Journal: Journal of Biological Chemistry

Article Title: Expression Profiles of Arabidopsis thaliana in Mineral Deficiencies Reveal Novel Transporters Involved in Metal Homeostasis

doi: 10.1074/jbc.m309338200

Figure Lengend Snippet: FIG. 6. Functional characterization of AtOPT3. A, hybridization of an 600-bp AtOPT3 cDNA probe, corresponding to the 3-terminal exon, to RNA extracted from Arabidopsis roots of plants grown in control conditions (Ctl), copper (-Cu), manganese (-Mn), zinc (-Zn), and iron (-Fe) deficiency. The ethidium bromide-stained RNA gel is shown for quantification. B, growth of ctr1 expressing AtOPT3 on YPG-Ura plates supplemented with 10 M CuSO4 compared with the mutant transformed with the vector alone. C, growth of smf1 expressing AtOPT3 on manganese-limited medium, with and without 1 mM EGTA compared with the growth of the mutant transformed with the vector alone.

Article Snippet: Genome-wide Analysis Provides Insight into Metal Transport—We have used Affymetrix Arabidopsis DNA chips containing 8,300 genes (which cover about one-third of the ge- FIG. 7.

Techniques: Functional Assay, Hybridization, Control, Staining, Expressing, Mutagenesis, Transformation Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) X-gal staining to detect Rcor2 expression patterns. Whole-embryo staining at E11.5 stage shows Rcor2 is mainly expressed in the CNS. Scale bar, 1 mm. ( b ) Western blot analysis of Rcor2 expression levels during brain development. The decreased expression of Rcor2 with embryonic development is noteworthy. β-Actin is used as an endogenous control. ( c ) In situ hybridization to detect endogenous Rcor2 mRNA expression patterns in cortical development at E11.5, E13.5, E15.5 and E17.5. Insets show high-magnification image of Rcor2 expression in the neocortex at E13.5. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate. Scale bar, 100 μm. ( d ) Confocal images of immunofluorescence to detect cellular localization of Rcor2 in the neocortex at E13.5. Rcor2 localized mainly in the nucleus at interphase and metaphase, and localized between separated chromosomes in anaphase of dividing cells in VZ. Dotted lines circle the shape of nuclei. Scale bar, 5 μm. ( e ) Western blot analysis of Rcor2 expression level in Rcor2 fl/fl and Rcor2 cko brains at E13.5 and E15.5, respectively. Rcor2 expression was depleted in Rcor2 cko brains. β-Actin is used as an endogenous control. ( f ) Representative images of Rcor2 fl/fl and Rcor2 cko brain size at different stages of development. Rcor2 cko mice show severe brain growth retardation at E13.5 and E15.5. Scale bar, 1 mm. ( g ) Representative images of Rcor2 fl/fl and Rcor2 cko cortex at E15.5 by Nissl staining. Structural abnormalities of lamination with reduced cortical thickness are observed in Rcor2 cko cortex. Scale bar, 200 μm. ( h ) RT–qPCR analysis of knockdown efficiencies of the two shRNAs targeting Rcor2. Transcripts were normalized to the control group. Data are shown as mean±s.e.m., t -test, **** P <0.0001, n =3. ( i ) Confocal images of E16.5 cortical sections electroporated with shControl (red), shRcor2-a (red) and shRcor2-b (red) plasmids at E13.5. Knockdown of Rcor2 results in impaired cortical development. IZ, intermediate zone. Scale bar, 20 μm. ( j ) Quantification of the percentage of RFP + cells in different regions of the developing neocortex after electroporation as shown in i . proportion of RFP + cells in different zones ( y axis). Data are shown as mean±s.e.m., t -test, * P <0.05, ** P <0.01, *** P <0.001 and **** P <0.0001, n =3 individual experiments.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Staining, Expressing, Western Blot, Control, In Situ Hybridization, Immunofluorescence, Quantitative RT-PCR, Knockdown, Electroporation

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Immunostaining images of Sox2 at E13.5. Sox2 is dramatically reduced on Rcor2 depletion. VZ, ventricular zone; SVZ, subventricular zone. Scale bar, 20 μm. ( b ) Quantification of Sox2 + cell ratios in VZ/SVZ regions shown in a . Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 individual experiments. ( c ) Confocal images of Tbr2 expression at E13.5. Tbr2 is dramatically reduced on Rcor2 depletion. Scale bar, 20 μm. ( d ) Quantification of Tbr2 + cell ratios in VZ/SVZ regions shown in c , respectively. Data are shown as mean±s.e.m., t -test, * P <0.05, n =3 individual experiments. ( e ) Immunostaining images of Nestin, Sox2 and Tbr2 in cultured Rcor2 fl/fl and Rcor2 cko NPCs, all of which exhibit significantly reduced expression in the Rcor2 cko NPCs. Scale bar, 20 μm. ( f ) Confocal images of immunofluorescence for Ki67 and PHH3 in Rcor2 fl/fl and Rcor2 cko cortex at E13.5 and E15.5. Ki67 signals (red), but not PHH3 signals (green), are dramatically reduced in Rcor2 cko developing brains. Scale bar, 20 μm. ( g ) Quantification of Ki67 + cells in the VZ/SVZ regions of the developing neocortex as shown in f . Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 separate stainings from three independent brains. ( h ) Confocal images of BrdU (green) and Ki67 (red) staining in Rcor2 fl/fl and Rcor2 cko cortex 24 and 48 h after BrdU incorporation. Scale bar, 100 μm. ( i ) Quantification of the cell cycle exit by percentage of BrdU + and Ki67 + NPCs divided by BrdU + cells shown in h . Data are shown as mean±s.e.m., t -test, *** P <0.001 and **** P <0.0001, n =3 individual experiments. ( j , k ) Representative images ( j ) and quantification of ( k ) of Rcor2 fl/fl and Rcor2 cko neurosphere sizes. The neurospheres'radius of Rcor2 cko are much smaller than those of Rcor2 fl/fl , t -test, **** P <0.0001, n =12. Scale bar, 50 μm. ( l ) Representative time-lapse imaging of the RGC dividing process in the sections of the cerebral cortex electroporated with RFP-shControl (upper panels) and RFP-shRcor2 (lower panels). The radial glial dividing pattern is abnormal on Rcor2 knockdown, resulting in cell death. Arrows, mother RGCs. Arrowheads, two daughter cells. Scale bar, 50 μm. ( m ) Representative time-lapse images of Rcor2 fl/fl cortex sections electroporated with EGFP-Control (upper panels) and EGFP-Cre (lower panels). Loss of cells is observed with Rcor2 knockout by Cre recombinase electroporation. Scale bar, 50 μm.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Immunostaining, Expressing, Cell Culture, Immunofluorescence, Staining, BrdU Incorporation Assay, Imaging, Knockdown, Control, Knock-Out, Electroporation

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Confocal images of Satb2 and Tbr1 expressions in Rcor2 fl/fl and Rcor2 cko cortex at E15.5, which exhibit significant reduction on Rcor2 knockout. Scale bar, 50 μm. ( b ) Quantification of Satb2 + and Tbr1 + cells in Rcor2 fl/fl and Rcor2 cko cortex at E15.5 in a indicates Satb2 and Tbr1 expressions are decreased on Rcor2 depletion during development. Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 individual experiments. ( c ) Western blot to analyse Dcx, Satb2 and Tbr1 expressions in Rcor2 fl/fl and Rcor2 cko cortex at E15.5. β-Actin is used as an endogenous control. ( d ) Representative images of Map2 and Tuj1 immunostaining in cultured neurons directly dissociated from Rcor2 fl/fl and Rcor2 cko brain cortex at E15.5. Decreased expression of both markers and reduced neurofilaments can be observed in Rcor2 cko cultured neurons. Scale bar, 20 μm. ( e ) Confocal images of in-vitro cultured Rcor2 fl/fl and Rcor2 cko NPCs 5 days post spontaneous differentiation using neuronal marker Map2 and Tuj1 antibodies, both of which are significantly reduced in the differentiated Rcor2 cko NPCs. Scale bar, 20 μm. ( f , g ) RT–qPCR analysis of neuronal markers expression in both Rcor2 fl/fl and Rcor2 cko neocortex at E15.5 stage ( f ) and in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs 5 days post spontaneous differentiation ( g ). Transcripts were normalized to Rcor2 fl/fl group. Data are shown as mean±s.d., t -test, * P <0.05, ** P <0.01 and *** P <0.001, n =3.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knock-Out, Western Blot, Control, Immunostaining, Cell Culture, Expressing, In Vitro, Marker, Quantitative RT-PCR

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Schematic overview of strategy to generate an Rcor2 Flag knock-in allele by CRISPR/Cas9. The sgRNA sequence site is shown as a green arrowhead. The start codon of Rcor2 is indicated and capitalized. The oligo donor contained 50 bp homologies on both sides flanking the DSB, in which 3 × Flag sequences are labelled as a red box. ( b ) Western blot analysis to validate FLAG, RCOR2 and LSD1 expressions in Rcor2 Flag knock-in neocortex using Flag-M2 antibody. β-Actin was used as an endogenous control. ( c ) Pie chart depicts distribution of Rcor2 occupancies in genome-wide scale in FLAG ChIP-seq results using Rcor2 Flag knock-in neocortex at E13.5. ( d ) WebLogos of consensus binding motifs of Rcor2 generated by Multiple EM for Motif Elicitation (MEME) motif analysis tool. ( e ) GO analysis for Rcor2-binding regions in genome-wide scale revealed by Flag ChIP-seq results using Rcor2 Flag brain. ( f ) GO analysis for LSD1 occupancy in genome-wide scale revealed by LSD1 ChIP-seq results using Rcor2 Flag brain. ( g ) Density plots analysis of H3K4me1 signal change in promoter regions (−2- to ∼0.5 kb from TSS) on Rcor2 depletion. Compared with all genes, the change of H3K4me1 signal is significantly ( P <0.0005, Kolmogorov–Smirnov test) enhanced in the promoter regions of Shh pathway-related genes on Rcor2 depletion. H3K4me1 signal change on Rcor2 depletion ( x axis); H3K4me1 signal density ( y axis). ( h ) Gene tracks of Rcor2, LSD1 and H3K4me1 enrichments by ChIP-seq analysis at core promoter regions of Dlx2 and upstream regulatory regions of Shh, which are closely related to Shh signalling. ( i ) ChIP–qPCR analysis of Rcor2 Flag and Rcor2 cko cortex at E13.5 using specific FLAG-M2 antibody. Significant enrichments of the Rcor2 at the regulatory regions of Dlx2 and Shh gene locus detected in g in the Rcor2 flag samples are worth noting. Fold enrichments of Rcor2 occupancy compared with input ( y axis). Data are shown as mean±s.d., t -test, *** P <0.001, n =3.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knock-In, CRISPR, Sequencing, Western Blot, Control, Genome Wide, ChIP-sequencing, Binding Assay, Generated, ChIP-qPCR

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a , b ) Scatter plot analysis of genome-wide expression profiles of Rcor2 cko versus Rcor2 fl/fl samples at E13.5 ( a ) and E15.5 ( b ). Dots above or below the dash line indicate upregulated or downregulated genes on Rcor2 depletion, respectively. Red dots or green dots highlight the significantly differentially expressed genes on Rcor2 depletion. Raw counts ( x axis); gene expression fold changes on Rcor2 depletion ( y axis). ( c ) Venn diagrams of upregulated genes (left) and downregulated genes (right) in Rcor2 cko samples compared with Rcor2 fl/fl samples. ( d ) The profiles of Rcor2 and H3k4me1 enrichments analysed in ChIP-seq results shown in in regulatory regions of genome-wide scale (red) and of the upregulated genes (purple) according to RNA-seq results. ( e ) Correlation network of overlapped upregulated genes in both E13.5 and E15.5 samples. Lines indicate the correlations between two connected genes with R >0.55. Genes were analysed by GO analysis and divided into different categories.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Genome Wide, Expressing, Gene Expression, ChIP-sequencing, RNA Sequencing

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) qPCR analysis of the expression of genes related to the Shh signalling pathway in the cortex of Rcor2 fl/fl and Rcor2 cko brains during development. Significant upregulation of these genes on Rcor2 depletion is noteworthy. Transcripts were normalized to Rcor2 fl/fl group. Data are shown as mean±s.d., t -test, * P <0.05, ** P <0.01 and *** P <0.001, n =3. ( b ) Confocal images of Shh and Ptch1 expressions in Rcor2 fl/fl and Rcor2 cko cortex. Enhanced Shh and Ptch1 signals are observed in Rcor2 cko compared with Rcor2 fl/fl neocortex at E13.5 and E15.5. Insets show high-magnification images of the outlined regions. Scale bars, 50 μm. ( c ) Dlx2 expression in Rcor2 fl/fl and Rcor2 cko cortex detected by immunofluorescence analysis at E15.5. Dlx2 + cells were observed in the neocortex on Rcor2 depletion. Scale bars, 50 μm. ( d ) Confocal images of Shh, Ptch1 and Dlx2 expressions in in-vitro -cultured Rcor2 cko NPCs. Scale bar, 20 μm. ( e ) Western blot analysis of expression levels of Dlx2, Shh and Ptch1 in Rcor2 fl/fl and Rcor2 cko cortex at E15.5. β-Actin is used as an endogenous control.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Expressing, Immunofluorescence, In Vitro, Cell Culture, Western Blot, Control

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Knockdown of Rcor2 impairs cortical neurogenesis, which can be partially rescued by knockdown of Shh during cortical development. In-utero electroporation with RFP-shControl (red)/GFP-shControl (green), RFP-shRcor2 (red)/GFP-shControl (green), RFP-shControl (red)/GFP-shShh (green) or RFP-shRcor2 (red)/GFP-shShh (green) plasmids was performed at E13.5. Cerebral sections were fixed and imaged at E16.5. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate. Scale bar, 50 μm. ( b ) Quantification of the percentage of RFP + /GFP + cells in different regions of the developing cortex after electroporation shown in a . Data are shown as mean±s.e.m., t -test, ** P <0.01 and *** P <0.001, n =3 individual experiments. ( c ) Inhibition of Shh by Cyclopamine can partially rescue neurogenesis defects caused by Rcor2 downregulation during cortical development. Rcor2 was knocked down at the lateral ventricle in the brain by in-utero electroporation with RFP-shRcor2 plasmids at E13.5. Cerebral sections were collected at E14.5 and then treated with cyclopamine to inhibit Shh activity for 48 h. Scale bar, 50 μm. ( d ) Quantification of the percentage of RFP + cells in different regions of the developing neocortex after knockdown of Rcor2 or inhibition of Shh shown in c . Data are shown as mean±s.e.m., t -test, * P <0.05, n =3 individual experiments. ( e ) Representative images depicting neurosphere size is partially rescued in the in-vitro -cultured Rcor2 cko NPCs after treatment with Cyclopamine. Scale bar, 50 μm. ( f ) Histogram depicting cell numbers of in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs with or without Cyclopamine treatment for 48 h. Cells (5 × 10 5 ) are seeded initially. Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3. ( g ) Confocal images of Tuj1 expression in the differentiated cells from in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs with or without Cyclopamine treatment. Tuj1 expressions are partially restored in Cyclopamine-treated Rcor2 cko cells. Scale bar, 20 μm. ( h ) Model of Rcor2 function in neurogenesis in the developing neocortex. Rcor2 safeguards cortical neurogenesis by recruiting LSD1 complex to the regulatory regions of Dlx2 and Shh genes, to inhibit the Shh pathway activation during development. The absence of Rcor2 leads to inhibition release of these genes and thus ectopic activation of Shh signalling in the developing neocortex, resulting in cortical neurogenesis defects.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knockdown, In Utero, Electroporation, Inhibition, Activity Assay, In Vitro, Cell Culture, Expressing, Activation Assay

Journal: bioRxiv

Article Title: Hepatocyte Cell Cycle Progression Depends on a Transcriptional Repressor Cascade Downstream of Wnt Signaling

doi: 10.1101/2021.10.15.464616

Figure Lengend Snippet: ( A ) TBX3 binds to E2f7 promoter and E2f8 enhancer , shown by Chromatin Immunoprecipitation-Sequencing (ChIP-seq) binding profiles (reads per million per base pair) for TBX3 at the E2f7 and E2f8 loci in primary hepatocytes. Red boxes indicate significant binding peaks. These sites are tested in (B) and (G). Profiles are representative of two independent biological replicates. ( B ) Validation of binding sites outlined in (A) by ChIP-qPCR. (C-E) E2f7 and E2f8 are ectopically upregulated upon deletion of Tbx3 . (C) mRNA in situ hybridization for E2f7 and E2f8 in Control (Axin2-rtTA; TetO-H2B-GFP; Tbx3 f/f ) and Tbx3 KO (Axin2-rtTA; TetO-H2B-GFP; TetO-Cre; Tbx3 f/f ) livers at postnatal week 4 show increased signal in the pericentral hepatocytes of Tbx3 KO livers. Arrowheads point to positive signals. (D) Quantitative real-time PCR (qRT-PCR) analysis of Tbx3, E2f7 and E2f8 transcripts from whole livers of the same samples as in (C) shows significantly increased levels of E2f7 and E2f8 in the absence of Tbx3 . (E) qRT-PCR analysis shows increased E2f7 and E2f8 transcription in Tbx3 KD cultured mouse primary hepatocytes. ( F ) E2f7 and E2f8 are downregulated upon overexpression of Tbx3 . qRT-PCR analysis of Tbx3, E2f7 and E2f8 transcripts in Control (EF1α-GFP) and Tbx3 OE (EF1 α-GFP-P2A-Tbx3) mouse primary hepatocytes. ( G ) TBX3 represses E2f7 and E2f8 expression by binding to their regulatory regions. Regions outlined in (A) as TBX3 binding sites were cloned into luciferase expression constructs for functional analyses in HepG2 cells and luciferase activity was measured. P-values noted via asterisks are comparisons of Control vs. E2f7 and Control vs. E2f8 in the Vector control condition. Scale bars, 100 μm. CV, Central Vein. KD, knockdown. Ns, not significant. Vec, Vector control. *P<0.05; **P < 0.01. Statistical significance was determined by Student’s t-test from n=3 independent experiments. Error bars indicate standard deviation.

Article Snippet: Gene Expression Assays used were Gapdh (Mm99999915_g1) as control, Tbx3 (Mm01195719_m1), E2f7 (Mm00618098_m1), and E2f8 (Mm01204165_g1).

Techniques: ChIP-sequencing, Binding Assay, Biomarker Discovery, ChIP-qPCR, In Situ Hybridization, Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Cell Culture, Over Expression, Expressing, Clone Assay, Luciferase, Construct, Functional Assay, Activity Assay, Plasmid Preparation, Knockdown, Standard Deviation

Journal: bioRxiv

Article Title: Hepatocyte Cell Cycle Progression Depends on a Transcriptional Repressor Cascade Downstream of Wnt Signaling

doi: 10.1101/2021.10.15.464616

Figure Lengend Snippet: ( A ) Tbx3 is expressed in the pericentral hepatocytes of human livers. TBX3 protein is detected by immunofluorescence in both human and mouse livers. ( B ) TBX3 binds to E2F7 and E2F8 in human hepatocytes. TBX3 Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) Signal Track from HepG2 Cells (ENCODE) for E2F7 and E2F8 loci. Regions underlined in red indicate significant binding peaks. Boxed regions are significant binding regions conserved in mouse. CV, Central Vein. PV, Portal Vein. Scale bars, 100 μm.

Article Snippet: Gene Expression Assays used were Gapdh (Mm99999915_g1) as control, Tbx3 (Mm01195719_m1), E2f7 (Mm00618098_m1), and E2f8 (Mm01204165_g1).

Techniques: Immunofluorescence, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Hepatocyte Cell Cycle Progression Depends on a Transcriptional Repressor Cascade Downstream of Wnt Signaling

doi: 10.1101/2021.10.15.464616

Figure Lengend Snippet: (A) Visualization of Axin2-rtTA-driven GFP expression in Control, Tbx3 KO, or Tbx3-E2f7-E2f8 TKO livers. GFP and TBX3 immunofluorescence in the pericentral zone in Control (Axin2-rtTA; TetO-H2B-GFP; Tbx3 f/f ; E2f7 f/f ; E2f8 f/f ), Tbx3 KO (Axin2-rtTA; TetO-H2B-GFP; TetO-Cre; Tbx3 f/f ) and Tbx3; E2f7; E2f8 TKO (Axin2-rtTA; TetO-H2B-GFP; TetO-Cre; Tbx3 f/f ; E2f7 f/f ; E2f8 f/f ) livers at postnatal week 4. (B-D) Deletion of E2f7 and E2f8 along with Tbx3 restores the balance of ploidy. (B) Bar graph summarizes nuclear ploidy distribution of GFP+ nuclei from each genotype at postnatal week 4. (C) Nuclear ploidy plots of GFP+ hepatocytes in Control and Tbx3-E2f7-E2f8 TKO mice, stained with Propidium Iodide (PI) and measured by flow cytometry. Plots shown are chosen as representatives of each genotype and noted percentages are averages from n=3 Control and n=4 Tbx3-E2f7-E2f8 TKO mice. (D) Measurements of nuclear area from GFP+ pericentral hepatocytes in Control (n=3 mice) and Tbx3-E2f7-E2f8 TKO (n=4 mice). Bars indicate mean and standard deviation. P=0.2249, t-test with Welch’s correction comparison of mean nuclear area from biological replicates. Scale bars, 100 μm. CV, Central Vein. ns, not significant.

Article Snippet: Gene Expression Assays used were Gapdh (Mm99999915_g1) as control, Tbx3 (Mm01195719_m1), E2f7 (Mm00618098_m1), and E2f8 (Mm01204165_g1).

Techniques: Expressing, Control, Immunofluorescence, Staining, Flow Cytometry, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Hepatocyte Cell Cycle Progression Depends on a Transcriptional Repressor Cascade Downstream of Wnt Signaling

doi: 10.1101/2021.10.15.464616

Figure Lengend Snippet: (A) Membrane and nuclear visualization of Control or Alb-CreERT2-driven Tbx3 KO or Tbx3-E2f7-E2f8 TKO livers. Representative HNF4α and β-ACTIN immunofluorescence images at postnatal week 4 are shown. (B-C) Deletion of E2f7 and E2f8 along with Tbx3 rescues the polyploidy phenotype of Tbx3 KO. (B) Representative flow cytometry plots of nuclear ploidy distribution from n=5 Control (Tbx3 f/f ; E2f7 f/f ; E2f8 f/f ) , n=4 Tbx3 KO (Alb-CreERT2; Tbx3 f/f ) and n=4 Tbx3-E2f7-E2f8 TKO (Alb-CreERT2; Tbx3 f/f ; E2f7 f/f ; E2f8 f/f ) livers at postnatal week 4. (C) Measurements of hepatocyte nuclear area in livers from (A). n=3 mice pre genotype. Bars indicate mean and standard deviation for all measured nuclei per genotype. Means of nuclear area were calculated for each animal. Means for each genotype were compared by one-way ANOVA. Ns= not significant (adjusted P=0.0626). Scale bars, 100μm. PI, propidium iodide.

Article Snippet: Gene Expression Assays used were Gapdh (Mm99999915_g1) as control, Tbx3 (Mm01195719_m1), E2f7 (Mm00618098_m1), and E2f8 (Mm01204165_g1).

Techniques: Membrane, Control, Immunofluorescence, Flow Cytometry, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Repression of TERRA Expression by Subtelomeric DNA Methylation Is Dependent on NRF1 Binding

doi: 10.3390/ijms20112791

Figure Lengend Snippet: The 29 bp repeats of the subtelomeric CpG islands are hypermethylated in HeLa cells. ( A ) Schematic representation of the subtelomeric region containing CpG island. Arrowheads indicate telomeric repeats, and blue box represents the subtelomeric 29 bp repeats. The subtelomeric telomere repeat containing RNA (TERRA) transcription start site (TSS) is indicated by a black arrow. The locations of the targets for RNA guides (sub1, sub2, and sub3) are indicated by green bars. The binding sites of the Reverse Primer (PR), Oligonucleotide Template (OT) (red), and the primer pairs used for the chromatin immunoprecipitation (ChIP) (ChIP-Foward/ChIP-Reverse (ChiP-F, ChiP-R)) and primers for the qPCR of TERRA, Foward (TF) and Reverse (TR) are indicated. Sequences amplified by these two latter primer pairs are indicated by dashed lines. ( B ) Schematic representation of the application of the method of analysis of the cytosine methylation at 29 bp repeats. In this example, two DNA molecules with two different patterns of cytosine methylation in the 29 bp repeats are represented (blue lines). Other 29 bp repeats are represented by dotted blue lines. (1) Genomic DNA is digested by the methylation-sensitive endonuclease HpaII (the HpaII recognition sites located within each 29 bp repeat are indicated by orange arrowheads). (2) After hybridization of the 3′ region of the oligonucleotide OT (red line) to the sequence adjacent to the HpaII recognition sites of the 29 bp repeats, DNA polymerase-catalyzed elongation (represented by dotted red lines) is performed in which the 5′ region of OT serves as template. OT is blocked at its 3′ end by dideoxycytidines to prevent its own elongation represented by crossed dotted red lines. (3) PCR is carried out with the Reverse Primer PR (black, complementary to a region upstream of the 29 bp repeats) and Forward Primer (PF) (purple, complementary to sequence added at the 3′ end of the cleaved DNA molecules). In this example, PCR products of two different sizes are obtained depending of the methylation pattern of the region. ( C ) Validation of the method was performed by analysis of 1 µg of genomic DNA extracted from HeLa cells untreated or treated with 5-aza-dC was undigested or digested by HpaII (methyl sensitive) or MspI (methyl insensitive). A sample without DNA (blank) was used as negative control. Elongation products (1/20 of the reaction) were amplified by PCR (35 PCR cycles). PCR products were run on a 1.5% BET-agarose gel. Ladder is the 50 bp DNA ladder (New England BioLabs).

Article Snippet: The fragment containing the coding sequence of TET1 served as template for two PCR with either assemb-TET1-F/TET1-mut-R oligonucleotides or assemb-TET1-R/TET1-mut-F oligonucleotides and using Phusion DNA polymerase (New England BioLabs).

Techniques: Binding Assay, Chromatin Immunoprecipitation, ChIP-chip, Amplification, Methylation, Hybridization, Sequencing, Negative Control, Agarose Gel Electrophoresis

Journal: Development (Cambridge, England)

Article Title: Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules

doi: 10.1242/dev.131664

Figure Lengend Snippet: β-catenin ChIP-seq analysis of Xenopus early gastrulae. (A) Experimental design of β-catenin ChIP-seq analysis. Early gastrulae were collected and fixed. Following chromatin shearing, β-catenin antibodies were used to selectively precipitate DNA fragments bound by β-catenin-containing protein complexes. Subsequently, the precipitated DNA fragments were sequenced. (B) Genome view of example β-catenin target gene hoxd1 . Note the clear β-catenin ChIP-seq peaks (β-peaks) downstream (to the left) of the hoxd1 locus. (C) Scatter plot combining peak calling analysis by SPP [considering signal strength, applying false discovery rate (FDR)≤0.1] and MACS2 (considering fold change, applying P ≤0.01) software, with black dots indicating 10,638 β-peaks reproducibly called [applying an irreproducible discovery rate (IDR)≤0.01]. (D,E) β-peaks are associated with sequences throughout the genome (D) but enriched close to and just upstream (putative promoter) of the transcriptional start site (TSS) of nearby genes (E; analysed in 500 bp bins). Pie chart (D) shows the percentage of β-peaks according to their location relative to TSS (within 1 kb, 1-5 kb, 5-10 kb, 10-50 kb, over 50 kb upstream or downstream of TSS). (F) Heat map illustrating genome-wide association of β-peaks with histone modifications and transcription co-factor binding sites indicative of cis-regulatory modules (CRMs; such as promoters and enhancers) in patterns that can be clustered into ten groups. Each horizontal line represents the 5 kb downstream and upstream regions of ChIP-seq data around a β-peak. (G) Enriched motifs from de novo motif search of sequences under β-peaks. Note the identification of consensus TCF/LEF binding but also other known transcription factor binding motifs. Statistical significance (e-values) and the number of β-peaks are indicated below each motif logo. The analysis of motif distribution shows central enrichment of motifs within β-peak regions (500 bp window).

Article Snippet: Two β-catenin antibodies, namely anti- Xenopus β-catenin antibody (1:28; ) and anti-β-catenin antibody [1:28 (2 μg); H-102; sc-7199, Santa Cruz Biotechnology], and normal rabbit IgG [1:56 (2 μg); sc-2027, Santa Cruz Biotechnology] were used for immunoprecipitation.

Techniques: ChIP-sequencing, Software, GWAS, Binding Assay

Journal: Development (Cambridge, England)

Article Title: Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules

doi: 10.1242/dev.131664

Figure Lengend Snippet: Integrating β-catenin ChIP-seq and RNA-seq analysis to identify direct Wnt8a/β-catenin target genes. (A) Venn diagram illustrating overlap between genes near β-peaks (red) and the wnt8a positively regulated genes (as in Fig. 2 B). Note that from among the longlist of 274 potential wnt8a -regulated genes, 179 are associated with identified β-peaks (amber border around lens-shaped area), representing the longlist of probable direct Wnt8a/β-catenin target genes. Also note that all but one ( xmcl2 ) of the validated shortlist of wnt8a positively regulated genes are among these and therefore represent the shortlist of 13 direct Wnt8a/β-catenin target genes (yellow). Also note that the majority of gene loci near β-peaks are not correlated with wnt8a -regulated genes and, conversely, that more than one-third of wnt8a -regulated genes in the longlist are not associated with identified β-peaks (most likely representing indirect wnt8a targets). (B) β-catenin ChIP-qPCR of identified β-peaks of our shortlist in chromatin extracted from control (uninjected) and wnt8a MO-injected embryos. Note that β-catenin association is reduced in the wnt8a loss-of-function experiment for most of the 15 β-peaks analysed. IgG antibodies were used as control. Error bars represent s.e.m. of three to five biological replicates. (C) Luciferase assays of reporter constructs containing sequences near identified β-peaks of wnt8a -regulated genes. Error bars represent s.d. of three biological replicates. (D) GO analysis suggests that β-peak-associated genes tend to encode transcription factors and also cell-to-cell signalling components, and to function in developmental processes, with different emphasis between wnt8a -regulated (purple and amber) and non-regulated genes (red). (E) DNA occupancy level of β - catenin around the peak summit shows higher enrichment in direct Wnt8a/β-catenin target gene loci [shortlist (purple) and longlist (amber)] compared with non- wnt8a -regulated genes (red). Read density was analysed using HOMER (bin size 100 bp). (F) TCF/LEF consensus motif is enriched under all 58 β-peaks associated with all 13 shortlisted Wnt8a/β-catenin target genes.

Article Snippet: Two β-catenin antibodies, namely anti- Xenopus β-catenin antibody (1:28; ) and anti-β-catenin antibody [1:28 (2 μg); H-102; sc-7199, Santa Cruz Biotechnology], and normal rabbit IgG [1:56 (2 μg); sc-2027, Santa Cruz Biotechnology] were used for immunoprecipitation.

Techniques: ChIP-sequencing, RNA Sequencing, ChIP-qPCR, Control, Injection, Luciferase, Construct

Journal: Development (Cambridge, England)

Article Title: Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules

doi: 10.1242/dev.131664

Figure Lengend Snippet: β-catenin recruitment is not sufficient for transcriptional regulation. (A,B) Maternally activated Wnt/β-catenin signalling regulates transcription of only context-specific maternal Wnt/β-catenin target genes. Experimental enhancement of maternal Wnt signalling, by injection of wnt8a mRNA at the two- to four-cell stage, increases expression of the maternal Wnt targets sia1 and nodal3.1 when analysed at the MBT, compared with the uninjected control (A). By contrast, expression levels of wnt8a target genes remain unchanged. However, β-catenin binding increases at both maternal Wnt target and zygotic wnt8a -regulated target loci at the 1000-cell stage (B). Note that the reduction of β-catenin binding following injection of axin mRNA indicates that maternally regulated endogenous β-catenin associates with not only maternal Wnt target genes but also zygotic wnt8a target genes. (C,D) Zygotically activated β-catenin controls the expression of only zygotic wnt8a targets. wnt8a MO or CSKA-wnt8a DNA were injected at the two- to four-cell stage and gene expression and β-catenin binding were analysed at the early gastrula stage. Knockdown of wnt8a reduces, and zygotic activation of Wnt8a signalling increases, expression of the wnt8a target hoxd1 , as a control. Whereas wnt8a knockdown or overexpression does not affect the expression of maternal Wnt-regulated genes (C), overactivation of Wnt8a signalling increases β-catenin binding to some maternal Wnt-regulated loci (D) but not to the well-characterised direct maternal Wnt target genes sia1 and nodal3.1 . Error bars indicate s.d. and s.e.m. of three biological replicates for RT-qPCR and ChIP-qPCR, respectively.

Article Snippet: Two β-catenin antibodies, namely anti- Xenopus β-catenin antibody (1:28; ) and anti-β-catenin antibody [1:28 (2 μg); H-102; sc-7199, Santa Cruz Biotechnology], and normal rabbit IgG [1:56 (2 μg); sc-2027, Santa Cruz Biotechnology] were used for immunoprecipitation.

Techniques: Injection, Expressing, Control, Binding Assay, Gene Expression, Knockdown, Activation Assay, Over Expression, Quantitative RT-PCR, ChIP-qPCR

Journal: Development (Cambridge, England)

Article Title: Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules

doi: 10.1242/dev.131664

Figure Lengend Snippet: BMP or FGF signalling is required for wnt8a target gene expression but not for β-catenin recruitment. (A) BMP signalling is required for context-specific transcriptional regulation by Wnt8a signalling, but only of some wnt8a target genes. Two- to four-cell stage embryos were injected with BMP antagonist noggin ( nog ) mRNA. CSKA-wnt8a DNA was injected additionally to reinstate Wnt8a expression (as endogenous wnt8a expression is itself regulated by BMP signalling). Expression was analysed by RT-qPCR at the early gastrula stage. When BMP signalling is blocked, expression of BMP-dependent genes remains reduced even when Wnt8a expression is reinstated. (B) FGF signalling is required for context-specific transcriptional regulation by Wnt8a signalling, but only of some wnt8a target genes. Embryos were treated with the FGFR inhibitor SU5402 from the 1000/2000-cell stage through the early gastrula and injected where indicated with CSKA-wnt8a DNA at the two- to four-cell stages (to reinstate Wnt8a expression, as endogenous wnt8a expression is itself regulated by FGF signalling). When FGF signalling is inhibited, expression of FGF-dependent genes remains reduced, even when Wnt8a expression is reinstated. (C) wnt8a targets can therefore be classified into BMP-dependent or FGF-dependent genes. Note that some genes belong to both groups and others are neither BMP nor FGF dependent. (D,E) In situ hybridisation shows expression of msx1 (D) and hoxd1 (E) in sagittal sections and lateral views (insets) of control uninjected and experimentally manipulated embryos as indicated (dorsal to the right). (F) BMP signalling is not required for wnt8a- regulated β-catenin recruitment to BMP-dependent wnt8a target gene loci. Embryos were treated as in A and analysed by β-catenin ChIP-qPCR at the early gastrula stage. (G) FGF signalling is not essential for wnt8a- regulated β-catenin recruitment to FGF-dependent wnt8a target gene loci. Embryos were treated as in B and analysed by β-catenin ChIP-qPCR at the early gastrula stage. Uninjected, untreated embryos were used as controls in A,B,D-G. * P <0.1, ** P <0.05; two-tailed Student's t -test. Error bars represent s.d. of four biological replicates (A,B) or s.e.m. of three biological replicates (F,G). Note that wnt8a gene expression itself was decreased by BMP or FGF pathway inhibition ( wnt8a blue bars in A,B) but restored by co-injection of CSKA-wnt8a DNA ( wnt8a orange bars in A,B) compared with controls ( wnt8a yellow bars in A,B), and that higher wnt8a expression levels with CSKA-wnt8a DNA ( wnt8a green bars in A,B) reflect both expression of endogenous wnt8a and expression from CSKA-wnt8a DNA, resulting in upregulation of several wnt8a target genes (in A,B).

Article Snippet: Two β-catenin antibodies, namely anti- Xenopus β-catenin antibody (1:28; ) and anti-β-catenin antibody [1:28 (2 μg); H-102; sc-7199, Santa Cruz Biotechnology], and normal rabbit IgG [1:56 (2 μg); sc-2027, Santa Cruz Biotechnology] were used for immunoprecipitation.

Techniques: Targeted Gene Expression, Injection, Expressing, Quantitative RT-PCR, In Situ, Hybridization, Control, ChIP-qPCR, Two Tailed Test, Gene Expression, Inhibition

Journal: Development (Cambridge, England)

Article Title: Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules

doi: 10.1242/dev.131664

Figure Lengend Snippet: Model for context-specific Wnt/β-catenin target gene regulation. (A) In the previous concept established from studies of individual genes, Wnt signalling specifically controls β-catenin recruitment to the Wnt-response element (WRE) of context-specific target genes and leads to their transcription (e.g. gene B in the context-X). (B) In the revised concept from our studies, Wnt-regulated β-catenin recruitment takes place at numerous loci. Transcriptional activation at those loci is conditional on context-specific mechanisms (e.g. a context-X-specific mechanism for gene B in context-X).

Article Snippet: Two β-catenin antibodies, namely anti- Xenopus β-catenin antibody (1:28; ) and anti-β-catenin antibody [1:28 (2 μg); H-102; sc-7199, Santa Cruz Biotechnology], and normal rabbit IgG [1:56 (2 μg); sc-2027, Santa Cruz Biotechnology] were used for immunoprecipitation.

Techniques: Activation Assay

Journal: Nature Communications

Article Title: The long non-coding RNA NEAT1 is a ΔNp63 target gene modulating epidermal differentiation

doi: 10.1038/s41467-023-39011-5

Figure Lengend Snippet: A Schematic illustration of the lncRNAs array-based approach utilized to identify lncRNAs regulated by p63. Briefly, the indicated primary (HEKn and hMEC) and cancer cell types (A253, FaDu, HCC1954) were transfected with scramble (SCR) or siRNA oligos targeting p63 mRNA (sip63), and the cDNA utilized for hybridization assay of a custom-made lncRNAs array. B Venn diagrams showing shared downregulated and upregulated lncRNAs in sip63-transfected cells. C Human primary keratinocytes (HEKn) were transfected with siRNA targeting p63 (sip63), ΔNp63 isoform (siΔNp63) or non-relevant mRNA (SCR). MALAT1 and NEAT1 RNA levels were quantified by RT-qPCR (left panel). Data shown are the mean of three ( n = 3) independent biological replicates ± SD. p value was calculated using two-tailed unpaired Student’s t test. In parallel, protein lysates from transfected cells were analyzed by western blotting using antibodies to the indicated proteins (right panel). D HEKn cells were transfected with siRNA targeting ΔNp63 isoform (siΔNp63) or non-relevant mRNA (SCR). NEAT1 long isoform (NEAT1_2) RNA levels were quantified by RT-qPCR. Data shown are the mean of three ( n = 3) independent biological replicates ± SD. p value was calculated using two-tailed unpaired Student’s t test. E ChIP-seq enrichment of endogenous p63 at MALAT1 and NEAT1 genomic loci in HEKn cells (GSM1446927). F ChIP-qPCR showing ΔNp63 occupancy at the p63 binding site of MALAT1 and NEAT1 genomic loci. Average values from n = 2 biological replicates measured using three technical replicates are plotted. G ChIP-qPCR showing endogenous HDAC1 occupancy at MALAT1 and NEAT1 genomic loci in HEKn cells. Average values from n = 2 biological replicates measured using three technical replicates are plotted. H ChIP-qPCR showing Histone H3 acetylated (H3ac) occupancy at MALAT1 and NEAT1 genomic loci in HEKn cells transfected with scramble (SCR) or siRNA oligo targeting p63 (sip63) (left panels). Average values from n = 2 biological replicates measured using three technical replicates are plotted. In parallel, protein lysates from transfected cells were analyzed by western blotting using antibodies to the indicated proteins (right panel). Source data are provided as a Source Data file.

Article Snippet: We utilized the following antibodies: rabbit monoclonal anti p63-α (Cell Signaling Technology, clone D2K8X, dilution: 1:1000), rabbit polyclonal anti‐KRT1 (BioLegend, PRB‐149P, dilution: 1:1000), rabbit polyclonal anti‐KRT10 (Covance PRB159P, dilution: 1:5000), rabbit polyclonal anti-ZNF750 (Sigma-Aldrich HPA023012, dilution: 1:200), rabbit polyclonal anti-PARP (Cell Signaling #9542, dilution: 1:1000) and mouse monoclonal anti β-actin (Sigma-Aldrich, #AC-15, dilution: 1:50000).

Techniques: Transfection, Hybridization, Quantitative RT-PCR, Two Tailed Test, Western Blot, ChIP-sequencing, ChIP-qPCR, Binding Assay

Journal: Nature Communications

Article Title: The long non-coding RNA NEAT1 is a ΔNp63 target gene modulating epidermal differentiation

doi: 10.1038/s41467-023-39011-5

Figure Lengend Snippet: Analysis of cell lysates ( A ) or total RNA ( B ) extracted from human primary keratinocytes (HEKn) at different time points (0, 3, 6 days) upon CaCl 2 treatment. The protein lysates were immunoblotted for the indicated proteins. MALAT1, NEAT1 and NEAT1_2 RNA levels were quantified by RT-qPCR. Data shown are the mean of three ( n = 3) independent biological replicates ± SD. p value was calculated using two-tailed unpaired Student’s t test. C MALAT1 and NEAT1 RNA-seq reads in proliferating keratinocytes (D0) or upon 7 days of CaCl 2 treatment (D7) (GSM1446880, GSM1446883). D RNA FISH of endogenous NEAT1 (magenta) and immunostaining of SFPQ (green) in proliferating (PROL) or differentiated (DIFF) HEKn cells. Nuclei were visualized by DAPI (blue) counterstaining. Scale bars, 25 µm. E Quantification of paraspeckle structures. Data shown are the mean of 170 nuclei ( n = 170) analysis ± SD. p value was calculated using two-tailed unpaired Student’s t test. The experiment was repeated twice with similar results ( n = 2). F Representative confocal images of MALAT1 and NEAT1 localization by RNA FISH in human skin tissue. Basal cells and nuclei were revealed by p63 immunostaining and DAPI counterstaining, respectively. Dotted white line demarcates the epidermal basement membrane. Scale bars, 50 µm. The experiment was repeated twice with similar results ( n = 2). Source data are provided as a Source Data file.

Article Snippet: We utilized the following antibodies: rabbit monoclonal anti p63-α (Cell Signaling Technology, clone D2K8X, dilution: 1:1000), rabbit polyclonal anti‐KRT1 (BioLegend, PRB‐149P, dilution: 1:1000), rabbit polyclonal anti‐KRT10 (Covance PRB159P, dilution: 1:5000), rabbit polyclonal anti-ZNF750 (Sigma-Aldrich HPA023012, dilution: 1:200), rabbit polyclonal anti-PARP (Cell Signaling #9542, dilution: 1:1000) and mouse monoclonal anti β-actin (Sigma-Aldrich, #AC-15, dilution: 1:50000).

Techniques: Quantitative RT-PCR, Two Tailed Test, RNA Sequencing, Immunostaining, Membrane

Journal: Nature Communications

Article Title: The long non-coding RNA NEAT1 is a ΔNp63 target gene modulating epidermal differentiation

doi: 10.1038/s41467-023-39011-5

Figure Lengend Snippet: A Volcano plots displaying gene expression Log2 fold changes and their respective statistical score ( p value) of negative LNA GapmeR control (NC) vs LNA GapmeR targeting MALAT1 (LNA-MALAT1) or NEAT1 (LNA-NEAT1) transfected differentiated HEKn cells. Differential expression analysis was carried out using DESeq2 package. Volcano plot was generated with Enhanced Volcano R package and statistical analyses were carried out using R software. P = FDR (False Discovery Rate) adjusted p value. B Venn diagram showing the shared downregulated genes and upregulated genes in LNA-NEAT1 or LNA-MALAT1 transfected HEKn cells. C Barplot showing the top gene ontology (GO) terms for Biological Process of the downregulated genes upon LNA-NEAT1 transfection of differentiated HEKn cells. GO terms were ordered by FDR (False Discovery Rate) adjusted p value calculated by ShinyGO version 0.76. D Heatmap showing the unsupervised hierarchical clustering of genes associated to the top GO terms in negative control (NC, green) or LNA-NEAT1 (violet) transfected differentiated HEKn cells. Color scheme: yellow (highest) blue (lowest) VSD score. E GSEA of genes downregulated in NEAT1 depleted keratinocytes against a calcium-differentiated keratinocyte gene set (GSE18590). F Bar plot showing the top GO DISEASE terms of genes downregulated in NEAT1 depleted keratinocytes. GO terms were ordered by FDR (False Discovery Rate) adjusted p value calculated by ShinyGO version 0.76. G GSEA of LNA-NEAT1 downregulated genes set against three keratinocytes differentiation signatures resulted by p63 (GSE33495), ZNF750 (GSE32685) or KLF4 (GSE32685) depletion. H IPA Upstream regulator analysis of NEAT1 profile RNA.

Article Snippet: We utilized the following antibodies: rabbit monoclonal anti p63-α (Cell Signaling Technology, clone D2K8X, dilution: 1:1000), rabbit polyclonal anti‐KRT1 (BioLegend, PRB‐149P, dilution: 1:1000), rabbit polyclonal anti‐KRT10 (Covance PRB159P, dilution: 1:5000), rabbit polyclonal anti-ZNF750 (Sigma-Aldrich HPA023012, dilution: 1:200), rabbit polyclonal anti-PARP (Cell Signaling #9542, dilution: 1:1000) and mouse monoclonal anti β-actin (Sigma-Aldrich, #AC-15, dilution: 1:50000).

Techniques: Gene Expression, Control, Transfection, Quantitative Proteomics, Generated, Software, Negative Control

Journal: Nature Communications

Article Title: The long non-coding RNA NEAT1 is a ΔNp63 target gene modulating epidermal differentiation

doi: 10.1038/s41467-023-39011-5

Figure Lengend Snippet: A Protein levels of epidermal differentiation markers, keratin 10 (K10), keratin 1 (K1) and ZNF750, were quantified by immunoblotting in differentiated keratinocytes transfected with negative LNA GapmeR control (NC), LNA GapmeR NEAT1 (LNA-NEAT1) or LNA GapmeR MALAT1 (LNA-MALAT1). B RNA levels of K10, p63 and NEAT1 were quantified by RT-qPCR in differentiated keratinocytes transfected with negative LNA GapmeR control (NC), LNA GapmeR NEAT1#1 (LNA-NEAT1#1) or LNA GapmeR NEAT1#2 (LNA-NEAT1#2). Data shown are the mean of three ( n = 3) independent biological replicates ± SD. p value was calculated using two-tailed unpaired Student’s t test. For ZNF750 mRNA levels quantification, average values from n = 2 biological replicates measured using three technical replicates are plotted. C Representative image of H&E staining of NEAT1-depleted (LNA-NEAT1) organotypic human epidermis (SKIN 3D) compared to control (NC) organotypic epidermis. The experiment was repeated twice with similar results ( n = 2). D Quantification of stratum corneum thickness in control (NC) and NEAT1-depleted organotypic human epidermis. Data shown are the mean of n = 59 (NC) and n = 29 (LNA-NEAT1#1) measurements ± SD. p value was calculated using two-tailed unpaired Student’s t test. E Quantification of Ki67 positive basal cells in control (NC) and NEAT1-depleted organotypic human epidermis. Data shown are the mean of n = 14 and n = 16 measurements ± SD for control and NEAT1 depleted epidermis, respectively. p value was calculated using two-tailed unpaired Student’s t test. F Representative images of the immunofluorescence analysis of control (NC) and NEAT1-depleted (LNA-NEAT1) organotypic skin cultures. Loricrin (LOR) and K10 are markers of differentiated layers, while keratin 14 (K14) and Laminin are markers of the basal layer. Immunofluorescence staining of p63 was used to confirm basal layer. DAPI (blue) was used to visualize nuclei. Dotted lines underline the keratinocyte-fibroblast border. Scale bars, 50 µm. The experiment was repeated twice with similar results ( n = 2). G Analysis of NEAT1 expression in lamellar Ichthyosis (GSE108640) and psoriasis (GSE13355) datasets. In the lamellar Ichthyosis dataset, NEAT1_2 expression value (DNA probe 227062_at) is shown as the mean ± SD of 14 ( n = 14, normal skin) and 6 ( n = 6 lamellar Ichthyosis lesions) samples. In the psoriasis dataset, NEAT1 expression value (DNA probe 224565_at) is shown as the mean ± SD of 64 ( n = 64, normal skin) and 58 ( n = 58, psoriatic lesions) samples. p value was calculated using two-tailed unpaired Student’s t test. H Schematic model of ΔNp63-NEAT1 axis function during keratinocytes differentiation. Source data are provided as a Source Data file.

Article Snippet: We utilized the following antibodies: rabbit monoclonal anti p63-α (Cell Signaling Technology, clone D2K8X, dilution: 1:1000), rabbit polyclonal anti‐KRT1 (BioLegend, PRB‐149P, dilution: 1:1000), rabbit polyclonal anti‐KRT10 (Covance PRB159P, dilution: 1:5000), rabbit polyclonal anti-ZNF750 (Sigma-Aldrich HPA023012, dilution: 1:200), rabbit polyclonal anti-PARP (Cell Signaling #9542, dilution: 1:1000) and mouse monoclonal anti β-actin (Sigma-Aldrich, #AC-15, dilution: 1:50000).

Techniques: Western Blot, Transfection, Control, Quantitative RT-PCR, Two Tailed Test, Staining, Immunofluorescence, Expressing