+; Brd4flox/flox embryonic hearts (same data appearing in Figure 4) with a subset of cardiac and Wnt-related genes annotated (see Methods for details). Isl1Cre/+; Brd4flox/+ (d,f) and Isl1Cre/+; Brd4flox/flox (e,g) E9.5 embryos at level of right ventricle stained with ISL1 (red, d-e), BRD4 (green, d-e), or AXIN2 (green, f-g). Note the expansion of ISL1- (arrow heads) and AXIN2- (dotted line) expressing cells into the RV from distal outflow tract in mutant embryos. (h-m) ISL1 and AXIN2 Immunohistochemistry of E10.5 Mef2c-AHF-Cre; Brd4flox/+ (h,k) and Mef2c-AHF-Cre; Brd4flox/flox (i,j,l,m) embryos at the level of outflow tract. (h-j, ISL1; k-m, AXIN2). Note the expansion of ISL1- (arrow heads) and AXIN2- (dotted line) expressing cells into the right ventricle from distal outflow tract in mutant embryos. (n-o) AXIN2 RNAscope of Brd4flox/flox (n,n′) and Isl1Cre/+; Brd4flox/flox (o,o′) E9.5 embryos at the level of the right ventricle (n′ and o′ are magnified images of n and o, respectively). (p-q) ISL1 representative immunofluorescence at day 8 of mESC-derived cardiac cultures treated with vehicle (DMSO; VEH) (p) or JQ1 (500 nM) (q) starting at day 5. (r) Isl1 expression in day 8–9 mESC-derived cardiac cultures treated with increasing doses of JQ1 (0–500 nM; JQ1 added at day 5; n=3 n=3 biologically independent samples per dose). (s-t) AXIN2 RNAscope of Mef2c-AHF-Cre; Brd4flox/+ (s,s′) and Mef2c-AHF-Cre; Brd4flox/flox (t,t′) E10.5 embryos at level of right ventricle (s′ and t′ are magnified images of s and t, respectively). For r, all comparisons are made relative to 0 nM compound. *** represents p=0.0007 (two-tailed unpaired t test). RV, right ventricle; OT, outflow tract. Scale Bar = 250 μm (a), 50 μm (d-m, n, o, s, t), 100 μm (p, q, n′, o′, s′, t′)

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: RNA Sequencing Assay, Staining, Expressing, Mutagenesis, Immunohistochemistry, RNAscope, Immunofluorescence, Derivative Assay, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: Inhibi (a) Expression of Myh6, Nkx2–5, and Tnnt2 at day 7 of mESCs cardiac differentiation treated with JQ1 (100 nM) starting day 5 of differentiation (n=3 biologically independent samples). (b) TNNT2+ cells quantified by flow cytometry at d9 of mESC differentiation in CMV-CreERT2; Brd4flox/flox cells treated with 4-hydroxytamoxifen (TAM), JQ1 (250 nM) or MZ3 (500 nM) starting at day 5 (n=3 biologically independent samples). (c-d) Immunofluorescence of TNNT2 at day 9 of mESC to CM differentiation in wild type cells treated with JQ1 (100 nM) or vehicle starting at day 5. (e-f) Immunofluorescence of BRD4 in vehicle (ethanol, e-e′′) and TAM (f-f′′) treated undifferentiated mESCs. (g) Volcano plot showing RNA-seq from CMV-CreERT2; Brd4flox/flox (TAM vs. vehicle [VEH]) mESCs and gene ontology analysis of downregulated and upregulated genes (see Methods for details). (h) Heatmap showing expression of select transcription factor and muscle structural protein genes from RNA-seq in CMV-CreERT2; Brd4flox/flox (TAM vs. VEH) mESC-derived cardiac tissues (day 10; TAM or VEH added at day 5). In all graphs, error bars represent ±1 SEM. For a, all comparisons are made relative to 0 nM compound for each gene; * represents p<0.0493, ** represents p<0.0085 (two-tailed unpaired t test). For b, all comparisons are made relative to VEH for each condition; * represents p<0.0188 (two-tailed unpaired t test). Scale Bars = 100 μm (c, d, e, e′, e′′, f, f′, f′′)

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, RNA Sequencing Assay, Derivative Assay, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: a-h, H&E staining (a,e) and immunohistochemistry (b-d, f-h) of Isl1Cre/+; Brd4flox/+ (b-d) and Isl1Cre/+; Brd4flox/flox (f-h) E14.5 hearts. Immunohistochemistry of Tnnt2 (red) and BRD4 (green). i,j, Isl1Cre/+; Brd4flox/+ (i,i′) and Isl1Cre/+; Brd4flox/flox (j,j′) E9.5 embryos. Regions in yellow boxes in i and j are shown in higher magnification in i′ and j′. k,l, H&E staining of a section through OFT and RV of Isl1Cre/+; Brd4flox/+ (k,k′) and Isl1Cre/+; Brd4flox/flox (l,l′) E9.5 embryos. Regions in yellow boxes in k and l are shown in higher magnification in k′ and l′. Scale bars, 200 μm (a,b,e,f), 100 μm (c,d,g,h,k,l), 250 μm (i,i′,j,j′) and 25 μm (k′,l′),

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Staining, Immunohistochemistry

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: (a) Lineage tracing of Isl1Cre/+; Brd4flox/+ and Isl1Cre/+; Brd4flox/flox with R26mTmG/+ allele. Immunohistochemistry of ISL1-derived cells (GFP) and TNNT2 or BRD4 (red) in heart and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes at E14 (5 sections from n=2 control embryos and 11 sections from n=4 mutant embryos). (b) Hematoxylin and eosin staining of a section through outflow tract and RV of Isl1Cre/+; Brd4flox/+ and Isl1Cre/+; Brd4flox/flox embryos at E12.5 and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes (10 sections from n=2 control embryos and 11 sections from n=2 mutant embryos). (c) Quantification of percentage phospho-histone H3-, cleaved caspase 3-, and TUNEL-positive cells in the RV of E12.5 and E14.5 embryos of indicated genotypes (n=2 biologically independent samples per genotype at E12.5; n=3–4 biologically independent samples per genotype at E14.5). (d) Hematoxylin and eosin staining of a section through outflow tract and RV of Isl1Cre/+; Brd4flox/+ and Isl1Cre/+; Brd4flox/flox embryos at E10 and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes (9 sections from n=3 control embryos and 8 sections from n=3 mutant embryos). (e) Quantification of percentage phospho-histone H3- and TUNEL-positive cells in the RV of E10 control (Isl1Cre/+; Brd4flox/+ or Brd4flox/flox) and Isl1Cre/+; Brd4flox/flox embryos (n=4 biologically independent samples per condition). (f) BRD4 and TNNT2 immunohistochemistry along with lineage tracing with R26mTmG/+ allele in Isl1Cre/+; Brd4flox/+ and Isl1Cre/+; Brd4flox/flox E9.5 embryos at level of right ventricle. Error bars represent ±1 SEM. All comparisons are made as indicated; * represents p=0.0091, ** represents p=0.0021, **** represents p<0.0001 (two-tailed unpaired t test). RV, right ventricle; LV, left ventricle; OT, outflow tract. Scale Bars = 100 μm (a, b, d, f)

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: In Vivo, Immunohistochemistry, Derivative Assay, Control, Mutagenesis, Staining, TUNEL Assay, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: a,b, H&E staining of Mef2c-AHF-Cre; Brd4flox/+ (a) and Mef2c-AHF-Cre; Brd4flox/flox (b) E12.5 hearts. Note the RV hypoplasia in the mutant compared to control. c,d, Immunohistochemistry of BRD4 in Mef2c-AHF-Cre; Brd4flox/+ (c) and Mef2c-AHF-Cre; Brd4flox/flox (d) E10.5 OFTs. e,f, Mef2c-AHF-Cre; Brd4flox/+ (e) and Mef2c-AHF-Cre; Brd4flox/flox (f) E10.5 embryos. Scale bars, 100 μm (a-d) and 250 μm (e,f)

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Staining, Mutagenesis, Control, Immunohistochemistry

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: (a) Hematoxylin and eosin staining of a section through outflow tract and RV of Mef2c-AHF-Cre; Brd4flox/+ and Mef2c-AHF-Cre; Brd4flox/flox embryos at E13.5 and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes (19 sections from n=3 control embryos and 18 sections from n=3 mutant embryos). (b) Quantification of percentage phospho-histone H3- and cleaved caspase 3-positive cells in the RV of E13.5 Mef2c-AHF-Cre; Brd4flox/+ and Mef2c-AHF-Cre; Brd4flox/flox embryos (n=3–4 biologically independent samples per genotype). (c) Hematoxylin and eosin staining of a section through outflow tract and RV of Mef2c-AHF-Cre; Brd4flox/+ and Mef2c-AHF-Cre; Brd4flox/flox embryos at E10.5 and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes (6 sections from n=3 control embryos and 6 sections from n=3 mutant embryos). (d) Quantification of percentage phospho-histone H3- and cleaved caspase 3-positive cells in the RV of E10.5 Mef2c-AHF-Cre; Brd4flox/+ and Mef2c-AHF-Cre; Brd4flox/flox embryos (n=3 biologically independent samples per genotype). Error bars represent ±1 SEM. All comparisons are made as indicated; * represents p=0.0489, ** represents p=0.0146, **** represents p<0.0001 (two-tailed unpaired t test). RV, right ventricle; LV, left ventricle. Scale Bars = 100 μm (a, c).

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: In Vivo, Staining, Control, Mutagenesis, Two Tailed Test

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: (a) Attenuating Wnt signaling at the CPC stage (day 5) in mESC to CM differentiation by doubling the normal concentration of the small molecule Wnt inhibitor XAV939 concomitant with Brd4 genetic deletion by 4-hydroxytamoxifen treatment (TAM) in CMV-CreERT2; Brd4flox/flox mESCs partially normalizes expression of CPC markers and Msx1/2 by qRT-PCR (n=3–4 biologically independent samples per genotype). (b-e) Attenuating Wnt signaling at the CPC stage (day 5) in mESC to CM differentiation by doubling the normal concentration of the small molecule Wnt inhibitor XAV939 concomitant with Brd4 genetic deletion by 4-hydroxytamoxifen treatment (TAM) in CMV-CreERT2; Brd4flox/flox mESCs partially normalizes TNNT2 staining by immunofluorescence at day 9 of CM differentiation (for e, n=3 biologically independent samples per condition). (f,g) Attenuation of Wnt signaling at the CPC stage (day 6) in hiPSC to CM differentiation by addition of the small molecule Wnt inhibitor IWP4 (5 μM for low dose and 10 μM high dose) concomitant with BET inhibition using JQ1 (25 nM for low dose and 50 nM for high dose) increases the number of TNNT2+ cells as assessed by flow cytometry (n=3 biologically independent samples per condition). Error bars represent ±1 SEM. For a,e,f,g all comparisons are made with p values as indicated (two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Concentration Assay, Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, Inhibition, Flow Cytometry

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: a, Heat maps showing enrichment of FLAG, BRD4 and H3K4Me3 CUT&RUN signals and H3K27Ac ChIP-seq34 enrichment from hiPSC-derived CPCs at day 6 ordered by FLAG intensity. b, Venn diagrams and table demonstrating co-occupancy of BRD4/FLAG and indicated histone modification (percentage of BRD4 peaks overlapping with indicated histone modification). c,d, Track view of AXIN2 (c) and CCND1 (d) loci showing indicated CUT&RUN factor occupancy or H3K27Ac ChIP-seq enrichment in hiPSC-derived CPCs. e, Metaplot demonstrating FLAG occupancy at genes associated with GO terms for Wnt signaling or contractility. ChIP-seq, chromatin immunoprecipitation followed by sequencing.

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Derivative Assay, Modification, ChIP-sequencing, Chromatin Immunoprecipitation, Sequencing

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: (a) Targeting strategy to introduce 3XFLAG epitope tag into the N-terminus of the endogenous BRD4 locus. (b) Karyotyping results of BRD4FLAG/FLAG hiPSC line. (c) Western blot analysis of protein lysates collected from BRD4FLAG/FLAG hiPSCs using FLAG antibody demonstrates expression of 3XFLAG-tagged BRD4 isoforms that are degraded upon addition of the PROTAC BET degrader dBET651. (d) Pearson correlation matrices demonstrating high reproducibility between replicate CUT&RUN datasets. (e-g) Track view of indicated loci showing CUT&RUN factor occupancy (FLAG, BRD4, H3K4Me3) or H3K27Ac ChIP-seq enrichment in hiPSC-derived CPCs.

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: Introduce, Western Blot, Expressing, ChIP-sequencing, Derivative Assay

Journal: Nature cardiovascular research

Article Title: A Genome-Wide CRISPR Screen Identifies BRD4 as a Regulator of Cardiomyocyte Differentiation

doi: 10.1038/s44161-024-00431-1

Figure Lengend Snippet: Mef2c-AHF-Cre; Brd4flox/+ (a) and Mef2c-AHF-Cre; Brd4flox/flox (b,c) E10.5 embryos at level of right ventricle stained with MSX1/2 (yellow); inset shows area indicated by arrowheads. RNAscope in Brd4flox/flox (d,e), Isl1Cre/+; Brd4flox/flox (f,g), Mef2c-AHF-Cre; Brd4flox/+ (h,i), and Mef2c-AHF-Cre; Brd4flox/flox (j,k) E9.5–10.5 embryos at level of right ventricle for MSX1 (d,f,h,j) or MSX2 (e,g,i,k). Regions in yellow boxes in d-k are shown in higher magnification in d′-k′. RV, right ventricle; OT, outflow tract. Scale Bars = 50 μm (a-c, d-k), 165 μm (d′, e′, f′, g′), 125 μm (h′, j′), 200 μm (i′, k′)

Article Snippet: Antibodies used were mouse anti-FLAG (Sigma-Aldrich, F1804), rabbit anti-BRD4 (EpiCypher, 13–2003), rabbit anti-H3K4Me3 (EpiCypher, 13–0041), rabbit anti-H3K27Me3 (EpiCypher, 13–0055) and rabbit IgG (EpiCypher, 13–0042).

Techniques: In Vivo, Staining, RNAscope

Journal: bioRxiv

Article Title: Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

doi: 10.1101/2022.09.26.509442

Figure Lengend Snippet: a , Numbers of cFos+ cells in the DMH during SD, RS and sleeping-control (SD-Cont, RS-Cont) detected by cFos immunohistochemistry. The total number of cFos+ cells in the DMH was counted at bregma -1.67 mm to, -1.79 mm and -1.91 mm and summed up (total three sections) each mouse (n=3). The third ventricle (3V) is shown. Values are shown as means ± S.E., *p<0.05, ***p<0.001, and non-significant (ns) by one-way ANOVA with Bonferroni’s post hoc test. b , c , Representative images of DMH sections at bregma -1.67 mm from mice under SD-Cont (left) and SD (right) with cFos. Boxed areas were shown at high magnification in c. d , Images of the ZsGreen signal including the DMH, amygdala (Amg) and tuberal nucleus (TN) at bregma -1.54, -1.79, -1.91 and -2.13 mm of Prdm13 -ZsGreen mice. e , Ratios of cFos + cells within Prdm13 + cells in young mice during SD and SD-Cont detected by RNAscope in situ hybridization (n=7-8). Values are shown as means ± S.E., *p<0.05 and **p<0.01 by two-way ANOVA with Bonferroni’s post hoc test. f , g , Representative images of DMH sections from young mice under SD-Cont (left) and SD (right) with Prdm13 (yellow) and cFos (red) visualized by RNAscope. Boxed areas were shown at high magnification in g . Cells were counterstained with DAPI (blue). Scale bars indicate 100 and 10 μm ( f and g , respectively).

Article Snippet: The 8% equivalent of each fraction by volume was run on a 4-15% TGX gel (Bio-Rad) for Western blotting using affinity-purified polyclonal rabbit anti-mouse Prdm13.

Techniques: Control, Immunohistochemistry, RNAscope, In Situ Hybridization

Journal: bioRxiv

Article Title: Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

doi: 10.1101/2022.09.26.509442

Figure Lengend Snippet: a , Breeding strategy to generate DMH-specific Prdm13 -knockout (Prdm13-KO) mice. After crossing Prdm13 fl/f ;Rosa26R ZsGreen/ZsGreen mice and Nkx2-1 CreERT2/+ ;Prdm13 fl/fl mice, Prdm13 fl/fl ;Nkx2-1 CreERT2/+ ;Rosa26R ZsGreen/+ mice were used as Prdm13-KO mice and Prdm13 fl/fl ;Nkx2-1 +/+ ;Rosa26R ZsGreen/+ mice were used as control (Cont) mice. b , Expression of Prdm13 in the DMH, tuberal nucleus (TN) and amygdala (Amg) of Prdm13-KO and Cont mice (n=5). Values are shown as means ± S.E., **p<0.01, ***p<0.001 and non-significant (ns) by two-way ANOVA with Bonferroni’s post hoc test. c , d , Number of episodes ( c ) or duration ( d ) of wakefulness (top), NREM sleep (middle) and REM sleep (bottom) every 3 hours through a day (left) and during the light (L) and dark (D) periods (right) in Prdm13-KO and Cont mice. Shading indicates dark period (n=6). Values are shown as means ± S.E., #p<0.05 by repeated measures ANOVA, listed p-values and *p<0.05 by repeated measures ANOVA with Bonferroni’s post hoc test (left) or unpaired t-test (right). e , Number of sleep attempts during SD from 6am to 8am (6-8), 8am to 9am (8-9), 9am to 10am (9-10), 10am to 11am (10-11) and 11am to 12pm (11-12) in Prdm13-KO and Cont mice (n=13-14). Values are shown as means ± S.E., *p<0.05, ***p<0.001 by repeated measures ANOVA with Bonferroni’s post hoc test. f , SWA during NREM sleep after SD. Normalized power is relative to the average of the 24-hour baseline day each group (n=6). Values are shown as means ± S.E. g , Total amount of wakefulness, NREM sleep and REM sleep during a 24-hour period (24h total), 12-hour light period (12h light) or 12-hour dark period (12h dark) (n=6). Values are shown as means ± S.E. h , EEG spectra of wakefulness (left), NREM sleep (middle) and REM sleep (right) during the light period (n=5-6). Values are shown as means ± S.E.

Article Snippet: The 8% equivalent of each fraction by volume was run on a 4-15% TGX gel (Bio-Rad) for Western blotting using affinity-purified polyclonal rabbit anti-mouse Prdm13.

Techniques: Knock-Out, Control, Expressing

Journal: bioRxiv

Article Title: Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

doi: 10.1101/2022.09.26.509442

Figure Lengend Snippet: a , b , Numbers of episodes ( a ) and duration ( b ) of wakefulness (top), NREM sleep (middle) and REM sleep (bottom) every 3 hours through a day (left) and during the light (L) and dark (D) periods (right) in old DMH-specific Prdm13 -knockout (Prdm13-KO) and control (Cont) mice (n=5-6). Values are shown as means ± S.E., #p<0.05 and ##p<0.01 by repeated measures ANOVA, *p<0.05 and **p<0.01 by repeated measures ANOVA with Bonferroni’s post hoc test (left) or unpaired t-test (right). c , EEG spectra of wakefulness (left), NREM sleep (middle) and REM sleep (right) during the light period (n=4-6). Values are shown as means ± S.E. d , Number of sleep attempts during SD from 6am to 8am (6-8), 8am to 9am (8-9), 9am to 10am (9-10), 10am to 11am (10-11) and 11am to 12pm (11-12) in old Prdm13-KO and Cont mice (n=5-6). Values are shown as means ± S.E., **p<0.01 by repeated measures ANOVA with Bonferroni’s post hoc test. e , SWA after SD of Prdm13-KO and Cont mice at 20 months of age. Normalized power is relative to the average of the 24-hour baseline day (n=5-6). Values are shown as means ± S.E., **p<0.01 by Bonferroni’s post hoc test. f , Body weight of old Prdm13-KO and Cont mice (n=5-7). Values are shown as means ± S.E., *p<0.05 by unpaired t-test. g , The level of wheel-running activity in old Prdm13-KO and Cont mice for six consecutive days (n=5-7). Values are shown as means ± S.E., #p<0.05 by repeated measures ANOVA. h , Kaplan-Meier curves of Prdm13-KO and Cont mice (n=13-18). Listed p-value was calculated by log-rank test.

Article Snippet: The 8% equivalent of each fraction by volume was run on a 4-15% TGX gel (Bio-Rad) for Western blotting using affinity-purified polyclonal rabbit anti-mouse Prdm13.

Techniques: Knock-Out, Control, Activity Assay

Journal: bioRxiv

Article Title: Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

doi: 10.1101/2022.09.26.509442

Figure Lengend Snippet: a , DR paradigm in C57BL/6J at 20 months of age. Mice at 20-months-old were fed under 60% diet or AL-diet for 14 to 28 days. b , c , Number of episodes ( b ) and duration ( c ) of wakefulness (top), NREM sleep (middle) and REM sleep (bottom) during the light (L) and dark (D) periods in AL and DR mice at 20 months of age (n=5). Values are shown as means ± S.E., listed p-value, *p<0.05 and **p<0.01 by unpaired t-test. d , EEG spectra of wakefulness (upper left), NREM sleep (upper right) and REM sleep (lower) during the light period (n=5). Values are shown as means ± S.E. e , Number of sleep attempts during SD from 6am to 8am (6-8), 8am to 9am (8-9), 9am to 10am (9-10), 10am to 11am (10-11) and 11am to 12pm (11-12) in AL and DR mice at 20 months of age (n=5-6). Values are shown as means ± S.E., **p<0.01 by repeated measures ANOVA with Bonferroni’s post hoc test. f , SWA after SD of AL and DR mice at 20 months of age. Normalized power is relative to the average of the 24-hour baseline day (n=5). Values are shown as means ± S.E., *p<0.05 by Bonferroni’s post hoc test. g , Number of sleep attempts during SD from 6am to 8am (6-8), 8am to 9am (8-9), 9am to 10am (9-10), 10am to 11am (10-11) and 11am to 12pm (11-12) in Prdm13-KO-AL and Prdm13-KO-DR mice (n=8). Values are shown as means ± S.E. h , Expression of Prdm13 in the hypothalamus of Prdm13 -overexpressing (Prdm13-OE) and control (Cont) mice (n=4-5). Values are shown as means ± S.E., *p<0.05 by unpaired t-test. i , j , Number of episodes ( i ) and duration ( j ) of wakefulness (top), NREM sleep (middle) and REM sleep (bottom) during the light (L) and dark (D) periods in Prdm13-OE and Cont mice (n=5). Values are shown as means ± S.E., *p<0.05 by unpaired t-test. k , Number of sleep attempts during SD from 6am to 8am (6-8), 8am to 9am (8-9), 9am to 10am (9-10), 10am to 11am (10-11) and 11am to 12pm (11-12) in Prdm13-OE and Cont mice (n=4). Values are shown as means ± S.E., *p<0.05 by repeated measures ANOVA with Bonferroni’s post hoc test. l , SWA after SD of Prdm13-OE and Cont mice. Normalized power is relative to the average of the 24-hour baseline day (n=4). Values are shown as means ± S.E.

Article Snippet: The 8% equivalent of each fraction by volume was run on a 4-15% TGX gel (Bio-Rad) for Western blotting using affinity-purified polyclonal rabbit anti-mouse Prdm13.

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

doi: 10.1101/2022.09.26.509442

Figure Lengend Snippet: a , Western blot of Prdm13 in DMH collected by laser microdissection from DMH-specific Prdm13-KO and Cont mice (n=4 mice/lane). The arrow indicates the band for Prdm13; asterisks (*) indicate non-specific bands. b , Schematic of fractionation protocol from mouse hypothalami (left). Western blot of Prdm13 in hypothalamic fractions of C57BL/6J mice (right). Hypothalami from two C57BL/6J female mice were combined for each lane, and 8% equivalent of each fraction was run on the gel. Cytoplasmic supernatant (S1), RNase-extractable supernatant (S2), DNase-extractable supernatant (S3), and insoluble pellet (P) were run each lane. The arrow indicates the band for Prdm13; asterisks (*) indicate non-specific bands. c , Western blot of Prdm13 in hypothalamic fractions of Prdm13 -PA-Tag (KI) and wild-type (WT) mice. Cytosolic and nuclear fractions were run each lane as indicated. d , Expression of Cck, Grp and Pmch mRNA in the DMH of DMH- Prdm13 -KO (Prdm13-KO) and control (Cont) mice (n=3-5). Values are shown as means ± S.E., listed p-value, **p<0.01 and ***p<0.001 by unpaired t-test. e , Transcriptional activity of Prdm13-202 and Prdm13-mutants for the luciferase reporter vector containing the promoter region of Cck, Grp and Pmch . Schematic representation of Prdm13-202 and Prdm13-mutants are shown above. NIH3T3 cells were co-transfected with 250 ng of luciferase reporter plasmid and plasmid expressing Prdm13-202 (Prdm13), Prdm13-Zif mutant (mutZif) or Prdm13-deltaPR mutant (deltaPR). Obtained luminescence was normalized to total protein concentration (n=3, four individual experiments). Values are shown as means ± S.E., listed p-value, *p<0.05, **p<0.01 and ***p<0.001 by one-way ANOVA with Bonferroni’s post hoc test, # p<0.05 and ## p<0.01 and ### p<0.001 by unpaired t-test. f , Transcriptional activity of hypothalamic Prdm13 (htPrdm13) for the luciferase reporter plasmid containing the promoter region of Cck . NIH3T3 cells were co-transfected with 250 ng of reporter plasmid and 10, 50 or 250 ng of htPrdm13 -expressing plasmid. Obtained luminescence was normalized to total protein concentrations (n=3, three individual experiments). Values are shown as means ± S.E., *p<0.05 by one-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: The 8% equivalent of each fraction by volume was run on a 4-15% TGX gel (Bio-Rad) for Western blotting using affinity-purified polyclonal rabbit anti-mouse Prdm13.

Techniques: Western Blot, Laser Capture Microdissection, Fractionation, Expressing, Control, Activity Assay, Luciferase, Plasmid Preparation, Transfection, Mutagenesis, Protein Concentration

Journal: bioRxiv

Article Title: Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

doi: 10.1101/2022.09.26.509442

Figure Lengend Snippet: a , Representative images of the DMH with Prdm13 (yellow) and one of the two genes, Cck or Grp (green) visualized by RNAscope. Cells were counterstained with DAPI (blue). White boxes show the DMH, which is divided into medial and lateral areas by dashed lines. White arrows show yellow+green+ cells. Scale bar indicates 100 μm. b , Ratios of Cck + or Grp + cells within Prdm13 + cells in medial, lateral or total (medial and lateral) DMH (n=3-5). Values are shown as means ± S.E., ***p<0.001 by unpaired t-test. c , Distribution of cFos + Prdm13 + cells (n=5). d , Representative images of the DMH from young mice under SD-Cont and SD with Prdm13 (yellow), Cck (green) and cFos (red) visualized by RNAscope. Cells were counterstained with DAPI (blue). White arrows show Prdm13+Cck+cFos+ cells. Scale bar indicates 100 μm. e , f , Ratios of cFos + cells within Prdm13 + Cck -(left) or Prdm13 + Cck + (right) cells in young ( e ) or old ( f ) mice during SD-Cont and SD (n=7-8). Values are shown as means ± S.E., *p<0.05, ***p<0.001 by two-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: The 8% equivalent of each fraction by volume was run on a 4-15% TGX gel (Bio-Rad) for Western blotting using affinity-purified polyclonal rabbit anti-mouse Prdm13.

Techniques: RNAscope

Journal: Neuron

Article Title: Edinger-Westphal peptidergic neurons enable maternal preparatory nesting

doi: 10.1016/j.neuron.2022.01.012

Figure Lengend Snippet:

Article Snippet: For circulating Progesterone titration, a drop of blood was sampled from the mouse cheek, and processed through a Mouse Progesterone ELISA kit according to the manufacturer’s instructions (Novus Biological NBP2-60125-1).

Techniques: Virus, Recombinant, RNAscope, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell reports

Article Title: Peptidergic and functional delineation of the Edinger-Westphal nucleus

doi: 10.1016/j.celrep.2023.112992

Figure Lengend Snippet:

Article Snippet: The cocktail included: 1 μM [D-p-Cl-Phe6,Leu17]-VIP to block VPACRs, 200 nM PACAP 6–38 to block PAC1R, 2 mM proglumide to block CCKRs, 300 nM astressin 2B to block CRFR2, and 1 μM NBI 35965 hydrochloride to block CRFR1 (all from Tocris).

Techniques: Virus, Plasmid Preparation, Recombinant, Electron Microscopy, RNAscope, Fluorescence, Multiplex Assay, Sequencing, Software

Journal: eLife

Article Title: The long noncoding RNA lnc-FANCI-2 intrinsically restricts RAS signaling in human papillomavirus type 16-infected cervical cancer cells

doi: 10.7554/eLife.102681

Figure Lengend Snippet: ( A ) RT-qPCR detection of lnc-FANCI-2 in HPV16 + cervical cancer cell lines SiHa, CaSki, and W12 20861 (integrated HPV16) and 20863 (episomal HPV16), HPV18 + cervical cancer cell lines HeLa and C4II, and HPV - cell lines C33A (cervical cancer cells with mutations of p53 and pRb), HCT116 (colorectal cancer cells), BCBL-1 (body cavity B lymphoma cells), HEK293 (Ad5 E1/E2-immortalized human kidney cells), and HaCaT (spontaneously immortalized human epidermal cells) in triplicates. ( B ) HeLa cells express no lnc-FANCI-2 when compared with C33A cells by northern blot. ( C ) lnc-FANCI-2 is mainly cytoplasmic in CaSki but nuclear in SiHa and C33A cells. Cytoplasmic and nuclear fractionation efficiency was blotted for nuclear SRSF3 (serine- and arginine-rich splicing factor 3) and cytoplasmic GAPDH. Total fractionated cytoplasmic and nuclear RNAs were quantified for lnc-FANCI-2 by RT-qPCR in triplicates, with GAPDH RNA serving as an internal control for RNA fractionation efficiency. ( D ) Subcellular lnc-FANCI-2 (red) localization in CaSki, SiHa, and C33A cells determined by RNAscope RNA in situ hybridization (RNA-ISH) analysis. Nuclei were stained with DAPI (blue). Scale bars: 25 μm in the top and 10 μm in the zoom. Figure 2—source data 1. Northern blot and western blot data for . Figure 2—source data 2. Northern blot and western blot data for .

Article Snippet: Antibody , Anti-p53 (Rabbit polyclonal) , ProteinTech , Cat# 10442-1-AP , Rabbit polyclonal; WB (1:1000).

Techniques: Quantitative RT-PCR, Northern Blot, Fractionation, Control, RNAscope, RNA In Situ Hybridization, Staining, Western Blot

Journal: eLife

Article Title: The long noncoding RNA lnc-FANCI-2 intrinsically restricts RAS signaling in human papillomavirus type 16-infected cervical cancer cells

doi: 10.7554/eLife.102681

Figure Lengend Snippet: ( A ) Effect of lnc-FANCI-2 KO in CaSki cells on the expression of HPV16 E6 and E7 and their downstream targets. Total cell extracts from parental CaSki, ΔPr-A9, and ΔPr-B3 cells were immunoblotted with the corresponding antibodies. Tubulin or GAPDH served as a protein loading control. The relative protein levels of E6, E7, p53, and E2F1 were calculated after normalizing to tubulin or GAPDH in the corresponding experiment. Expt I or II = experiment I or II. ( B ) KO of lnc-FANCI-2 expression enhances cell senescence in β-gal analysis. Figure 3—figure supplement 2—source data 1. lnc-FANCI-2 KO in CaSki cells and the expression of HPV16 E6 and E7 and their downstream targets. Figure 3—figure supplement 2—source data 2. lnc-FANCI-2 KO in CaSki cells and the expression of HPV16 E6 and E7 and their downstream targets.

Article Snippet: Antibody , Anti-p53 (Rabbit polyclonal) , ProteinTech , Cat# 10442-1-AP , Rabbit polyclonal; WB (1:1000).

Techniques: Expressing, Control