Journal: Development (Cambridge, England)

Article Title: Examining the NEUROG2 lineage and associated gene expression in human cortical organoids.

doi: 10.1242/dev.202703

Figure Lengend Snippet: Fig. 1. NEUROG1 and NEUROG2 expression in human fetal cortices. (A) Pseudo-bulk analysis of NEUROG1 and NEUROG2 transcript counts in scRNA-seq data collected from post-conception weeks (PCW) 5-14 human cortices (Braun et al., 2023), showing log2 counts per million (CPM). (B) Distribution of NEUROG1/NEUROG2 single and double-positive cells in scRNA-seq datasets from human fetal cortices between gestational week (GW) 8 and 26 (Zhong et al., 2018).

Article Snippet: We mined scRNA seq data in human COs available from GSE137877 (Sivitilli et al., 2020) and scRNA seq data of human fetal cortices was available from https://singlecell.broadinstitute.org/single_cell/study/ SCP1290/molecular-logic-of-cellular-diversification-in-the-mammalian-cerebralcortex (available in GEO under accession number GSE153164) and downloaded the human data from https://cellxgene.cziscience.com/collections/4d8fed08-2d6d4692-b5ea-464f1d072077.

Techniques: Expressing

Journal: Development (Cambridge, England)

Article Title: Examining the NEUROG2 lineage and associated gene expression in human cortical organoids.

doi: 10.1242/dev.202703

Figure Lengend Snippet: Fig. 7. NEUROG2 engages with PPP1R17-regulatory elements and is sufficient to induce PPP1R17 transcription. (A) Bar graph showing log2FC values of DEGs involved in neurogenesis in mCherry-high and in mCherry-low CO cells. (B) Pseudo-bulk analysis of NEUROG2 and PPP1R17 transcript counts in scRNA-seq data collected from PCW 5-14 human cortices (Braun et al., 2023), showing log2CPM. (C) Single-cell ATAC-seq profiling of the PPP1R17 locus, showing accessible chromatin in regions and cell types in the developing human brain. Conserved accessible chromatin regions were identified in an upstream enhancer (yellow box) and surrounding the TSS (green box). A primate-specific HAR (red box) is mainly accessible in glutamatergic cortical lineages. A phyloP score was derived from multiple mammalian species, with negative scores indicative of accelerated evolution for the PPP1R17- HAR element in chimps and rhesus monkeys. (D) qPCR to validate FACS-enrichment of PPP1R17 transcripts in mCherry-high versus mCherry-low cells (n=3 each). (E) NEUROG2 ChIP-qPCR (n=3), or mock control ChIP-qPCR (n=3), using day 45 COs. qPCR was used to quantify PPP1R17-HAR and -TSS binding sites in the eluted chromatin. (F) Transcriptional reporter assay in SHSY-5Y human neuroblastoma cells using pCIG2-Neurog2 or pCIG2-GFP (negative control) expression vectors and luciferase (LUC) constructs with a minimal promoter carrying the PPP1R17-HAR or -TSS elements. (G) Neurog2 gain-of-function assay, using AAV5-GFAP-iCre (control) and AAV5-GFAP-Neurog2-iCre to transduce day 90 COs (n=3 each). COs were harvested after 14 days and the expression of PPP1R17 was analyzed by qPCR. (H) NEUROG2 silencing in day 60 COs using lentiviral shRNA constructs, with a scrambled control sequence (shScr) or targeting NEUROG2 (-A and -C) (n=7 each). COs were harvested after 72 h and PPP1R17 expression was analyzed by qPCR. Graphs show mean±s.e.m. Unpaired Student’s t-tests were used for pairwise comparisons. Significance was defined as P<0.05.

Article Snippet: We mined scRNA seq data in human COs available from GSE137877 (Sivitilli et al., 2020) and scRNA seq data of human fetal cortices was available from https://singlecell.broadinstitute.org/single_cell/study/ SCP1290/molecular-logic-of-cellular-diversification-in-the-mammalian-cerebralcortex (available in GEO under accession number GSE153164) and downloaded the human data from https://cellxgene.cziscience.com/collections/4d8fed08-2d6d4692-b5ea-464f1d072077.

Techniques: Derivative Assay, ChIP-qPCR, Control, Binding Assay, Reporter Assay, Negative Control, Expressing, Luciferase, Construct, Functional Assay, Transduction, shRNA, Sequencing

Journal: Nature Communications

Article Title: Oncoprotein SET-associated transcription factor ZBTB11 triggers lung cancer metastasis

doi: 10.1038/s41467-024-45585-5

Figure Lengend Snippet: a Silver staining and mass spectrometry (MS) analysis of the protein complex purified from control- or SET-overexpressing stable H1299 cells identified ZBTB11. An empty vector (EV) or a Flag-HA-tagged SET (FH-SET)-expressing construct was stably transfected into H1299 cells, and the protein complex from the nuclear fraction of the indicated cells was tandemly purified by immobilized anti-Flag and anti-HA agarose. 7 unique out of 9 peptides corresponding to ZBTB11 were identified from the FH-SET-containing protein complex. b Coimmunoprecipitation (Co-IP)-Western blot (WB) analysis of the SET-ZBTB11 complex in H1299-EV or H1299-FH-SET cells purified by anti-Flag agarose. c Co-IP-WB analysis of the SET-ZBTB11 interaction in HEK293T cells transiently transfected with Myc-tagged SET (Myc-SET) with or without S protein-Flag-streptavidin-binding protein-tagged ZBTB11 (SFB-ZBTB11). Co-IP-WB analysis of the interaction between endogenous SET and ZBTB11 in H1299 cells by anti-SET ( d ) or anti-ZBTB11 ( e ) antibody. f WB analysis of ZBTB11 and SET in the cytoplasmic or nuclear fraction of H1299 cells. g Immunofluorescence assay of endogenous SET and ZBTB11 in H1299 cells. DAPI was used to counterstain the nucleus. h In vitro pull-down analysis of the direct binding between purified full-length SET and ZBTB11. In vitro pull-down analysis of the domain(s) of SET ( i ) or ZBTB11 ( j ) responsible for mediating their physical interaction. Source data are provided as a Source Data file. * indicates GST or GST-fusion protein.

Article Snippet: Antibodies: ZBTB11 (Bethyl, A303-240A; 2 μg for ChIP; 5 μg for ChIP-seq; 2 μg for Co-IP; 1:1000 for WB; 1:100 for IF; 1:100 for IHC); Rabbit IgG (Invitrogen, TF272445A; 2 μg for Co-IP; 5 μg for ChIP-seq); SET (Homemade; 2 μl for Co-IP; 10 μl for ChIP); SET (Bethyl, A302-262A; 1:1000 for WB; 1:100 for IHC); SET (Santa Cruz, sc-133138; 1:1000 for WB); SET (Sigma, WH0006418M1-100UG; 1:100 for IF); Flag (MBL, PM020; 1:5000 for WB); Myc (Santa Cruz, sc-40; 1:1000 for WB); HA (Roche, 11867423001; 1:2000 for WB); Vinculin (Sigma, V9131; 1:10000 for WB); YAP1 (Santa Cruz, sc-376830; 1:1000 for WB; 1:100 for IHC); MMP9 (CST, 13667 S; 1:1000 for WB); MMP9 (ABclonal, A11521; 1:100 for IHC); PRRG2 (Abcam, ab228870; 1:1000 for WB; 1:100 for IHC); p-YAP1-S127 (ABclonal, AP0489; 1:1000 for WB; 1:100 for IHC); p-YAP1-S397 (ABclonal, AP0922; 1:1000 for WB); β-actin (Proteintech, 60008-1-lg; 1:1000 for WB); HDAC1 (Santa Cruz, sc-81598; 1:1000 for WB); Collagen IV (NOVUS, NB120-6586S; 1:100 for IHC).

Techniques: Silver Staining, Mass Spectrometry, Purification, Control, Plasmid Preparation, Expressing, Construct, Stable Transfection, Transfection, Co-Immunoprecipitation Assay, Western Blot, Binding Assay, Immunofluorescence, In Vitro

Journal: Nature Communications

Article Title: Oncoprotein SET-associated transcription factor ZBTB11 triggers lung cancer metastasis

doi: 10.1038/s41467-024-45585-5

Figure Lengend Snippet: a WB analysis of ZBTB11 knockdown efficiency in H1299 cells transiently transfected with control siRNA (si-Ctr) or two individual siRNAs targeting ZBTB11 (si-ZBTB11) for 96 h. b Volcano plot revealing the differentially expressed genes upon ZBTB11 knockdown in H1299 cells ( n = 2 biologically independent samples). The p -values were determined by Wald test. c Pie-plot showing the category of the differentially expressed genes upon ZBTB11 knockdown in H1299 cells. d Distribution of ZBTB11 ChIP-seq reads and heatmap of binding signals around the 10-kb windows centered on the transcription start site (TSS) of genes. e The ZBTB11 binding motif discovered de novo from ZBTB11-high peaks in ChIP-seq. f Venn diagram of the genes with ZBTB11 enrichment analyzed by ChIP-seq. ChIP with normal IgG served as a negative control. g Venn diagram showing putative ZBTB11 direct target genes by combinational analyses of both RNA-seq and ChIP-seq datasets. h Heatmap of putative ZBTB11 direct target genes with upregulation or downregulation upon ZBTB11 depletion. i Gene Ontology (GO) analysis of the top 10 biological processes enriched by the differentially expressed putative ZBTB11 direct target genes. The p -value was determined by one-sided hypergeometric test. Source data are provided as a Source Data file.

Article Snippet: Antibodies: ZBTB11 (Bethyl, A303-240A; 2 μg for ChIP; 5 μg for ChIP-seq; 2 μg for Co-IP; 1:1000 for WB; 1:100 for IF; 1:100 for IHC); Rabbit IgG (Invitrogen, TF272445A; 2 μg for Co-IP; 5 μg for ChIP-seq); SET (Homemade; 2 μl for Co-IP; 10 μl for ChIP); SET (Bethyl, A302-262A; 1:1000 for WB; 1:100 for IHC); SET (Santa Cruz, sc-133138; 1:1000 for WB); SET (Sigma, WH0006418M1-100UG; 1:100 for IF); Flag (MBL, PM020; 1:5000 for WB); Myc (Santa Cruz, sc-40; 1:1000 for WB); HA (Roche, 11867423001; 1:2000 for WB); Vinculin (Sigma, V9131; 1:10000 for WB); YAP1 (Santa Cruz, sc-376830; 1:1000 for WB; 1:100 for IHC); MMP9 (CST, 13667 S; 1:1000 for WB); MMP9 (ABclonal, A11521; 1:100 for IHC); PRRG2 (Abcam, ab228870; 1:1000 for WB; 1:100 for IHC); p-YAP1-S127 (ABclonal, AP0489; 1:1000 for WB; 1:100 for IHC); p-YAP1-S397 (ABclonal, AP0922; 1:1000 for WB); β-actin (Proteintech, 60008-1-lg; 1:1000 for WB); HDAC1 (Santa Cruz, sc-81598; 1:1000 for WB); Collagen IV (NOVUS, NB120-6586S; 1:100 for IHC).

Techniques: Knockdown, Transfection, Control, ChIP-sequencing, Binding Assay, Negative Control, RNA Sequencing

Journal: Nature Communications

Article Title: Oncoprotein SET-associated transcription factor ZBTB11 triggers lung cancer metastasis

doi: 10.1038/s41467-024-45585-5

Figure Lengend Snippet: a WB analysis of SET knockdown efficiency in H1299 cells transiently transfected with control siRNA (si-Ctr) or siRNAs targeting SET (si-SET) for 96 h. b Volcano map of the differentially expressed genes upon SET knockdown in H1299 cells ( n = 2 biologically independent samples). The p -value was determined by Wald test. c Pie-plot showing the category of the differentially expressed genes upon SET knockdown in H1299 cells. d Venn diagram of the ZBTB11 target genes coregulated by SET. e Correlation analysis of SET/ZBTB11-regulated genes revealing that SET and ZBTB11 synergistically modulate transcription. Pearson’s correlation analysis was performed to calculate correlation coefficients and p -values. The genes related to extracellular matrix organization were highlighted with red. f GO analysis of the top 10 biological processes enriched by the SET-regulated ZBTB11 direct target genes. The p -values were determined by hypergeometric test. g RT–qPCR analysis of representative genes in H1299 cells with SET or ZBTB11 knockdown, individually or together. Data were shown as the mean ± S.E.M., n = 2 experimental replicates. Source data are provided as a Source Data file.

Article Snippet: Antibodies: ZBTB11 (Bethyl, A303-240A; 2 μg for ChIP; 5 μg for ChIP-seq; 2 μg for Co-IP; 1:1000 for WB; 1:100 for IF; 1:100 for IHC); Rabbit IgG (Invitrogen, TF272445A; 2 μg for Co-IP; 5 μg for ChIP-seq); SET (Homemade; 2 μl for Co-IP; 10 μl for ChIP); SET (Bethyl, A302-262A; 1:1000 for WB; 1:100 for IHC); SET (Santa Cruz, sc-133138; 1:1000 for WB); SET (Sigma, WH0006418M1-100UG; 1:100 for IF); Flag (MBL, PM020; 1:5000 for WB); Myc (Santa Cruz, sc-40; 1:1000 for WB); HA (Roche, 11867423001; 1:2000 for WB); Vinculin (Sigma, V9131; 1:10000 for WB); YAP1 (Santa Cruz, sc-376830; 1:1000 for WB; 1:100 for IHC); MMP9 (CST, 13667 S; 1:1000 for WB); MMP9 (ABclonal, A11521; 1:100 for IHC); PRRG2 (Abcam, ab228870; 1:1000 for WB; 1:100 for IHC); p-YAP1-S127 (ABclonal, AP0489; 1:1000 for WB; 1:100 for IHC); p-YAP1-S397 (ABclonal, AP0922; 1:1000 for WB); β-actin (Proteintech, 60008-1-lg; 1:1000 for WB); HDAC1 (Santa Cruz, sc-81598; 1:1000 for WB); Collagen IV (NOVUS, NB120-6586S; 1:100 for IHC).

Techniques: Knockdown, Transfection, Control, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Oncoprotein SET-associated transcription factor ZBTB11 triggers lung cancer metastasis

doi: 10.1038/s41467-024-45585-5

Figure Lengend Snippet: a WB analysis of ZBTB11 knockdown efficiency in H1299-EV or H1299-FH-SET stable cells transiently transfected with control siRNA (si-Ctr) or siRNA targeting ZBTB11 (si-ZBTB11) for 96 h. Cell migration ( b , c ) or invasion ( d , e ) of H1299-EV or H1299-FH-SET stable cells depleted with or without ZBTB11. Data were shown as the mean ± S.E.M., n = 3 biologically independent samples. The p -value was determined by two-sided t -test. f WB analysis of SET knockdown efficiency in H1299-EV or H1299-ZBTB11-SFB stable cells transiently transfected with control siRNA (si-Ctr) or siRNA targeting SET (si-SET) for 96 h. Cell migration ( g , h ) or invasion ( i , j ) of H1299-EV or H1299-ZBTB11-SFB cells depleted with or without SET. Data were shown as the mean ± S.E.M., n = 3 biologically independent samples. The p -value was determined by two-sided t -test. k WB analysis of SET and ZBTB11 knockdown efficiency in H1299 cells transiently transfected with control siRNA (si-Ctr) or siRNA against SET or ZBTB11 (si-SET or si-ZBTB11) for 96 h. Cell migration ( l , m ) or invasion ( n , o ) of H1299 cells upon SET and/or ZBTB11 depletion. Data were shown as the mean ± S.E.M., n = 3 biologically independent samples. The p -value was determined by two-sided t -test. p Schematic diagram (created with BioRender) of the workflow for analyzing tumor metastasis in vivo. q WB analysis of SET and ZBTB11 knockdown efficiency in H1299-Luc2-tdT-2 cells stably transfected with control shRNA (sh-Ctr) or shRNA targeting SET and/or ZBTB11 (sh-SET and/or sh-ZBTB11). r Bioluminescent image of lung metastasis from the primary tumors in a mouse model where H1299-Luc2-tdT-2 cells with or without SET/ZBTB11 knockdown were subcutaneously inoculated into the flanks of immunodeficient B-NDG (NSG) mice. s Quantitative analysis of the metastasis of cancer cells in the lung based on ( r ). Data were shown as the mean ± S.E.M., n = 5 mice per group. The p -value was determined by two-sided t -test. Source data are provided as a Source Data file.

Article Snippet: Antibodies: ZBTB11 (Bethyl, A303-240A; 2 μg for ChIP; 5 μg for ChIP-seq; 2 μg for Co-IP; 1:1000 for WB; 1:100 for IF; 1:100 for IHC); Rabbit IgG (Invitrogen, TF272445A; 2 μg for Co-IP; 5 μg for ChIP-seq); SET (Homemade; 2 μl for Co-IP; 10 μl for ChIP); SET (Bethyl, A302-262A; 1:1000 for WB; 1:100 for IHC); SET (Santa Cruz, sc-133138; 1:1000 for WB); SET (Sigma, WH0006418M1-100UG; 1:100 for IF); Flag (MBL, PM020; 1:5000 for WB); Myc (Santa Cruz, sc-40; 1:1000 for WB); HA (Roche, 11867423001; 1:2000 for WB); Vinculin (Sigma, V9131; 1:10000 for WB); YAP1 (Santa Cruz, sc-376830; 1:1000 for WB; 1:100 for IHC); MMP9 (CST, 13667 S; 1:1000 for WB); MMP9 (ABclonal, A11521; 1:100 for IHC); PRRG2 (Abcam, ab228870; 1:1000 for WB; 1:100 for IHC); p-YAP1-S127 (ABclonal, AP0489; 1:1000 for WB; 1:100 for IHC); p-YAP1-S397 (ABclonal, AP0922; 1:1000 for WB); β-actin (Proteintech, 60008-1-lg; 1:1000 for WB); HDAC1 (Santa Cruz, sc-81598; 1:1000 for WB); Collagen IV (NOVUS, NB120-6586S; 1:100 for IHC).

Techniques: Knockdown, Transfection, Control, Migration, In Vivo, Stable Transfection, shRNA

Journal: Nature Communications

Article Title: Oncoprotein SET-associated transcription factor ZBTB11 triggers lung cancer metastasis

doi: 10.1038/s41467-024-45585-5

Figure Lengend Snippet: a WB analysis of ZBTB11 in H1299-EV or H1299-MMP9-Flag stable cells upon endogenous ZBTB11 depletion. Cell migration ( b , c ) or invasion ( d , e ) of H1299-EV or H1299-MMP9-Flag stable cells upon ZBTB11 depletion (mean ± S.E.M., n = 3 biologically independent samples, two-sided t -test). f WB analysis of SET in H1299-EV or H1299-MMP9-Flag stable cells upon endogenous SET depletion. Cell migration ( g , h ) or invasion ( i , j ) of H1299-EV or H1299-MMP9-Flag stable cells upon SET depletion (mean ± S.E.M., n = 3 biologically independent samples, two-sided t -test). k ChIP–qPCR analysis of the enrichment of ZBTB11 and SET at MMP9 loci in H1299 cells (mean ± S.E.M., n = 3 experimental replicates, two-sided t -test). l Re-ChIP–qPCR analysis of SET binding to MMP9 loci following primary ChIP with anti-ZBTB11 antibody in H1299 cells (mean ± S.E.M., n = 3 experimental replicates, two-sided t -test). m ChIP–qPCR analysis of SET enrichment at MMP9 loci in H1299-ZBTB11-iKO cells upon doxycycline (Doxy) treatment (mean ± S.E.M., n = 3 experimental replicates, two-sided t -test). n ChIP–qPCR analysis of ZBTB11 enrichment at MMP9 loci in H1299-EV or H1299-FH-SET stable cells (mean ± S.E.M., n = 3 experimental replicates, two-sided t -test). o Luciferase assays of SET/ZBTB11-driven transcriptional of MMP9 . The EV or FH-ZBTB11 construct was transfected into H1299-EV or H1299-FH-SET stable cells, together with luciferase reporter and Renilla control vector, for 24 h (mean ± S.E.M., n = 3 experimental replicates, two-sided t -test). p Schematic diagram (created with BioRender) of the workflow for analyzing tumor metastasis in vivo. q WB analysis of ZBTB11 and MMP9 in H1299-Luc2-tdT-2 cells stably transfected with the ZBTB11-expressing construct (ZBTB11-OE) and/or shRNA targeting MMP9 (MMP9-KD). r Bioluminescent image of lung metastasis from the primary tumors in a mouse model where H1299-Luc2-tdT-2 cells with ZBTB11-OE/MMP9-KD were subcutaneously inoculated into the flanks of immunodeficient B-NDG (NSG) mice. s Quantitative analysis of the metastasis of cancer cells in the lung based on ( r ) (mean ± S.E.M., n = 4 mice per group, two-sided t -test). c , h shared the same si-Ctr group for quantitative analysis; ( e ) and ( j ) shared the same si-Ctr group for quantitative analysis. Source data are provided as a Source Data file.

Article Snippet: Antibodies: ZBTB11 (Bethyl, A303-240A; 2 μg for ChIP; 5 μg for ChIP-seq; 2 μg for Co-IP; 1:1000 for WB; 1:100 for IF; 1:100 for IHC); Rabbit IgG (Invitrogen, TF272445A; 2 μg for Co-IP; 5 μg for ChIP-seq); SET (Homemade; 2 μl for Co-IP; 10 μl for ChIP); SET (Bethyl, A302-262A; 1:1000 for WB; 1:100 for IHC); SET (Santa Cruz, sc-133138; 1:1000 for WB); SET (Sigma, WH0006418M1-100UG; 1:100 for IF); Flag (MBL, PM020; 1:5000 for WB); Myc (Santa Cruz, sc-40; 1:1000 for WB); HA (Roche, 11867423001; 1:2000 for WB); Vinculin (Sigma, V9131; 1:10000 for WB); YAP1 (Santa Cruz, sc-376830; 1:1000 for WB; 1:100 for IHC); MMP9 (CST, 13667 S; 1:1000 for WB); MMP9 (ABclonal, A11521; 1:100 for IHC); PRRG2 (Abcam, ab228870; 1:1000 for WB; 1:100 for IHC); p-YAP1-S127 (ABclonal, AP0489; 1:1000 for WB; 1:100 for IHC); p-YAP1-S397 (ABclonal, AP0922; 1:1000 for WB); β-actin (Proteintech, 60008-1-lg; 1:1000 for WB); HDAC1 (Santa Cruz, sc-81598; 1:1000 for WB); Collagen IV (NOVUS, NB120-6586S; 1:100 for IHC).

Techniques: Migration, ChIP-qPCR, Binding Assay, Luciferase, Construct, Transfection, Control, Plasmid Preparation, In Vivo, Stable Transfection, Expressing, shRNA

Journal: Nature Communications

Article Title: Oncoprotein SET-associated transcription factor ZBTB11 triggers lung cancer metastasis

doi: 10.1038/s41467-024-45585-5

Figure Lengend Snippet: a WB analysis of ectopic PRRG2 in H1299-EV or H1299-PRRG2-Flag stable cell lines. Cell migration ( b , c ) or invasion ( d , e ) of H1299-EV or H1299-PRRG2-Flag stable cells (mean ± S.E.M., n = 3 biologically independent samples, two-sided t -test). f RT–qPCR analysis of ZBTB11 or PRRG2 expression in H1299 cells depleted with or without ZBTB11 or PRRG2 for 96 h (mean ± S.E.M., n = 2 experimental replicates). Cell migration ( g , h ) or invasion ( i , j ) assays of H1299 cells with ZBTB11 or PRRG2 depletion, individually or together (mean ± S.E.M., n = 3 biologically independent samples, two-sided t -test). k ChIP-seq and ChIP–qPCR analysis of ZBTB11 enrichment on the PRRG2 promoter in H1299 cells (mean ± S.E.M., n = 2 experimental replicates). l Luciferase assays of ZBTB11-driven transcriptional regulation of PRRG2 (mean ± S.E.M., n = 2 experimental replicates). The luciferase reporter containing the ZBTB11-binding element of the PRRG2 promoter and Renilla control were transfected into the cells as indicated for 24 h. For ZBTB11 depletion, siRNA against ZBTB11 was used. For overexpression of ZBTB11, the H1299-ZBTB11-SFB stable cell line was used. The ZBTB11 knockdown efficiency or the expression of ectopic ZBTB11 was validated by WB assay. m Schematic diagram (created with BioRender) of the workflow for analyzing tumor metastasis in vivo. n WB analysis of ZBTB11 and PRRG2 in H1299-Luc2-tdT-2 cells stably knocked down with ZBTB11 (ZBTB11-KD) and/or PRRG2 (PRRG2-KD). o Bioluminescent image of lung metastasis from the primary tumors in a mouse model where H1299-Luc2-tdT-2 cells with ZBTB11-KD and/or PRRG2-KD were subcutaneously inoculated into the flanks of immunodeficient B-NDG (NSG) mice. p Quantitative analysis of the metastasis of cancer cells in the lung based on ( o ) (mean ± S.E.M., n = 5 mice per group, two-sided t -test). Source data are provided as a Source Data file.

Article Snippet: Antibodies: ZBTB11 (Bethyl, A303-240A; 2 μg for ChIP; 5 μg for ChIP-seq; 2 μg for Co-IP; 1:1000 for WB; 1:100 for IF; 1:100 for IHC); Rabbit IgG (Invitrogen, TF272445A; 2 μg for Co-IP; 5 μg for ChIP-seq); SET (Homemade; 2 μl for Co-IP; 10 μl for ChIP); SET (Bethyl, A302-262A; 1:1000 for WB; 1:100 for IHC); SET (Santa Cruz, sc-133138; 1:1000 for WB); SET (Sigma, WH0006418M1-100UG; 1:100 for IF); Flag (MBL, PM020; 1:5000 for WB); Myc (Santa Cruz, sc-40; 1:1000 for WB); HA (Roche, 11867423001; 1:2000 for WB); Vinculin (Sigma, V9131; 1:10000 for WB); YAP1 (Santa Cruz, sc-376830; 1:1000 for WB; 1:100 for IHC); MMP9 (CST, 13667 S; 1:1000 for WB); MMP9 (ABclonal, A11521; 1:100 for IHC); PRRG2 (Abcam, ab228870; 1:1000 for WB; 1:100 for IHC); p-YAP1-S127 (ABclonal, AP0489; 1:1000 for WB; 1:100 for IHC); p-YAP1-S397 (ABclonal, AP0922; 1:1000 for WB); β-actin (Proteintech, 60008-1-lg; 1:1000 for WB); HDAC1 (Santa Cruz, sc-81598; 1:1000 for WB); Collagen IV (NOVUS, NB120-6586S; 1:100 for IHC).

Techniques: Stable Transfection, Migration, Quantitative RT-PCR, Expressing, ChIP-sequencing, ChIP-qPCR, Luciferase, Binding Assay, Control, Transfection, Over Expression, Knockdown, In Vivo

Journal: Nature Communications

Article Title: Oncoprotein SET-associated transcription factor ZBTB11 triggers lung cancer metastasis

doi: 10.1038/s41467-024-45585-5

Figure Lengend Snippet: a Co-IP-WB assay of the interaction between YAP1 and PRRG2 in H1299 cells transfected with or without Myc-YAP1 and PRRG2-Flag, as indicated. b WB analysis of YAP1 knockdown in H1299-EV or H1299-PRRG2-Flag cells transfected with control siRNA (si-Ctr) or siRNA against YAP1 (si-YAP1) for 96 h. Cell migration ( c , d ) or invasion ( e , f ) of H1299-EV or H1299-PRRG2-Flag cells with or without YAP1 depletion (mean ± S.E.M., n = 3 biologically independent samples, two-sided t -test). g WB analysis of YAP1 phosphorylation in H1299-EV and H1299-PRRG2-Flag stable cells with the indicated antibodies. h WB analysis of YAP1 phosphorylation in H1299 cells with or without ZBTB11 and/or PRRG2 depletion, as indicated, for 48 h. i Schematic diagram of the workflow for analyzing tumor metastasis in vivo. j WB analysis of PRRG2 and YAP1 in H1299-Luc2-tdT-2 cells stably expressing PRRG2 (PRRG2-OE) and/or the YAP1-S127A construct (YAP1-S127A-OE). k Bioluminescent image of lung metastasis from the primary tumors in a mouse model where H1299-Luc2-tdT-2 cells with PRRG2-OE and/or YAP1-S127A-OE were subcutaneously inoculated into the flanks of immunodeficient B-NDG (NSG) mice. l Quantitative analysis of the metastasis of cancer cells in the lung based on ( k ) (mean ± S.E.M., n = 5 mice per group, two-sided t -test). m WB analysis of ZBTB11, PRRG2 and YAP1 in H1299-Luc2-tdT-2 cells stably expressing the ZBTB11 (ZBTB11-OE) and/or PRRG2 (PRRG2-OE) construct and/or depleted of YAP1 (YAP-KD), as indicated. n Bioluminescent image of lung metastasis from the primary tumors in a mouse model where H1299-Luc2-tdT-2 cells with ZBTB11-OE, PRRG2-OE and/or YAP1-KD were subcutaneously inoculated into the flanks of immunodeficient B-NDG (NSG) mice. o Quantitative analysis of the metastasis of cancer cells in the lung based on ( n ) (mean ± S.E.M., n = 5 mice per group, two-sided t -test). Source data are provided as a Source Data file.

Article Snippet: Antibodies: ZBTB11 (Bethyl, A303-240A; 2 μg for ChIP; 5 μg for ChIP-seq; 2 μg for Co-IP; 1:1000 for WB; 1:100 for IF; 1:100 for IHC); Rabbit IgG (Invitrogen, TF272445A; 2 μg for Co-IP; 5 μg for ChIP-seq); SET (Homemade; 2 μl for Co-IP; 10 μl for ChIP); SET (Bethyl, A302-262A; 1:1000 for WB; 1:100 for IHC); SET (Santa Cruz, sc-133138; 1:1000 for WB); SET (Sigma, WH0006418M1-100UG; 1:100 for IF); Flag (MBL, PM020; 1:5000 for WB); Myc (Santa Cruz, sc-40; 1:1000 for WB); HA (Roche, 11867423001; 1:2000 for WB); Vinculin (Sigma, V9131; 1:10000 for WB); YAP1 (Santa Cruz, sc-376830; 1:1000 for WB; 1:100 for IHC); MMP9 (CST, 13667 S; 1:1000 for WB); MMP9 (ABclonal, A11521; 1:100 for IHC); PRRG2 (Abcam, ab228870; 1:1000 for WB; 1:100 for IHC); p-YAP1-S127 (ABclonal, AP0489; 1:1000 for WB; 1:100 for IHC); p-YAP1-S397 (ABclonal, AP0922; 1:1000 for WB); β-actin (Proteintech, 60008-1-lg; 1:1000 for WB); HDAC1 (Santa Cruz, sc-81598; 1:1000 for WB); Collagen IV (NOVUS, NB120-6586S; 1:100 for IHC).

Techniques: Co-Immunoprecipitation Assay, Transfection, Knockdown, Control, Migration, Phospho-proteomics, In Vivo, Stable Transfection, Expressing, Construct

Journal: Nature Communications

Article Title: Oncoprotein SET-associated transcription factor ZBTB11 triggers lung cancer metastasis

doi: 10.1038/s41467-024-45585-5

Figure Lengend Snippet: a Schematic diagram (created with BioRender) of the strategy to establish a metastatic lung tumor mouse model. b IVIS of the primary lung tumors in KLLE and KLLE-Zbtb11 Fl/Fl mice at approximately 9 weeks after Ad-Cre inhalation. c Ex vivo bioluminescent assays of biopsied lung tissues derived from KLLE or KLLE-Zbtb11 Fl/Fl mice showing primary lung tumor formation. d Quantitative analysis of primary lung tumor formation in ( c ). Data were shown as boxplots with medians, interquartile ranges and lower/upper whiskers, n = 5 mice per group. The p -values were determined by two-way ANOVA. e Ex vivo bioluminescent assays of biopsied metastatic tumors in the liver, kidney, spleen and intestine derived from KLLE or KLLE-Zbtb11 Fl/Fl mice. Quantitative analysis of metastatic tumors in the liver ( f ), kidney ( g ), spleen ( h ) and intestine ( i ) based on ( e ). Data were shown as boxplots with medians, interquartile ranges and lower/upper whiskers, n = 5 mice per group. The p -values were determined by two-way ANOVA. j Overall survival (OS) analysis of KLLE or KLLE-Zbtb11 Fl/Fl mice with lung tumor onset induced by Ad-Cre virus inhalation. The p -value was determined by log-rank test. Source data are provided as a Source Data file.

Article Snippet: Antibodies: ZBTB11 (Bethyl, A303-240A; 2 μg for ChIP; 5 μg for ChIP-seq; 2 μg for Co-IP; 1:1000 for WB; 1:100 for IF; 1:100 for IHC); Rabbit IgG (Invitrogen, TF272445A; 2 μg for Co-IP; 5 μg for ChIP-seq); SET (Homemade; 2 μl for Co-IP; 10 μl for ChIP); SET (Bethyl, A302-262A; 1:1000 for WB; 1:100 for IHC); SET (Santa Cruz, sc-133138; 1:1000 for WB); SET (Sigma, WH0006418M1-100UG; 1:100 for IF); Flag (MBL, PM020; 1:5000 for WB); Myc (Santa Cruz, sc-40; 1:1000 for WB); HA (Roche, 11867423001; 1:2000 for WB); Vinculin (Sigma, V9131; 1:10000 for WB); YAP1 (Santa Cruz, sc-376830; 1:1000 for WB; 1:100 for IHC); MMP9 (CST, 13667 S; 1:1000 for WB); MMP9 (ABclonal, A11521; 1:100 for IHC); PRRG2 (Abcam, ab228870; 1:1000 for WB; 1:100 for IHC); p-YAP1-S127 (ABclonal, AP0489; 1:1000 for WB; 1:100 for IHC); p-YAP1-S397 (ABclonal, AP0922; 1:1000 for WB); β-actin (Proteintech, 60008-1-lg; 1:1000 for WB); HDAC1 (Santa Cruz, sc-81598; 1:1000 for WB); Collagen IV (NOVUS, NB120-6586S; 1:100 for IHC).

Techniques: Ex Vivo, Derivative Assay, Virus

Journal: Nature Communications

Article Title: Oncoprotein SET-associated transcription factor ZBTB11 triggers lung cancer metastasis

doi: 10.1038/s41467-024-45585-5

Figure Lengend Snippet: a The positive correlation of high expression of both ZBTB11 and SET with LUAD based on the TCGA database . b The relationship of ZBTB11 and SET expression with the stages of LUAD based on the TCGA database . c The relationship of ZBTB11 and SET expression with the lymph node metastatic status of LUAD based on the TCGA database . N0: no regional lymph node metastasis; N1: 1–3 axillary lymph node metastases; N2: 4–9 axillary lymph node metastases; N3: ≥10 axillary lymph node metastases. Data were shown as boxplots with medians, interquartile ranges and lower/upper whiskers in ( a – c ). The p -values were determined by two-sided t -test. d The positive correlation of the expression between ZBTB11 and SET in lung tissues based on TCGA database . Pearson’s correlation analysis was performed to determine correlation coefficients and p -values. The gray band represents the 95% confidence interval band. e Representative and quantitative IHC of ZBTB11 and SET in LUAD tissue arrays containing primary lung tumors with paired adjacent normal lung tissues and long-distance metastatic tumors from primary LUAD. Data were shown as mean±S.E.M. The p -values were determined by one-sided t -test. f The positive correlation of the expression between ZBTB11 and SET in lung tissue arrays. Pearson’s correlation analysis was performed to determined correlation coefficients and p -values. The gray band represents the 95% confidence interval band. g Kaplan–Meier plots of lung cancer patients stratified by ZBTB11 , SET , MMP9, and PRRG2 expression levels, based on GEO dataset (GSE30219) . The p -values were determined by log-rank test. Source data are provided as a Source Data file.

Article Snippet: Antibodies: ZBTB11 (Bethyl, A303-240A; 2 μg for ChIP; 5 μg for ChIP-seq; 2 μg for Co-IP; 1:1000 for WB; 1:100 for IF; 1:100 for IHC); Rabbit IgG (Invitrogen, TF272445A; 2 μg for Co-IP; 5 μg for ChIP-seq); SET (Homemade; 2 μl for Co-IP; 10 μl for ChIP); SET (Bethyl, A302-262A; 1:1000 for WB; 1:100 for IHC); SET (Santa Cruz, sc-133138; 1:1000 for WB); SET (Sigma, WH0006418M1-100UG; 1:100 for IF); Flag (MBL, PM020; 1:5000 for WB); Myc (Santa Cruz, sc-40; 1:1000 for WB); HA (Roche, 11867423001; 1:2000 for WB); Vinculin (Sigma, V9131; 1:10000 for WB); YAP1 (Santa Cruz, sc-376830; 1:1000 for WB; 1:100 for IHC); MMP9 (CST, 13667 S; 1:1000 for WB); MMP9 (ABclonal, A11521; 1:100 for IHC); PRRG2 (Abcam, ab228870; 1:1000 for WB; 1:100 for IHC); p-YAP1-S127 (ABclonal, AP0489; 1:1000 for WB; 1:100 for IHC); p-YAP1-S397 (ABclonal, AP0922; 1:1000 for WB); β-actin (Proteintech, 60008-1-lg; 1:1000 for WB); HDAC1 (Santa Cruz, sc-81598; 1:1000 for WB); Collagen IV (NOVUS, NB120-6586S; 1:100 for IHC).

Techniques: Expressing

Journal: Cell

Article Title: Exit from Pluripotency Is Gated by Intracellular Redistribution of the bHLH Transcription Factor Tfe3

doi: 10.1016/j.cell.2013.03.012

Figure Lengend Snippet: Genome-wide Tfe3-Target Determination Identifies Esrrb as a Downstream Effector of Flcn-Fnip1/2-Tfe3 (A) Gene tracks of loci identified by Tfe3 ChIP-Seq with control (ctrl.2), Flcn shRNA (Flcn.4), and Tfe3 shRNA (Tfe3.3) cell lines, overlaid with Nanog-, Oct4- and Tcf3-bound regions. (B) ESCs expressing indicated constructs were treated for 3 hr with 0.1 μM Tam in 2i. Average mRNA fold changes relative to EtOH treatment and SD are from two independent experiments with three different cell lines per genotype. ( ∗ ) indicates Student’s t test values < 0.005. (C) Average mRNA changes in ESCs transfected with the indicated siRNA combinations. Average relative expression normalized to no and negative siRNA treatments and SD are of three independent experiments. ( ∗ ) indicates Student’s t test values < 0.05. (D) O4GIP ESCs expressing Tfe3-ERT2 were transfected with indicated siRNAs, differentiated for 3 days in the presence of 0.1 μM Tam or EtOH, and switched back to 2i with puromycin selection, and remaining ESC colonies were quantified with a cell-viability assay. Average fold changes over negative siRNA-transfected, EtOH-treated cells and SD are from two independent experiments. ( ∗∗ ) and ( ∗ ) indicate Student’s t test values < 0.001 and 0.03, respectively. (E) Commitment of O4GIP cells transfected with indicated siRNA combinations, including two independent Esrrb siRNAs. Exit from pluripotency was quantified with a cell-viability assay and normalized to negative siRNA treatment. Average and SD are of two technical replicates. See also Figure S6 .

Article Snippet: ChiP-Seq libraries were generated using NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (NEB) using 10 amplification cycles.

Techniques: Genome Wide, ChIP-sequencing, shRNA, Expressing, Construct, Transfection, Selection, Viability Assay

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. wtEGFR and EGFRvIII bands are marked with * and **, respectively. ( b ) Densitometric quantification of galectin1 protein level normalized to tubulin in different BTSC lines is shown. ( c-d ) EGFR / EGFRvIII KD (si EGFR ) and control BTSCs (siCTL) were analyzed by immunoblotting as described in a. ( e-h ) BTSCs were treated with 1 or 5 µM lapatinib and galectin1 expression was assessed by immunoblotting (e-f) and immunostaining (g-h). Nuclei were stained with DAPI. Scale bar = 10 μm. ( i ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. ( j ) Pearson correlation analysis of pSTAT3-Y705 and galectin1 protein expression in different BTSCs is shown. ( k-l ) STAT3 KD (si STAT3 ) and siCTL BTSCs were analyzed by immunoblotting as described above. ( m-p ) BTSCs were subjected to immunoblotting or immunostaining following treatment with 25 or 50 µM of the STAT3 inhibitor, S3I-201. Scale bar = 10 μm. ( q-s ) EGFRvIII-expressing BTSCs were subjected to ChIP using an antibody to STAT3 or IgG control followed by qPCR using two different pairs of primers ( LGALS1 -a and LGALS1 -b). OSMR , and HPRT loci were used as positive and negative controls, respectively. ( t-u ) Luciferase reporter assay was performed in BTSC73 following KD of STAT3 using siRNA (t) or treatment with STAT3 inhibitors, 5 µM WP1066 or 50 μM S3I-201 (u). Data are presented as the mean□±□SEM, n ≥ 3. Unpaired two-tailed t -test (q, r and s); one-way ANOVA followed by Dunnett’s test (b) or Tukey’s test (t and u),*p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S1 and S2.

Article Snippet: Second, the transgenic LGALS1 KD BTSCs were generated via lentivirus carrying two different LGALS1 shRNA plasmids (OriGene, #TL311756).

Techniques: Western Blot, Expressing, Immunostaining, Staining, Luciferase, Reporter Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) Cell viability was assessed by CellTiter-Glo assay in LGALS1 CRISPR and CTL BTSCs. ( c ) Population growth curves for LGALS1 CRISPR and CTL BTSC73 are shown. ( d-f ) Cell viability assay (d-e) and population growth curves (f) of BTSC73 treated with 1 or 10 µM OTX008 are shown. ( g ) Representative images of EdU staining in LGALS1 CRISPR and CTL BTSC73 are shown. ( h ) The number of EdU positive cells was quantified using Fiji software. ( i ) EdU incorporation was analyzed by flow cytometry in LGALS1 CRISPR and CTL BTSC73. Representative scatter plots of flow cytometry analyses are shown. Data are presented as the mean□±□SEM, n = 3. Unpaired two-tailed t -test (a, b, c and h); one-way ANOVA followed by Dunnett’s test (d, e and f), **p < 0.01, ***p < 0.001. See also Figures S3 and S4.

Article Snippet: Second, the transgenic LGALS1 KD BTSCs were generated via lentivirus carrying two different LGALS1 shRNA plasmids (OriGene, #TL311756).

Techniques: Glo Assay, CRISPR, Viability Assay, Staining, Software, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) LGALS1 CRISPR or CTL BTSC73 were subcutaneously injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (a). Graph represents tumour mass (b). ( c-f ) BTSC73 or BTSC147 were injected subcutaneously into SCID mice and treated with 10 mg/kg OTX008. Representative bioluminescence real-time images tracing tumour growth are shown (c, e). Graphs represent tumour mass (d, f). ( g-j ) LGALS1 CRISPR or CTL BTSC73 were intracranially injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (g). Intensities of luciferase signal were quantified at different time points using Xenogen IVIS software (h). Graph represents quantification of animal weight (i). KM survival plot was graphed to evaluate mice lifespan in each group (j). Data are presented as the mean□±μSEM, n ≥ 4 mice. Unpaired two-tailed t -test (b, d, f, h and i); log-rank test (j), **p < 0.01, ***p < 0.001.

Article Snippet: Second, the transgenic LGALS1 KD BTSCs were generated via lentivirus carrying two different LGALS1 shRNA plasmids (OriGene, #TL311756).

Techniques: CRISPR, Injection, Luciferase, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) Volcano plot representing LGALS1 differentially regulated genes is shown. ( b-c ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal (b) and proneural (c) subtypes of glioblastoma. ( d ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal-like meta-module (MES1-like) signature. ( e-f ) GSEA analysis demonstrates enrichment for gene sets corresponding to recruitment of NuMA to mitotic centrosomes (e) and mitotic G2−G2/M phases (f). ( g-h ) RNA-seq data was validated by RT-qPCR in BTSC73 and BTSC147. ( i-j ) Cell cycle distribution was assessed by flow cytometry after PI staining in LGALS1 CRISPR BTSCs. Data are presented as the mean□±□SEM, n = 3. One-way ANOVA followed by Dunnett’s test (g and h); unpaired two- tailed t -test (i and j), *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.

Article Snippet: Second, the transgenic LGALS1 KD BTSCs were generated via lentivirus carrying two different LGALS1 shRNA plasmids (OriGene, #TL311756).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Flow Cytometry, Staining, CRISPR, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-d ) LGALS1 CRISPR and CTL EGFRvIII-expressing BTSCs were subjected to LDA (a-b) or ELDA (c-d). ( e-f ) EGFRvIII-expressing LGALS1 CRISPR and CTL BTSCs were subjected to clonogenicity assay performed by culturing one single cell per well. ( g-h ) BTSCs that don’t harbour the EGFRvIII mutation were electroporated with siCTL or si LGALS1 and subjected for ELDA analysis. ( i-p ) EGFRvIII-expressing BTSCs were subjected to LDA (i, j, m and n) or ELDA (k, l, o and p) following the treatment with 1 or 10 µM OTX008. ( q-t ) BTSCs that don’t harbour the EGFRvIII mutation were subjected to LDA (q-r) or ELDA (s-t) following the treatment with 1 or 10 µM OTX008. *p < 0.05, **p < 0.01, ***p < 0.001; unpaired two-tailed t -test (a, b, e and f); one-way ANOVA followed by Dunnett’s test (i, j, m and n), n = 3. Data are presented as the mean□±□SEM. See also Figure S6.

Article Snippet: Second, the transgenic LGALS1 KD BTSCs were generated via lentivirus carrying two different LGALS1 shRNA plasmids (OriGene, #TL311756).

Techniques: CRISPR, Expressing, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) ELDA was performed following 4 Gy of IR in LGALS1 CRISPR or CTL BTSCs. ( b-c ) LGALS1 CRISPR and CTL BTSC73 were subjected to IR (8□Gy). Apoptosis analysis was performed by flow cytometry 48□h following IR using annexin V and PI double staining. Representative scatter plots of flow cytometry analyses are shown (b). The percentage of cell death (annexin V positive cells) is presented in the histogram (c), n□=□3. ( d ) Schematic diagram of the experimental procedure is shown. BTSC73 were intracranially injected into SCID mice and then treated with OTX008, 4□Gy of IR or a combination of OTX008 and IR. ( e ) Representative bioluminescence real-time images tracing tumour growth are shown, n□=□6 mice. ( f ) Coronal sections of mouse brains were stained with hematoxylin and eosin on day 22 after injection. Representative images of 3 different tumour sections are shown. Scale bar = 1□mm, scale bar (inset) = 0.2 mm. ( g ) Intensities of luciferase signal were quantified at different time points, n = 6 mice. ( h ) KM survival plot was graphed to assess animal lifespan, n□=□6 mice. ( i ) Survival extension of mice bearing BTSC-derived tumours treated with OTX008, IR, or OTX008 + IR relative to those treated with the vehicle control. Data are presented as the mean□±□SEM. One-way ANOVA followed by Tukey’s test (c and i); log-rank test (h), *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Second, the transgenic LGALS1 KD BTSCs were generated via lentivirus carrying two different LGALS1 shRNA plasmids (OriGene, #TL311756).

Techniques: CRISPR, Flow Cytometry, Double Staining, Injection, Staining, Luciferase, Derivative Assay

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) LGALS1 -differentially regulated genes were subjected to enrichment analysis of TF binding motifs using oPOSSUM-3 software. ( b ) Volcano plot representing the HOXA5 target genes among the LGALS1 -differentially-regulated genes is shown. ( c ) BTSCs were analyzed by immunoblotting using the antibodies indicated on the blots. ( d ) Pearson correlation analysis of HOXA5 and galectin1 protein expression is shown. ( e ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients is shown. ( f ) Relative positions of HOXA5 ChIP-seq peaks to the adjacent TSS of LGALS1 -differentially regulated genes are shown. The x-axis indicates the distance between peak centers and the TSS of adjacent LGALS1 -differentially regulated genes. The y-axis denotes the expression ratios (log2) of the LGALS1 -differentially regulated gene. Circle size indicates HOXA5 peak height, and color denotes the conservation score of HOXA5 peaks. ( g-h ) HOXA5 KD (si HOXA5 ) and siCTL BTSCs were subjected to RT-qPCR analysis. ( i ) ELDA was performed following 4LGy of IR in si HOXA5 vs. siCTL. ( j - m ) Endogenous Co-IP experiments were performed in different BTSC lines using an anti-HOXA5 antibody, followed by immunoblotting with galectin1 and HOXA5 antibodies. ( n ) Co-IP experiment was performed using anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-HOXA5 antibodies. ( o - r ) PLA of galectin1 and HOXA5 were performed in different BTSC lines. Primary antibodies were omitted for the controls. Nuclei were stained with DAPI. Scale bar = 10 μm. ( s ) LGALS1 CRISPR and CTL BTSC73 were subjected to ChIP using an antibody to HOXA5 followed by qPCR for HOXA5 candidate target genes. HBB locus was used as a negative control. ( t-u ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients treated with radiotherapy (microarray G4502A Agilent, level 3, n = 489). Data are presented as the meanL±LSEM, n = 3. Log-rank test (e, t and u); one-way ANOVA followed by Dunnett’s test (g and h); unpaired two-tailed t -test (s). *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S7.

Article Snippet: Second, the transgenic LGALS1 KD BTSCs were generated via lentivirus carrying two different LGALS1 shRNA plasmids (OriGene, #TL311756).

Techniques: Binding Assay, Software, Western Blot, Expressing, ChIP-sequencing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Staining, CRISPR, Negative Control, Microarray, Two Tailed Test

Journal: Nature Communications

Article Title: EPOP restricts PRC2.1 targeting to chromatin by directly modulating enzyme complex dimerization

doi: 10.1038/s41467-025-68280-5

Figure Lengend Snippet: a Re-expression of WT and mutant EPOP in mESCs. Representative of two biological replicates . b Co-IP assay of re-expressed EPOP. The re-expressed 3 × FLAG-EPOP-HA protein was used as the bait, and the bound endogenous SUZ12 was detected. Representative of two replicates. c Schematic of the EpiLC differentiation. Some parts of the figure were created in BioRender. Liu, X. (2026) ( https://biorender.com/gvraoqv ). d Heatmaps of ChIP-seq replicates. Using the FDR < 0.05 threshold, the gain-of-signal MTF2 (purple) and H3K27me3 (green) peaks from individual replicates (WT-1, WT-2, D5-1, and D5-2) are shown in the heatmaps. The centers of the consensus binding sites are aligned. The number of the consensus binding sites is labeled. e Metaplots of ChIP-seq replicates. The mean ChIP-seq signal of the differential MTF2 (upper panel) and H3K27me3 (lower panel) peaks shown in ( d ) is plotted. The individual replicates are color-coded. f Genome browser tracks of selected gene loci. Tracks were generated by SparK ( https://github.com/harbourlab/SparK ). Genomic coordinates are provided. The difference between the replicates, calculated as the standard deviation, is indicated as the shaded areas surrounding the tracks. g Scatter plots of MTF2 and H3K27me3. MTF2 (left panel) and H3K27me3 (right panel) reads were normalized by the total reads. The mean read concentration corresponding to log2 (normalized ChIP-seq reads with input reads subtracted) was calculated by DiffBind.

Article Snippet: For the shRNA KD ChIP-seq data sequenced by Novogene, the raw sequencing reads were trimmed by Cutadapt and then mapped by Bowtie 2 with the “--very-sensitive” parameter.

Techniques: Expressing, Mutagenesis, Co-Immunoprecipitation Assay, ChIP-sequencing, Binding Assay, Labeling, Generated, Standard Deviation, Concentration Assay

Journal: Nature Communications

Article Title: EPOP restricts PRC2.1 targeting to chromatin by directly modulating enzyme complex dimerization

doi: 10.1038/s41467-025-68280-5

Figure Lengend Snippet: a Heatmaps of ChIP-seq replicates. The differential MTF2 peaks between the EPOP WT and EPOP D5 EpiLCs were grouped into three categories based on the response to shRNA KD, using the FDR < 0.05 threshold. Differential peaks shared by the control and Elongin B KD are labeled as n1, differential peaks unique to the control KD are labeled as n2, and differential peaks unique to the Elongin B KD are labeled as n3. b Genome browser tracks of selected gene loci. Tracks were generated by SparK. Two gene loci associated with the shared differential MTF2 peaks between the control KD and the Elongin B KD are shown. c Venn diagram of the differential MTF2 peaks in the three categories.

Article Snippet: For the shRNA KD ChIP-seq data sequenced by Novogene, the raw sequencing reads were trimmed by Cutadapt and then mapped by Bowtie 2 with the “--very-sensitive” parameter.

Techniques: ChIP-sequencing, shRNA, Control, Labeling, Generated

Journal: Nature Communications

Article Title: EPOP restricts PRC2.1 targeting to chromatin by directly modulating enzyme complex dimerization

doi: 10.1038/s41467-025-68280-5

Figure Lengend Snippet: a Volcano plot of differential gene expression. RNA-seq results of the EPOP D5 EpiLCs in triplicates were compared to those of the EPOP WT EpiLCs. The number of upregulated and downregulated genes is indicated. Data passing the FDR < 0.05, FC > 1.5, and average TPM of WT or mutant > 0.5 thresholds were analyzed. b Correlation of RNA-seq and ChIP-seq. One ChIP-seq replicate is shown here, and the other replicate is shown in the supplemental materials. The differential gene expression was aligned with the differential MTF2 enrichment around the transcription start site (TSS). The corresponding H3K27me3 signals are also displayed. Compared to the WT counterpart, 130 genes in the EPOP D5 EpiLCs were downregulated and associated with enhanced MTF2 signals around the TSS. The other 318 downregulated genes were not associated with EPOP-regulated MTF2 targeting. 187 upregulated genes are shown as well. c Gene ontology analysis. The PRC2.1-repressed, EPOP-maintained genes were subjected to gene ontology analysis on the DAVID server. The top 5 overrepresented terms in molecular function are shown. The p -value is one-tail Fisher Exact probability value used for gene-enrichment analysis by the DAVID server. d Schematic model of developmental gene repression by PRC2. On the left, the transient intrinsic dimer of the PRC2 core complex is illustrated. In the middle, distinct oligomerization states of various PRC2.1 and PRC2.2 holocomplexes are highlighted. MTF2 mediates direct chromatin binding, and it also stabilizes the intrinsic dimer, promoting chromatin targeting of the dimeric PRC2.1, likely via an avidity effect. EPOP disrupts the dimeric architecture of PRC2.1, containing MTF2, restricts PRC2.1 targeting, and thereby maintains the limited expression of PRC2.1-repressed developmental regulators. On the right, the PRC2.1-dependent role of EPOP in early development is illustrated. Black solid curve: during the ESC differentiation, a set of key gene regulators is repressed by PRC2.1, with limited expression being maintained by the EPOP-mediated inhibition of PRC2.1 targeting, which is followed by upregulation of the same set of gene regulators, leading to cell fate 1, e.g., PGCLCs. Gray dotted curve: the absence of EPOP results in the over-repression of these gene regulators by PRC2.1, which may change stem cell differentiation trajectories and result in an alternative cell fate 2. Some parts of the figure were created in BioRender. Liu, X. (2026) ( https://biorender.com/l3ji249 ).

Article Snippet: For the shRNA KD ChIP-seq data sequenced by Novogene, the raw sequencing reads were trimmed by Cutadapt and then mapped by Bowtie 2 with the “--very-sensitive” parameter.

Techniques: Gene Expression, RNA Sequencing, Mutagenesis, ChIP-sequencing, Binding Assay, Expressing, Inhibition, Cell Differentiation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: E3 Ubiquitin Ligases Cbl-b Regulates Thymic- Derived CD4 + CD25 + Regulatory T Cell Development by Targeting Foxp3 for Ubiquitination

doi: 10.4049/jimmunol.1402434

Figure Lengend Snippet: Cbl-b inducibly associates with Foxp3. (A) BALB/c T cells were stimulated with anti-CD3 and anti-CD28 for 15 min or for 15 and 30 min, and lysed in 0.5% NP-40 lysis buffer. The cell lysates were immunoprecipitated with anti-Foxp3, and blotted with anti-Cbl-b (upper panel) or anti-Stub1 (lower panel). The cell lysates from the unstimulated sample were used as a positive control. The cell lysates immunoprecipitated with rabbit IgG (IgG) were used as a negative control. (B) Schematic design of Cbl-b mutant constructs. (C) HEK293T cells were transfected with HA-tagged Cbl-b, Cbl-b N1/3, Cbl-b C2/3, Cbl-b ΔUBA, together with Flag-tagged Foxp3, and lysed. The cell lysates were immunoprecipitated with anti-Flag, and blotted with anti-HA. (D) Naïve CD4+CD25+ T cells from BALB/c mice were nucleofected with siRNA specific for Stub1, stimulated with anti-CD3 and anti-CD28, and lysed. The cell lysates were immunoprecipitated with anti-Foxp3 and blotted with anti-Cbl-b. The data shown are one representative of two independent experiments.

Article Snippet: GFP-tagged Stub1 plasmid was obtained from OriGene (Rockville, MD).

Techniques: Lysis, Immunoprecipitation, Positive Control, Negative Control, Mutagenesis, Construct, Transfection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: E3 Ubiquitin Ligases Cbl-b Regulates Thymic- Derived CD4 + CD25 + Regulatory T Cell Development by Targeting Foxp3 for Ubiquitination

doi: 10.4049/jimmunol.1402434

Figure Lengend Snippet: Stub1 and Cbl-b sequentially ubiquitinates Foxp3 upon TCR/CD28 stimulation. (A) CD4+CD25+ T cells from WT mice were stimulated with anti-CD3 and anti-CD28, or left unstimulated, and lysed in RIPA buffer. The cell lysates were immunoprecipitated with anti-Foxp3, and blotted with anti-ubiquitin. (B) HEK293T cells were transfected with HA-tagged Cbl-b or Cbl-b C373A, Flag-tagged Foxp3, and His-tagged ubiquitin. The Cell lysates were immunoprecipitated with anti-Flag, and blotted with anti-HA. (C and D) CD4+CD25+ T cells from WT, Cblb−/− or CblbC373A mice, and stimulated with anti-CD3 and anti-CD28, and lysed. The Foxp3 ubiquitination was determined. (E) Cytoplasmic and nuclear fractions of WT CD4+ T cells stimulated with anti-CD3 and anti-CD28 were separated, and immunoprecipitated with anti-Foxp3, and blotted with anti-ubiquitin. (F) WT CD4+ T cells were stimulated with anti-CD3 and anti-CD28 for 1, 2, and 4 hrs in the presence or absence of MG-132. Foxp3 expression was determined. (G) BALB/c CD4+CD25+ T cells were transfected with siRNAs specific for Stub1, Cbl-b, or a scrambled siRNA, and stimulated with anti-CD3 and anti-CD28, and lysed in RIPA buffer. The cell lysates were immunoprecitated with anti-Foxp3, and blotted with anti-ubiquitin. The fold changes of Foxp3 ubiquitination bands in arbitrary densitometric units were determined by the ImageJ 1.48. The data shown are one representative of three independent experiments.

Article Snippet: GFP-tagged Stub1 plasmid was obtained from OriGene (Rockville, MD).

Techniques: Immunoprecipitation, Transfection, Expressing