sox2 inducible lentiviral small hairpin rna shrna  (Addgene inc)

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SnRNA-seq reveals cellular diversity and dynamic differentiation in NOTCH-driven CP tumors characterized by a glial progenitor-like signature. (A) Major cell types of a NOTCH-driven CPP from an adult Lcre;NICD1 mouse. Uniform Manifold Approximation and Projection (UMAP) of 6428 single-nucleus profiles, colored by post hoc annotated cell type; also see . (B) UMAP showing mesenchymal, epithelial, and NOTCH-activated profiles from snRNA-seq in NOTCH-driven CPP, colored by expression of Wnt5a (mesenchymal marker, left ), Hes1 (NOTCH pathway target, middle ), and Otx2 (CP epithelial marker, right ); also see . (C) GO analysis of differentially expressed genes in the epithelial-like tumor cell compartment in NOTCH-driven CPP. (D) The expression of markers for glial progenitors in the rhombic lip in NOTCH-driven CPP. UMAP shows 6428 single-nucleus profiles from snRNA-seq in NOTCH-driven CPP, colored by expression of genes associated with glial progenitors in the rhombic lip in the hindbrain ( Rspo1 , Zfp423 , Zic3 , Msx1 , Sox2 , and Slc1a3 ). (E) Western blot analysis of MSX1 expression in the CP of wild-type mice, and NOTCH-driven CPP in Lcre;NICD1 animals ( n = 3 per genotype, mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). Data were generated from 2 independent experiments. (F) RT-qPCR analysis of Rspo1 and Gdf7 mRNA levels in NOTCH-driven CPP and wild-type CP ( n = 3 per time point per genotype, mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). Three independent experiments were conducted. (G) UMAP showing single-nucleus profiles in the epithelial-like compartment in (A) colored by subgroups; also see . (H) Cell trajectory analysis of single-nucleus profiles of subgroups of the epithelial-like compartment, and the glia-like compartment in NOTCH-driven CPP. (I) Violin plots for the expression of Rspo1 , Zfp423 , Msx1 , and Zic4 in subgroups of the epithelial-like compartment in NOTCH-driven CPP.

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Expressing, Marker, Western Blot, Generated, Quantitative RT-PCR

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: Increased SOX2 expression in CP tumors in humans and mice. (A) Immunohistochemistry of SOX2 is shown in the upper rhombic lip/roof plate (red dotted lines) and hindbrain CP (arrow) in wild-type mice, NOTCH-driven CPP and CPC (arrowheads) in Lcre;NICD1 and Lcre;Ptch cko ;NICD1 animals at embryonic (E) day 14.5 (E14.5). Scale bar, 50 µm. Black dotted line marks the border of the ventricle with SOX2-expressing ependymal cells. (B) Immunofluorescence of SOX2 and OTX2 is shown in Rb1/Trp53 -deficient CPC in adult Lcre;p53 cko ;Rb cko mice. Nuclei are labeled with DAPI. Scale bar, 50 µm. (C) RT-qPCR analysis of Sox2 expression in wild-type CP, NOTCH-driven CPP, and Rb1/Trp53 -deficient CPC ( n = 5 per tissue type, mean ± SEM, 1-way ANOVA, **** P < 0.0001). (D) RNAscope of Sox2 and Myc expression in hindbrain CP (black arrow) in adult wild-type mice, and NOTCH-driven CPP (arrowhead) in adult Lcre;NICD1 animals. The dotted line marks ventricular walls with ependymal cells (red arrow). Scale bar, 50 µm. (E) Immunofluorescence of SOX2 and ARL13B is shown in NOTCH-driven CPP in adult Lcre;NICD1 animals. DAPI labels nuclei. Scale bar, 20 µm. (F) Immunofluorescence of SOX2 is shown in NOTCH-driven CP tumor cells treated with DMSO or RIN-1 (10 µM). DAPI labels nuclei. Scale bar, 50 µm. Quantification of fluorescence intensity is shown ( n = 333 [DMSO]; n = 101 [RIN-1], mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). (G) Immunohistochemistry of SOX2 in human CP tumors are shown. Scale bar, 50 µm. (CPC: n = 14; CPP and atypical CPP: n = 26). All results were obtained from 3 independent experiments. (H) RT-qPCR analysis of gene expression in human CP organoids and CP tumors ( n = 6 [CP organoid], n = 9 [CPP], n = 13 [CPC], mean ± SEM, 1-way ANOVA, * P < 0.05; ** P < 0.01; *** P < 0.001; NS, nonsignificant). (I) CosMx analysis of the expression of NOTCH1 , NOTCH2 , NOTCH3 , and SOX2 in a human CPC sample. Boxed region is shown in higher magnification on the right. (J) RNAscope studies of SOX2 and HES5 expression in a human CPP. Scale bar, 200 µm.

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Labeling, Quantitative RT-PCR, RNAscope, Fluorescence, Gene Expression

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SOX2 is essential for the glial progenitor-like signature and NOTCH-driven CP tumor development. (A) Immunofluorescence of Ki-67 is shown in NOTCH-driven CPP cells treated with control scrambled siRNAs or siRNAs against Sox2 at different concentrations. DAPI labels nuclei. Scale bar, 50 µm. Quantification of Ki-67 expression is shown ( n = 3 per group, mean ± SEM, 1-way ANOVA, **** P < 0.0001). Data were obtained from 3 independent experiments; see also . (B) Bisected brain hemispheres and hematoxylin and eosin (H&E) staining of the hindbrain CP in adult wild-type mice, and CPP in adult Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals. Red arrows point to wild-type CP, arrowheads point to CPP, black arrows point to SOX2-deficient CPP. Scale bar, 50 µm. (C) Immunofluorescence of Ki-67 and GFP is shown in CPP in Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals at day E13.5. Nuclei are labeled with DAPI. Scale bar, 50 µm. Quantification of Ki-67 expression is shown ( n = 3 per group, mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). Results were obtained from 3 independent experiments; also see . (D) Principal component analysis (PCA) of CP in wild-type mice, CPP in Lcre;NICD1 mice, and Sox2 -deficient CPP in Lcre;NICD1;Sox2 cko animals; also see . (E) Gene set enrichment analysis (GSEA) of the effect of Sox2 loss on NOTCH-driven CP tumors. Pathways regulating pluripotency of the stem cells are shown as an example; also see . NES, normalized enrichment score. FDR, false discovery rate. (F) RT-qPCR analysis of gene expression in CP in wild-type mice, and CPP from Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals ( n = 8 [CP], n = 6 [CPP], mean ± SEM, 1-way ANOVA, ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, nonsignificant); also see .

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Immunofluorescence, Control, Expressing, Staining, Labeling, Quantitative RT-PCR, Gene Expression

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SOX2 transcriptional targets in CP tumors are enriched in markers and transcriptional regulators of progenitors in the rhombic lip. (A) Pie chart illustrating the distribution of SOX2-binding sites in relation to genes in NOTCH-driven CPP in Lcre;NICD1 animals; also see . (B) Heatmap of tag densities of SOX2 (left) or H3K23Ac (right) ChIP-seq signals at all of the binding regions identified in ChIP-seq experiments. In each heat map the tag density is plotted for 10 Kb at either side of its binding peak summit. (C) Comparison of SOX2 and H3K27Ac signals generated from ChIP-seq fragment counts in the 20 Kb genomic regions surrounding SOX2 peaks in NOTCH-driven CPP. (D) Logos for the motif enriched in SOX2-binding sequences identified by motif analysis in NOTCH-driven CPP; also see . TF: transcription factor; FDR: false discovery rate. (E) Venn diagram shows the overlap of SOX2-associated genes and differentially expressed genes in NOTCH-driven CPPs at days P0 and P21, respectively. (F) GO analysis of candidate SOX2 transcriptional targets in NOTCH-driven CPP. (G) Venn diagram shows the overlap of SOX2 candidate transcriptional targets in (E) and significantly downregulated genes in Sox2 -deficient tumors. (H) Hierarchical clustering of the expression of 52 candidate SOX2 transcriptional targets identified in (G, FDR < 0.05) in wild-type CP, and Sox2 -wild-type or Sox2 -deficient NOTCH-driven CPP. Lmx1b and Hes5 on the heatmap are marked by arrows. (I) The peak density plot of fragment counts is shown in genomic regions that encompass Lmx1b , Lmx1a , Hes5 , and Zic4 , and bound by SOX2 and H3K27Ac, respectively. Genes are labeled in black with sequence in a single exon as a rectangle; also see . (J) RT-qPCR analysis of gene expression in CP in wild-type mice, and CPP from Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals ( n = 8 [CP], n = 6 [CPP], mean ± SEM, 1-way ANOVA, *** P < 0.001; **** P < 0.0001; NS, nonsignificant).

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Binding Assay, ChIP-sequencing, Comparison, Generated, Expressing, Labeling, Sequencing, Quantitative RT-PCR, Gene Expression

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SOX2 regulates transcription factors LMX1A and LMX1B in NOTCH-driven CP tumors. (A) UMAP of 6428 single-nucleus profiles from a NOTCH-driven CPP colored by Lmx1a and Lmx1b expression, respectively. (B) t-distributed stochastic neighbor embedding (t-SNE) plot shows the annotated scATAC-seq profiles of different cell populations in NOTCH-driven CPP. Different subclusters of cells are marked by different colors. (C) Violin plots show the activity of different genes in each subcluster of cells. (D) Western blot analysis of the expression of SOX2, LMX1A, and LMX1B in CP in wild-type mice, and NOTCH-driven CPP in Lcre;NICD1 animals ( n = 3 per group, mean ± SEM, 2-tailed unpaired t test, ** P < 0.01; *** P < 0.001). Three independent experiments were conducted; also see . (E, F) Immunofluorescence of LMX1A (E) and LMX1B (F) is shown in Rb1/Trp53 -deficient CPC cells infected with viruses expressing SOX2. SOX2 labels infected cells. Scale bars, 50 µm. Three independent experiments were conducted. (G) Immunofluorescence of LMX1A is shown in TP53 -deficient human CPC cells infected with viruses expressing SOX2. SOX2 labels infected cells. Scale bar, 50 µm. Three independent experiments were conducted. (H, I) Immunofluorescence of LMX1A (H) LMX1B (I) is shown in CPP in Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals at postnatal (P) day 7 (P7). Arrowheads point to tumor cells, arrows point to SOX2-deficient tumor cells. DAPI labels nuclei. Scale bars, 50 µm. Fluorescence intensity is quantified ( n = 1101 [LMX1A], n = 754 [LMX1B] for NOTCH-driven CPP cells; n = 457 [LMX1A], n = 563 [LMX1B] for Sox2 -deficient tumor cells; mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). The experiments were repeated 3 times independently; also see and . (J) RT-qPCR analysis of the expression of Lmx1a and Lmx1b in CP in wild-type mice, and CPP from Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals ( n = 8 [CP], n = 6 [CPP], mean ± SEM, 1-way ANOVA, *** P < 0.001; **** P < 0.0001; NS, nonsignificant). Results were obtained from 1 experiment. (K) t-SNE plots show that motifs of LMX1A and LMX1B are enriched in tumor cell subclusters in NOTCH-driven CPP. Colors represent average gene activity score of cells in each subcluster. Dark red means high gene activity score, blue means low gene activity score.

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Expressing, Activity Assay, Western Blot, Immunofluorescence, Infection, Fluorescence, Quantitative RT-PCR

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: LMX1A and LMX1B mediate SOX2 functions to support tumor cell proliferation. (A) Immunofluorescence of Ki-67 is shown in NOTCH-driven CPP treated with control scrambled siRNAs or siRNAs against Lmx1a and/or Lmx1b (40 nM). DAPI labels nuclei. Scale bar, 50 µm. Quantification of Ki-67 expression is shown ( n = 3 per group, mean ± SEM, 1-way ANOVA, ** P < 0.01). The experiments were repeated 3 times independently; also see . (B) Immunofluorescence of Ki-67 is shown in NOTCH-driven CPP treated with control scrambled siRNAs or siRNAs against Sox2 (40 nM), and infected with viruses expressing HA-tagged Lmx1a , Lmx1b , or control viruses. DAPI labels nuclei. Scale bar, 50 µm. Quantification of Ki-67 expression is shown ( n = 3 per group, mean ± SEM, 1-way ANOVA, **** P < 0.0001; NS, nonsignificant). Three independent experiments were conducted; also see . (C) Immunofluorescence of LMX1B is shown in NOTCH-driven CPP cells treated with DMSO or RIN-1 (10 µM). DAPI labels nuclei. Scale bar, 50 µm. Quantification of fluorescence intensity is shown ( n = 233 [DMSO]; n = 201 [RIN-1], mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). (D) RT-qPCR analysis of the expression of SOX2 , LMX1A , and LMX1B in human CP organoids and CP tumors ( n = 6 [CP organoid], n = 9 [CPP], n = 13 [CPC]; mean ± SEM, NS, nonsignificant). The experiments were repeated 1 time independently. (E) RNAscope studies of LMX1A and SOX2 expression in human CP tumors. Scale bar, 200 µm. (F) Immunofluorescence of LMX1A in human CP tumor samples is shown. DAPI labels nuclei. Scale bar, 50 µm. Three independent experiments were conducted. (G) RNAscope studies of LMX1A and HES5 expression in a human CPP. Scale bar, 200 µm.

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Immunofluorescence, Control, Expressing, Infection, Fluorescence, Quantitative RT-PCR, RNAscope

Journal: Frontiers in Molecular Biosciences

Article Title: SOX2 induces LPCAT1 expression to promote cholesterol metabolic reprogramming-mediated invasion and metastasis in osteosarcoma

doi: 10.3389/fmolb.2025.1679244

Figure Lengend Snippet: Expression levels of SOX2 and LPCAT1 in osteosarcoma. (A,B) qRT-PCR analysis of SOX2 and LPCAT1 expression in tumor tissues (n = 20) and adjacent normal tissues (n = 20) from osteosarcoma patients. (C,D) Western blot analysis of SOX2 and LPCAT1 protein levels in osteosarcoma tissues and paired adjacent normal tissues. (E–H) qRT-PCR and Western blot detection of SOX2 and LPCAT1 expression in normal osteoblast cell line hFOB1.19 and osteosarcoma cell lines (MG63, 143B, U2OS, and MNNG/HOS). Data are presented as mean ± SD. ** P < 0.01, *** P < 0.001 vs. PT or hFOB1.19.

Article Snippet: Short hairpin RNAs (shRNAs) targeting LPCAT1 (shLPCAT1, sc-91777-SH, target sequence: 5′-GGAACTCTGATCCAGTATATA-3′) and SOX2 (shSOX2, sc-38408-SH, target sequence: 5′-AGGAGCACCCGGATTATAAAT-3′) were purchased from Santa Cruz (United States).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Molecular Biosciences

Article Title: SOX2 induces LPCAT1 expression to promote cholesterol metabolic reprogramming-mediated invasion and metastasis in osteosarcoma

doi: 10.3389/fmolb.2025.1679244

Figure Lengend Snippet: SOX2 mediated transcriptional regulation of LPCAT1. (A) JASPAR prediction of SOX2 binding sites in the LPCAT1 promoter region. (B,C) Dual-luciferase reporter assay validating the interaction between SOX2 and LPCAT1 promoter. (D) ChIP assay demonstrating the binding of SOX2 to LPCAT1 promoter region. (E,F) qRT-PCR confirmation of SOX2 overexpression and knockdown efficiency. (G–I) Western blot and qRT-PCR analysis of LPCAT1 expression following SOX2 modulation. Data are representative of three independent experiments (mean ± SD). ** P < 0.01, *** P < 0.001 vs. NC, anti-IgG or shNC.

Article Snippet: Short hairpin RNAs (shRNAs) targeting LPCAT1 (shLPCAT1, sc-91777-SH, target sequence: 5′-GGAACTCTGATCCAGTATATA-3′) and SOX2 (shSOX2, sc-38408-SH, target sequence: 5′-AGGAGCACCCGGATTATAAAT-3′) were purchased from Santa Cruz (United States).

Techniques: Binding Assay, Luciferase, Reporter Assay, Quantitative RT-PCR, Over Expression, Knockdown, Western Blot, Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: SOX2 induces LPCAT1 expression to promote cholesterol metabolic reprogramming-mediated invasion and metastasis in osteosarcoma

doi: 10.3389/fmolb.2025.1679244

Figure Lengend Snippet: In vitro validation of SOX2/LPCAT1 axis in regulating osteosarcoma cell biological behaviors and cholesterol metabolism. MG63 cells were co-transfected with oe-LPCAT1 and/or shSOX2, while U2OS cells were co-transfected with shLPCAT1 and/or SOX2. (A,B) Cell viability was determined by CCK-8 assay. (C,D) Migration ability was assessed by wound healing assay. (E,F) Invasion capacity was evaluated by Transwell assay. (G,H) Intracellular cholesterol level measurement. (I,J) Western blot and qRT-PCR analysis of metastasis- and cholesterol metabolism-related proteins. Data are representative of three independent experiments (mean ± SD). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. NC+shNC, shLPCAT1+NC or LPCAT1+shNC, SOX2+shNC or shSOX2+NC.

Article Snippet: Short hairpin RNAs (shRNAs) targeting LPCAT1 (shLPCAT1, sc-91777-SH, target sequence: 5′-GGAACTCTGATCCAGTATATA-3′) and SOX2 (shSOX2, sc-38408-SH, target sequence: 5′-AGGAGCACCCGGATTATAAAT-3′) were purchased from Santa Cruz (United States).

Techniques: In Vitro, Biomarker Discovery, Transfection, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Assay, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Molecular Biosciences

Article Title: SOX2 induces LPCAT1 expression to promote cholesterol metabolic reprogramming-mediated invasion and metastasis in osteosarcoma

doi: 10.3389/fmolb.2025.1679244

Figure Lengend Snippet: In vivo validation of SOX2/LPCAT1 axis in osteosarcoma progression and cholesterol metabolism. For (A–D) , MG63 cells were transfected with oe-LPCAT1 and/or shSOX2, and then subcutaneously injected into nude mice to construct a xenograft model. (A) Representative images of xenograft tumors. (B) Tumor volume. (C) Cholesterol content measurement in tumor tissues. (D,E) Western blot analysis of metastasis- and cholesterol metabolism-related proteins in xenograft tumors. (F) MG63 cells transfected with oe-LPCAT1 and/or shSOX2 were injected into the tail vein of nude mice to construct a lung metastasis model. The number of pulmonary nodules was counted. Data are representative of three independent experiments (mean ± SD). ** P < 0.01, *** P < 0.001 vs. NC+shNC, LPCAT1+shNC, and shSOX2+NC.

Article Snippet: Short hairpin RNAs (shRNAs) targeting LPCAT1 (shLPCAT1, sc-91777-SH, target sequence: 5′-GGAACTCTGATCCAGTATATA-3′) and SOX2 (shSOX2, sc-38408-SH, target sequence: 5′-AGGAGCACCCGGATTATAAAT-3′) were purchased from Santa Cruz (United States).

Techniques: In Vivo, Biomarker Discovery, Transfection, Injection, Construct, Western Blot

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SnRNA-seq reveals cellular diversity and dynamic differentiation in NOTCH-driven CP tumors characterized by a glial progenitor-like signature. (A) Major cell types of a NOTCH-driven CPP from an adult Lcre;NICD1 mouse. Uniform Manifold Approximation and Projection (UMAP) of 6428 single-nucleus profiles, colored by post hoc annotated cell type; also see . (B) UMAP showing mesenchymal, epithelial, and NOTCH-activated profiles from snRNA-seq in NOTCH-driven CPP, colored by expression of Wnt5a (mesenchymal marker, left ), Hes1 (NOTCH pathway target, middle ), and Otx2 (CP epithelial marker, right ); also see . (C) GO analysis of differentially expressed genes in the epithelial-like tumor cell compartment in NOTCH-driven CPP. (D) The expression of markers for glial progenitors in the rhombic lip in NOTCH-driven CPP. UMAP shows 6428 single-nucleus profiles from snRNA-seq in NOTCH-driven CPP, colored by expression of genes associated with glial progenitors in the rhombic lip in the hindbrain ( Rspo1 , Zfp423 , Zic3 , Msx1 , Sox2 , and Slc1a3 ). (E) Western blot analysis of MSX1 expression in the CP of wild-type mice, and NOTCH-driven CPP in Lcre;NICD1 animals ( n = 3 per genotype, mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). Data were generated from 2 independent experiments. (F) RT-qPCR analysis of Rspo1 and Gdf7 mRNA levels in NOTCH-driven CPP and wild-type CP ( n = 3 per time point per genotype, mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). Three independent experiments were conducted. (G) UMAP showing single-nucleus profiles in the epithelial-like compartment in (A) colored by subgroups; also see . (H) Cell trajectory analysis of single-nucleus profiles of subgroups of the epithelial-like compartment, and the glia-like compartment in NOTCH-driven CPP. (I) Violin plots for the expression of Rspo1 , Zfp423 , Msx1 , and Zic4 in subgroups of the epithelial-like compartment in NOTCH-driven CPP.

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Expressing, Marker, Western Blot, Generated, Quantitative RT-PCR

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: Increased SOX2 expression in CP tumors in humans and mice. (A) Immunohistochemistry of SOX2 is shown in the upper rhombic lip/roof plate (red dotted lines) and hindbrain CP (arrow) in wild-type mice, NOTCH-driven CPP and CPC (arrowheads) in Lcre;NICD1 and Lcre;Ptch cko ;NICD1 animals at embryonic (E) day 14.5 (E14.5). Scale bar, 50 µm. Black dotted line marks the border of the ventricle with SOX2-expressing ependymal cells. (B) Immunofluorescence of SOX2 and OTX2 is shown in Rb1/Trp53 -deficient CPC in adult Lcre;p53 cko ;Rb cko mice. Nuclei are labeled with DAPI. Scale bar, 50 µm. (C) RT-qPCR analysis of Sox2 expression in wild-type CP, NOTCH-driven CPP, and Rb1/Trp53 -deficient CPC ( n = 5 per tissue type, mean ± SEM, 1-way ANOVA, **** P < 0.0001). (D) RNAscope of Sox2 and Myc expression in hindbrain CP (black arrow) in adult wild-type mice, and NOTCH-driven CPP (arrowhead) in adult Lcre;NICD1 animals. The dotted line marks ventricular walls with ependymal cells (red arrow). Scale bar, 50 µm. (E) Immunofluorescence of SOX2 and ARL13B is shown in NOTCH-driven CPP in adult Lcre;NICD1 animals. DAPI labels nuclei. Scale bar, 20 µm. (F) Immunofluorescence of SOX2 is shown in NOTCH-driven CP tumor cells treated with DMSO or RIN-1 (10 µM). DAPI labels nuclei. Scale bar, 50 µm. Quantification of fluorescence intensity is shown ( n = 333 [DMSO]; n = 101 [RIN-1], mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). (G) Immunohistochemistry of SOX2 in human CP tumors are shown. Scale bar, 50 µm. (CPC: n = 14; CPP and atypical CPP: n = 26). All results were obtained from 3 independent experiments. (H) RT-qPCR analysis of gene expression in human CP organoids and CP tumors ( n = 6 [CP organoid], n = 9 [CPP], n = 13 [CPC], mean ± SEM, 1-way ANOVA, * P < 0.05; ** P < 0.01; *** P < 0.001; NS, nonsignificant). (I) CosMx analysis of the expression of NOTCH1 , NOTCH2 , NOTCH3 , and SOX2 in a human CPC sample. Boxed region is shown in higher magnification on the right. (J) RNAscope studies of SOX2 and HES5 expression in a human CPP. Scale bar, 200 µm.

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Labeling, Quantitative RT-PCR, RNAscope, Fluorescence, Gene Expression

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SOX2 is essential for the glial progenitor-like signature and NOTCH-driven CP tumor development. (A) Immunofluorescence of Ki-67 is shown in NOTCH-driven CPP cells treated with control scrambled siRNAs or siRNAs against Sox2 at different concentrations. DAPI labels nuclei. Scale bar, 50 µm. Quantification of Ki-67 expression is shown ( n = 3 per group, mean ± SEM, 1-way ANOVA, **** P < 0.0001). Data were obtained from 3 independent experiments; see also . (B) Bisected brain hemispheres and hematoxylin and eosin (H&E) staining of the hindbrain CP in adult wild-type mice, and CPP in adult Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals. Red arrows point to wild-type CP, arrowheads point to CPP, black arrows point to SOX2-deficient CPP. Scale bar, 50 µm. (C) Immunofluorescence of Ki-67 and GFP is shown in CPP in Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals at day E13.5. Nuclei are labeled with DAPI. Scale bar, 50 µm. Quantification of Ki-67 expression is shown ( n = 3 per group, mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). Results were obtained from 3 independent experiments; also see . (D) Principal component analysis (PCA) of CP in wild-type mice, CPP in Lcre;NICD1 mice, and Sox2 -deficient CPP in Lcre;NICD1;Sox2 cko animals; also see . (E) Gene set enrichment analysis (GSEA) of the effect of Sox2 loss on NOTCH-driven CP tumors. Pathways regulating pluripotency of the stem cells are shown as an example; also see . NES, normalized enrichment score. FDR, false discovery rate. (F) RT-qPCR analysis of gene expression in CP in wild-type mice, and CPP from Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals ( n = 8 [CP], n = 6 [CPP], mean ± SEM, 1-way ANOVA, ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, nonsignificant); also see .

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Immunofluorescence, Control, Expressing, Staining, Labeling, Quantitative RT-PCR, Gene Expression

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SOX2 transcriptional targets in CP tumors are enriched in markers and transcriptional regulators of progenitors in the rhombic lip. (A) Pie chart illustrating the distribution of SOX2-binding sites in relation to genes in NOTCH-driven CPP in Lcre;NICD1 animals; also see . (B) Heatmap of tag densities of SOX2 (left) or H3K23Ac (right) ChIP-seq signals at all of the binding regions identified in ChIP-seq experiments. In each heat map the tag density is plotted for 10 Kb at either side of its binding peak summit. (C) Comparison of SOX2 and H3K27Ac signals generated from ChIP-seq fragment counts in the 20 Kb genomic regions surrounding SOX2 peaks in NOTCH-driven CPP. (D) Logos for the motif enriched in SOX2-binding sequences identified by motif analysis in NOTCH-driven CPP; also see . TF: transcription factor; FDR: false discovery rate. (E) Venn diagram shows the overlap of SOX2-associated genes and differentially expressed genes in NOTCH-driven CPPs at days P0 and P21, respectively. (F) GO analysis of candidate SOX2 transcriptional targets in NOTCH-driven CPP. (G) Venn diagram shows the overlap of SOX2 candidate transcriptional targets in (E) and significantly downregulated genes in Sox2 -deficient tumors. (H) Hierarchical clustering of the expression of 52 candidate SOX2 transcriptional targets identified in (G, FDR < 0.05) in wild-type CP, and Sox2 -wild-type or Sox2 -deficient NOTCH-driven CPP. Lmx1b and Hes5 on the heatmap are marked by arrows. (I) The peak density plot of fragment counts is shown in genomic regions that encompass Lmx1b , Lmx1a , Hes5 , and Zic4 , and bound by SOX2 and H3K27Ac, respectively. Genes are labeled in black with sequence in a single exon as a rectangle; also see . (J) RT-qPCR analysis of gene expression in CP in wild-type mice, and CPP from Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals ( n = 8 [CP], n = 6 [CPP], mean ± SEM, 1-way ANOVA, *** P < 0.001; **** P < 0.0001; NS, nonsignificant).

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Binding Assay, ChIP-sequencing, Comparison, Generated, Expressing, Labeling, Sequencing, Quantitative RT-PCR, Gene Expression

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SOX2 regulates transcription factors LMX1A and LMX1B in NOTCH-driven CP tumors. (A) UMAP of 6428 single-nucleus profiles from a NOTCH-driven CPP colored by Lmx1a and Lmx1b expression, respectively. (B) t-distributed stochastic neighbor embedding (t-SNE) plot shows the annotated scATAC-seq profiles of different cell populations in NOTCH-driven CPP. Different subclusters of cells are marked by different colors. (C) Violin plots show the activity of different genes in each subcluster of cells. (D) Western blot analysis of the expression of SOX2, LMX1A, and LMX1B in CP in wild-type mice, and NOTCH-driven CPP in Lcre;NICD1 animals ( n = 3 per group, mean ± SEM, 2-tailed unpaired t test, ** P < 0.01; *** P < 0.001). Three independent experiments were conducted; also see . (E, F) Immunofluorescence of LMX1A (E) and LMX1B (F) is shown in Rb1/Trp53 -deficient CPC cells infected with viruses expressing SOX2. SOX2 labels infected cells. Scale bars, 50 µm. Three independent experiments were conducted. (G) Immunofluorescence of LMX1A is shown in TP53 -deficient human CPC cells infected with viruses expressing SOX2. SOX2 labels infected cells. Scale bar, 50 µm. Three independent experiments were conducted. (H, I) Immunofluorescence of LMX1A (H) LMX1B (I) is shown in CPP in Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals at postnatal (P) day 7 (P7). Arrowheads point to tumor cells, arrows point to SOX2-deficient tumor cells. DAPI labels nuclei. Scale bars, 50 µm. Fluorescence intensity is quantified ( n = 1101 [LMX1A], n = 754 [LMX1B] for NOTCH-driven CPP cells; n = 457 [LMX1A], n = 563 [LMX1B] for Sox2 -deficient tumor cells; mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). The experiments were repeated 3 times independently; also see and . (J) RT-qPCR analysis of the expression of Lmx1a and Lmx1b in CP in wild-type mice, and CPP from Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals ( n = 8 [CP], n = 6 [CPP], mean ± SEM, 1-way ANOVA, *** P < 0.001; **** P < 0.0001; NS, nonsignificant). Results were obtained from 1 experiment. (K) t-SNE plots show that motifs of LMX1A and LMX1B are enriched in tumor cell subclusters in NOTCH-driven CPP. Colors represent average gene activity score of cells in each subcluster. Dark red means high gene activity score, blue means low gene activity score.

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Expressing, Activity Assay, Western Blot, Immunofluorescence, Infection, Fluorescence, Quantitative RT-PCR

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: LMX1A and LMX1B mediate SOX2 functions to support tumor cell proliferation. (A) Immunofluorescence of Ki-67 is shown in NOTCH-driven CPP treated with control scrambled siRNAs or siRNAs against Lmx1a and/or Lmx1b (40 nM). DAPI labels nuclei. Scale bar, 50 µm. Quantification of Ki-67 expression is shown ( n = 3 per group, mean ± SEM, 1-way ANOVA, ** P < 0.01). The experiments were repeated 3 times independently; also see . (B) Immunofluorescence of Ki-67 is shown in NOTCH-driven CPP treated with control scrambled siRNAs or siRNAs against Sox2 (40 nM), and infected with viruses expressing HA-tagged Lmx1a , Lmx1b , or control viruses. DAPI labels nuclei. Scale bar, 50 µm. Quantification of Ki-67 expression is shown ( n = 3 per group, mean ± SEM, 1-way ANOVA, **** P < 0.0001; NS, nonsignificant). Three independent experiments were conducted; also see . (C) Immunofluorescence of LMX1B is shown in NOTCH-driven CPP cells treated with DMSO or RIN-1 (10 µM). DAPI labels nuclei. Scale bar, 50 µm. Quantification of fluorescence intensity is shown ( n = 233 [DMSO]; n = 201 [RIN-1], mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). (D) RT-qPCR analysis of the expression of SOX2 , LMX1A , and LMX1B in human CP organoids and CP tumors ( n = 6 [CP organoid], n = 9 [CPP], n = 13 [CPC]; mean ± SEM, NS, nonsignificant). The experiments were repeated 1 time independently. (E) RNAscope studies of LMX1A and SOX2 expression in human CP tumors. Scale bar, 200 µm. (F) Immunofluorescence of LMX1A in human CP tumor samples is shown. DAPI labels nuclei. Scale bar, 50 µm. Three independent experiments were conducted. (G) RNAscope studies of LMX1A and HES5 expression in a human CPP. Scale bar, 200 µm.

Article Snippet: Viruses expressing HA-tagged LMX1A or LMX1B (269200540200, 269210540200, Applied Biological Materials Inc.), and viruses expressing SOX2, shRNAs against Sox2 (shADV-272885) or Lmx1a (shADV-263539, all from Vector Biolabs) were amplified and purified from AD-293 cells (Agilent Technologies).

Techniques: Immunofluorescence, Control, Expressing, Infection, Fluorescence, Quantitative RT-PCR, RNAscope

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: The expression of TGIF2 and SOX2 in human PC and adjacent pancreas. A. TGIF2 and SOX2 expression in PC and paired pancreas specimens by IHC. B and C. TGIF2, SOX2, EGFR and E-cad expression in two PC specimens (#21 and #37). D. The mRNA level of TGIF2 in 20 cases of human PC and adjacent pancreas by qRT-PCR (T: PC; N: paired pancreas) and SOX2. E. The mRNA level of SOX2 in our cohort. F and G. High and low expression of TGIF2 and SOX2 against prognosis. H. Combination of TGIF2 and SOX2 against prognosis.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: The relationship among TGIF2, SOX2, EGFR and E-cad expression in 88 cases of clinical PC samples

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Expressing

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: Univariate and Multivariate analysis in survival time

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: Coordination of TGIF2/SOX2 promotes EMT of PC in vitro . A and B. TGIF2 and SOX2 protein (A) and mRNA (B) level in 4 PC cell lines. C. The protein expression of TGIF2, SOX2 and EMT signaling in Scramble, TGIF2-OE, siSOX2 and TGIF2-OE/siSOX2 groups in PANC-1 cells. D. The protein expression of TGIF2, SOX2 and EMT signaling in Scramble, siTGIF2, SOX2-OE, and siTGIF2/SOX2-OE groups in BxPC-3 cells. E. The EMT phenotype in Scramble, TGIF2-OE, siSOX2 and TGIF2-OE/siSOX2 groups in PANC-1 cells. F. The EMT phenotype in Scramble, SOX2-OE, siTGIF2 and siTGIF2/SOX2-OE groups in BxPC-3 cells. G and H. Cell invasion (G) and migration (H) in Scramble, TGIF2-OE, siSOX2 and TGIF2-OE/siSOX2 groups in PANC-1 cells. I and J. Cell invasion (I) and migration (J) in Scramble, siTGIF2, SOX2-OE and siTGIF2/SOX2-OE groups Scramble, SOX2-OE, siTGIF2 and siTGIF2/SOX2-OE in BxPC-3 cells. Bars indicate ± S.E.*, P <0.05; **, P <0.01 compared with the control.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: In Vitro, Expressing, Migration, Control

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: Coordination of TGIF2/SOX2 promotes CSCs and drug resistance of PC in vitro . A and B. Sphere formation in PANC-1 cells (A) and BxPC-3 (B) cells following the activation of TGIF2, SOX2, CD133, CD44 and EGFR/MAPK signaling. C and D. The sphere number (C) and the protein expression of TGIF2, SOX2, CD133, CD44 and EGFR/MAPK signaling (D) in Scramble, TGIF2-OE, siSOX2 and TGIF2-OE/siSOX2 groups in PANC-1 cells. E and F The sphere number (E) and the protein expression of TGIF2, SOX2, CD133, CD44 and EGFR/MAPK signaling (F) in Scramble, siTGIF2, SOX2-OE and siTGIF2/SOX2-OE groups in BxPC-3 cells. ( G-H ) the spheroids number of PC in wildtype (treated with EGF to activate EGFR/ERK signaling) and Kras-mutant (constant activation of EGFR/ERK signaling) mice. ( I ) The overexpression of TGIF2, SOX2, CD133 and p-ERK were in pancreatic intraepithelial neoplasia (PanIN) of KC mice. J. Cell growth in Scramble, TGIF2-OE, siSOX2, TGIF2-OE/siSOX2 and TGIF2-OE/Erlotinib groups of PANC-1 cells and in Scramble, siTGIF2, SOX2-OE and siTGIF2/SOX2-OE of BxPC-3 cells by MTT. K. Under various concentration of Gemcitabine treatment, Cell proliferation rate in Scramble, TGIF2-OE, siSOX2, TGIF2-OE/siSOX2 and TGIF2-OE/Erlotinib groups of PANC-1 cells and in Scramble, siTGIF2, SOX2-OE and siTGIF2/SOX2-OE of BxPC-3 cells by MTT. Bars indicate ± S.E.*, P <0.05; **, P <0.01 compared with the control.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: In Vitro, Activation Assay, Expressing, Mutagenesis, Over Expression, Concentration Assay, Control

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: TGIF2 is a transactivation factor of SOX2 and interacts with Smad2 to co-regulate SOX2. A. The mRNA level of TGIF2 and SOX2 in TGIF2 overexpressing PANC-1 and TGIF2 silencing BxPC-3 cells, respectively. B. The binding site of TGIF2 in its zinc fingers domain was obtained from the JASPAR database. C. The predicted potential binding site of TGIF2 to SOX2 promoter, as well as wild-type/mutant SOX2 promoter plasmids (SOX2-WT or SOX2-Mut) designed accordingly. D. Chip assays in PANC-1 and BxPC-3 cells. E. Luciferase assay in 293 T cells co-transfected with SOX2-WT or SOX2-Mut promoter plasmid and TGIF2 overexpression plasmid. F. The protein interaction network of TGIF2 via String database. G. Co-IP was performed in PANC-1 and BxPC-3 cells. H and I. The protein and mRNA level of Smad2 and SOX2 in Smad2 silencing BxPC-3 cells. J. The binding site of Smad2 in its zinc fingers domain was obtained from the JASPAR database. K. The predicted potential binding site of Smad2 to SOX2 promoter, as well as wild-type/mutant SOX2 promoter plasmids (SOX2-WT or SOX2-Mut2) designed accordingly. L. Chip assays in BxPC-3 cells. M. Luciferase assay in 293 T cells co-transfected with SOX2-WT or SOX2-Mut2 promoter plasmid and Smad2 overexpression plasmid (or empty vector scramble). N. The protein level of Smad2 and SOX2 in Scramble, TGIF2-OE, si2-Smad2 and TGIF2-OE/si2-Smad2 groups in PANC-1 cells. Bars indicate ± S.E.*, P <0.05; **, P <0.01 in contrast with the control.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Binding Assay, Zinc-Fingers, Mutagenesis, Luciferase, Transfection, Plasmid Preparation, Over Expression, Co-Immunoprecipitation Assay, Control

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: SOX2 is a transactivation factor of EGFR and Slug. A. The mRNA level of SOX2, EGFR and Slug in SOX2 overexpressing PANC-1 and SOX2 silencing BxPC-3 cells, respectively. B. The DNA binding site of SOX2 was obtained from the JASPAR database. C. The predicted potential binding site of SOX2 to Slug promoter. D and E. Chip assays in PANC-1 (D) and BxPC-3 (E) cells. F. The predicted potential binding site of SOX2 to EGFR promoter, as well as wild-type/mutant EGFR promoter plasmids designed accordingly. G. Chip assays in PANC-1 and BxPC-3 cells. H. Luciferase assay in 293 T cells co-transfected with EGFR-WT (or EGFR-Mut) promoter plasmid and SOX2 overexpression plasmid (or empty vector corresponding to scramble group). I. The roles of SOX2-OE and Erlotinib treatment to TGIF2 nuclear translocation. J. IF staining of TGIF2 and SOX2 in Scramble, SOX2-OE and SOX2-OE plus Erlotinib groups of PANC-1. Bars indicate ± S.E.*, P <0.05; **, P <0.01 in contrast with the control.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Binding Assay, Mutagenesis, Luciferase, Transfection, Plasmid Preparation, Over Expression, Translocation Assay, Staining, Control

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: Coordination of TGIF2/SOX2 promotes subcutaneous tumor size in vivo . A. Tumor volumes in Scramble, TGIF2-OE, TGIF2-OE/shSOX2, and TGIF2-OE plus Erlotinib groups implanted with PANC-1 cells. B. Tumor growth curve in above group. C. HE staining of harvested tumor. D. The statistical data of IHC assays. E. The different expression of TGIF2, SOX2, E-cad, EGFR and Vimentin in Scramble, TGIF2-OE, and TGIF2-OE/shSOX2 groups by IHC. Bars indicate ± S.E. *, P<0.05; **, P<0.01 compared with control.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: In Vivo, Staining, Expressing, Control

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: Coordination of TGIF2/SOX2 promotes liver metastasis in vivo . A . Liver metastasis in Scramble, shTGIF2 and shTGIF2/SOX2-OE groups. B-C. The Liver body ratio and the number of liver metastases in above groups. D. The different expression of TGIF2, SOX2, E-cad, EGFR and Vimentin in Scramble, shTGIF2 and shTGIF2/SOX2-OE groups by IHC. E. The statistical data of IHC assays. Bars indicate ± S.E. *, P<0.05; **, P<0.01 compared with control.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: In Vivo, Expressing, Control

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: Smad2 cooperating with TGIF2 contributes to CSC and EMT via co-targeting SOX2. TGIF2 activates SOX2 promoter via interacting with Smad2, which stimulates EMT and EGFR/MAPK signaling by transactivating Slug and EGFR, and promoting EMT and CSCs function. Moreover, the stimulation of EGFR/MAPK signaling by SOX2 promotes TGIF2 nuclear translocation, forming a positive feedback loop.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Translocation Assay

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: The clinicopathological significance of TGIF2 and SOX2 expression in 88 cases of clinical PC samples

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Expressing