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: bioRxiv

Article Title: Enhancing Sox/Oct cooperativity induces higher-grade developmental reset

doi: 10.1101/2022.09.23.509242

Figure Lengend Snippet: a , Brightfield and Oct4-GFP merged overview images showing retroviral reprogramming of MEFs carrying Oct4-GFP (OG2) reporter on 21 dpi (scale=l mm). b , Schematic representation of Sox2 and Sox17 structures and chimeric transcription factors (TFs) generated by swapping nonconserved residues from Sox17 into Sox2. Sequence from Sox2 in blue and from Sox17 in red. c , Protein sequence alignment of DNA binding domains of mouse and human Sox2, Sox17 as well as the most crucial chimeric Sox factors of this study. d-g , Reprogramming of Oct4-GFP MEFs by retroviral vectors carrying Klf4, Sox2-Sox17 chimeric TFs, and wild-type Oct4 ( d ), Oct4 L80A linker mutant ( e-f ), or Brn4 ( g ). Error bars represent SD; n = 3. Statistical significance was calculated with Student’s t-test. h , Representative phase-contrast and Oct4-GFP merged microscopy images of primary iPSCs colonies generated with retroviral vectors carrying different POU factors combined with Sox2 AV and Klf4 (Scale=200μm). i , Cell proliferation assay, where 2×10 3 MEFs were transduced with the indicated tet-inducible polycistronic constructs in 96-well plates. Error bars represent SD; n = 3. The cells were counted after 2, 4, and 6 dpi. 61V was compared with 61A for each construct to calculate the statistical significance with Student’s t-test.

Article Snippet: The pMX-Sox2/Sox17 chimeric TF vectors were based on Addgene ID 13367 and the tet-inducible pHAGE2-tetO-Oct4-P2A-Sox2-17-T2A-Klf4-E2A-cMyc (OS*KM) and pHAGE-tetO-Sox2-17-T2A-Klf4-E2A-cMyc (S*KM) vectors were based on Addgene ID 136551 and 136541, respectively .

Techniques: Generated, Sequencing, Binding Assay, Mutagenesis, Microscopy, Proliferation Assay, Transduction, Construct

Journal: bioRxiv

Article Title: Enhancing Sox/Oct cooperativity induces higher-grade developmental reset

doi: 10.1101/2022.09.23.509242

Figure Lengend Snippet: a, OSK reprogramming of Oct4-GFP reporter MEFs by retroviral monocistronic Sox2 or Sox17 EK , combined with Klf4 and Oct4 L80A linker mutant. Error bars represent SD; n = 3. Statistical significance was calculated with Student’s t-test. b, Representative brightfield and Oct4-GFP merged overview images showing OG2 MEFs reprogrammed with Oct4 L80A linker mutant, Klf4, and wild-type Sox2 versus Sox17 E57K mutant, 21 dpi, scale=l mm. c-d , qPCR titration of the retroviral vectors from .

Article Snippet: The pMX-Sox2/Sox17 chimeric TF vectors were based on Addgene ID 13367 and the tet-inducible pHAGE2-tetO-Oct4-P2A-Sox2-17-T2A-Klf4-E2A-cMyc (OS*KM) and pHAGE-tetO-Sox2-17-T2A-Klf4-E2A-cMyc (S*KM) vectors were based on Addgene ID 136551 and 136541, respectively .

Techniques: Mutagenesis, Titration

Journal: bioRxiv

Article Title: Enhancing Sox/Oct cooperativity induces higher-grade developmental reset

doi: 10.1101/2022.09.23.509242

Figure Lengend Snippet: a, Western blot of whole-cell lysates of HEK293 overexpressing flagged POU factors used in ( b ). b, EMSAs of whole-cell lysates from (a) on the Nanog promoter locus labeled with Cy5. White arrow heads indicate nonspecific bands (ns) and black arrow heads indicate free DNA or DNA bound by Oct (O/DNA), Sox (S/DNA), or both (O/S/DNA). c-d , Representative brightfield and Oct4-GFP merged overview images showing MEFs reprogrammed with Oct4 mutant without the C- ( c , ΔCTD) or N- ( d , ΔNTD) terminal transactivator domain, 21 dpi, scale=l mm. e , A primary iPSC colony generated by the Oct4 mutant with the POU HD domain removed (except the NLS), scale=l00 μm. f , PCR genotyping confirming the identity of two Oct4ΔPOU HD /Sox2 AV /Klf4 iPSC lines, g , PCR genotyping of chimeric mice generated by aggregation with Oct4ΔPOU HD /Sox2 AV /K- generated iPSCs. h , Brightfield and Oct4-GFP merged image of embryonic day 13.5 gonad dissected from chimeric embryo from (g). i , Coomassie stained SDS-polyacrylamide gel of mouse Sox2, Sox2 AV , and Oct4 from insect cells used in and . j , EMSAs of insect cell-purified Sox2 (S, blue), Sox2 AV (S AV , light red), and wild-type Oct4 on the Nanog promoter, Utf1 and Fgf4 enhancer DNA elements labeled with Cy5. Arrow heads indicate free DNA or DNA bound by Oct4 (O/DNA), Sox2 (S/DNA), or the heterodimer (O/S/DNA). k , Representative kinetic off-rate EMSAs using whole-cell lysates overexpressing full-length Oct4, Oct4 L80A , Oct4 GL19 , or Brn4 combined Sox2 versus Sox2 AV lysates on the Nanog promoter locus labeled with Cy5. Following the binding reaction, half-life was determined by adding excess unlabeled Nanog element for the indicated time. White arrow heads indicated nonspecific bands (ns) and black arrow heads indicate free DNA or DNA bound by POU/Sox heterodimer.

Article Snippet: The pMX-Sox2/Sox17 chimeric TF vectors were based on Addgene ID 13367 and the tet-inducible pHAGE2-tetO-Oct4-P2A-Sox2-17-T2A-Klf4-E2A-cMyc (OS*KM) and pHAGE-tetO-Sox2-17-T2A-Klf4-E2A-cMyc (S*KM) vectors were based on Addgene ID 136551 and 136541, respectively .

Techniques: Western Blot, Labeling, Mutagenesis, Generated, Staining, Purification, Binding Assay

Journal: bioRxiv

Article Title: Enhancing Sox/Oct cooperativity induces higher-grade developmental reset

doi: 10.1101/2022.09.23.509242

Figure Lengend Snippet: a-c , OSK reprogramming of Oct4-GFP MEFs with monocistronic retroviral vectors carrying Oct4 domain deletion mutants where the linker domain was replaced with a synthetic poly-Glycine linker (GL) of different length (3-30 residues) ( a ), Oct4’s N- or C-terminal transactivator domain (NTD or CTD) was removed ( b ); POU S or POU HD (except for NLS) was removed ( c ) combined with wild-type Sox2 or Sox2-Sox17 chimeric factors. Error bars represent SD; n = 3. Statistical significance was calculated with Student’s t-test. d , Western blot of whole-cell lysates from HEK293 used in . e , Electrophoretic mobility shift assays (EMSAs) of whole-cell lysates of HEK293 cells transfected with Sox2 (S, blue), Sox2 AV (S AV , light red) and wild-type, or mutant Oct4 in which the POU S or POU HD domain was removed on the Nanog promoter and HoxB1 enhancer SoxOct DNA elements labeled with Cy5. White arrow heads indicate nonspecific bands (ns) and black arrow heads indicate free DNA or DNA bound by Oct4, Sox2, or the heterodimer. f , Representative kinetic off-rate EMSAs of whole-cell lysates from HEK293 overexpressing Oct4, Sox2 (blue), Sox2 A61V (light red) or Sox2 – , , – c17 (Sox2-17, S*, dark red) on Oct4 distal enhancer ( Oct4DE), Nanog promoter, or Fgf4 enhancer DNA elements labeled with Cy5. White arrow heads indicated nonspecific bands (ns) and black arrow heads indicate free DNA or DNA bound by Oct4 (O/DNA), Sox (S/DNA), or Oct4/Sox heterodimer (O/S/DNA). Error bars on graphs represent SD; n = 3. g , Representative kinetic off-rate EMSAs using purified proteins bound to the Utf1 enhancer and Nanog promoter loci labeled with Cy5. Following the binding reaction, halflife was determined by adding excess unlabeled Nanog element for the indicated time. The error bars on quantitation graphs represent SD, n =3. Ternary complex half-life t 1/2 . l , A schematic of the enabling effect of highly cooperative Sox2 AV mutant on reprogramming with tissue specific POU factors or incapable Oct4 mutants.

Article Snippet: The pMX-Sox2/Sox17 chimeric TF vectors were based on Addgene ID 13367 and the tet-inducible pHAGE2-tetO-Oct4-P2A-Sox2-17-T2A-Klf4-E2A-cMyc (OS*KM) and pHAGE-tetO-Sox2-17-T2A-Klf4-E2A-cMyc (S*KM) vectors were based on Addgene ID 136551 and 136541, respectively .

Techniques: Western Blot, Electrophoretic Mobility Shift Assay, Transfection, Mutagenesis, Labeling, Purification, Binding Assay, Quantitation Assay

Journal: bioRxiv

Article Title: Enhancing Sox/Oct cooperativity induces higher-grade developmental reset

doi: 10.1101/2022.09.23.509242

Figure Lengend Snippet: a, qPCR titration of tet-inducible lentiviral vectors from after 24h of Dox induction. Error bars represent SD; n = 3. b , Bisulfite sequencing analysis of DNA methylation in Oct4, Nanog , and Col1a1 promoters in MEFs and an iPSC line generated by inducing tetO-OS*KM for 24h. c , H&E staining of teratoma sections generated with 24h OS*KM iPSCs with representation of the three germ layers (ectoderm - Ec: keratinizing epithelium; mesoderm - M: striated muscles; endoderm - En: cuboidal epithelium). d , Bright-field and Oct4-GFP merged images of the gonads from E13.5 24h OS*KM iPSC chimeric embryos. e , All-iPSC pups generated by tetraploid (4N) complementation assays with 24h OS*KM iPSC#l line. 12 aggregates were transferred to a pseudopregnant CD-1 (white) female. f , Representative brightfield and Oct4-GFP merged overview images showing Oct4-GFP MEFs reprogrammed with tet-inducible lentiviral Sox-T2A-Klf4 vectors carrying wild-type Sox2, Sox2c17, Sox17 EK , or Sox2-17, 21 dpi, scale<1 mm. g , Phase-contrast and Oct4-GFP merged microscopy image of two-factor mouse S*K iPSC clonal line generated in (f) at passage three, scale = 100 μm. h , PCR genotyping of two mouse S*K iPSC lines from (f-g). i , Immunostaining of a mouse S*K iPSC line for pluripotency markers Nanog and SSEA-1. Nuclei were stained with Hoechst 33342, scale = 100 μm. j , H&E staining of teratoma sections generated with S*K mouse iPSC line with representation of three germ layers (ectoderm - Ec: keratinizing epithelium; mesoderm - M: striated and smooth muscles; endoderm - En: cuboidal epithelium). k , Western blot of whole-cell lysates from HEK293T transfected with pCXLE-Oct4-P2A-Sox-T2A-KIf4-E2A-cMyc, pCXLE-Oct4-P2A-KIf4-IRES-Sox episomal vectors carrying mouse Sox2, Sox2 AV , or Sox2-17. l , Phase-contrast microscopy image of two-factor human iPSC line generated with monocistronic retroviral OCT4 and SOX2-17 at passage two, scale = 200 μm. m , Immunostaining of a human OS* iPSC line for pluripotency markers NANOG and TRA1-81. Nuclei were stained with Hoechst 33342, scale = 100 μm. n , H&E staining of teratoma sections generated with OS* human iPSC line with representation of three germ layers (ectoderm - Ec: neural rosettes; mesoderm - M: cartilage, bone, endothelium; endoderm - En: gut and lung epithelium). o , Western blot of whole-cell lysates from HEK293T transfected with the original episomal pCXLE-SOX2-F2A-KLF4 construct, as well as generated in this study P2A vectors: pCXLE-SOX2-P2A-KLF4, pCXLE-SOX2 AV -P2A-KLF4 and pCXLE-SOX2-17-P2A-KLF4. p, PCR genotyping of episomal iPSC lines generated from cynomolgus macaque fibroblasts at passage three. q , Chromosomal spreads of two integration-free cynomolgus macaque iPSC lines. r, Phase-contrast image of integration-free cynomolgus macaque iPSC#11 from panel o at passage seven, scale = 200μm. s , Immunostaining of cynomolgus integration-free iPSC line for NANOG and OCT4. Nuclei were stained with Hoechst 33342, scale = 100 μm. t , H&E staining of teratoma sections generated with cynomolgus integration-free iPSC line with representation of three germ layers (ectoderm - Ec: neural rosettes; mesoderm - M: cartilage, smooth muscles; endoderm - En: cuboidal epithelium). u , A representative whole-well scan of alkaline phosphatase (AP) staining for episomal reprogramming of bovine fetal fibroblasts on day 21 after nucleofection with episomal OSKML (omitting P53 knockdown). v , PCR genotyping of episomal OSKML bovine iPSC lines from (u) at passage six. w , Phase-contrast image of integration-free bovine iPSC line at passage eight from (v), scale = 200μm. x , A representative chromosomal spread of integration-free bovine iPSC line from (v). y , Immunostaining of bovine integration-free iPSC line for SOX2 and OCT4. Nuclei were stained with DAPI, scale = 200 μm. z , PCR genotyping of episomal iPSC lines generated from dermal fibroblasts of aged male (AG04148) at passage three. aa-ab, Karyotyping of human integration-free iPSC lines generated from newborn foreskin fibroblasts (young, Y) or 56-year-old male fibroblast (old, O) using chromosomal spreads ( aa ) or ekaryotyping based on RNA-seq data (ab).

Article Snippet: The pMX-Sox2/Sox17 chimeric TF vectors were based on Addgene ID 13367 and the tet-inducible pHAGE2-tetO-Oct4-P2A-Sox2-17-T2A-Klf4-E2A-cMyc (OS*KM) and pHAGE-tetO-Sox2-17-T2A-Klf4-E2A-cMyc (S*KM) vectors were based on Addgene ID 136551 and 136541, respectively .

Techniques: Titration, Methylation Sequencing, DNA Methylation Assay, Generated, Staining, Microscopy, Immunostaining, Western Blot, Transfection, Construct, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Enhancing Sox/Oct cooperativity induces higher-grade developmental reset

doi: 10.1101/2022.09.23.509242

Figure Lengend Snippet: a, Schematic representation of time course tet-inducible lentiviral reprogramming experiment. b, Time course reprogramming of Oct4-GFP MEFs induced with OSKM or SKM carrying either Sox2 (S) or Sox2-17 (S*) for the given number of days; the iPSC colonies were counted on 9 dpi. c , Representative brightfield and Oct4-GFP merged overview images of MEFs induced for 4 days with OSKM or OS*KM, images taken on 9 dpi, scale = 2 mm. d , Reprogramming of Oct4-GFP MEFs using episomal OKS (pCXLE-Oct4-P2A-KIf4-IRES-Sox) carrying either wild-type Sox2 or Sox2-17. 1.5×10 5 cells were transfected with Fugene6. e , All-iPSC pups generated by the tetraploid (4N) complementation assay with epi-OKS* iPSC#l. 20 aggregates were transferred to two pseudopregnant CD-1 (white) females. f , Percentage of 4N-aggregated embryos that gave rise to adult healthy mice (survived till at least 3 months), including our previously published results . Only XY lines were plotted. Data are represented as the mean of all tested lines. g , Reprogramming of human fetal fibroblasts (CRL-2097) with monocistronic retroviral OSKM carrying either wild-type SOX2 or SOX2 – , , – c17 (SOX2-17, S*). The volumes of viral supernatants were adjusted according to the qPCR titration. 10 4 transduced cells were plated on a feeder layer of 6 well-plates. TRA1-60+ colonies were counted after two weeks of infection. Error bars represent SD; n = 3. h , Representative whole-well scan of (g). i , Two-factor (OS) reprogramming of human fibroblasts with monocistronic retroviral vectors. TRA1-60+ colonies were counted after four weeks of infection. Error bars represent SD; n = 3. j , Representative whole-well scan of (i). k, TRA1-60 staining of human fetal (CRL-2097) fibroblasts reprogrammed with self-replicating RNA (VEE) vectors carrying either OKSiG or OKS*iG reprogramming cassettes. l-o , Whole-well scans of alkaline phosphatase (AP) staining for episomal reprogramming of (l) 56-year-old human male dermal fibroblasts on day 25, ( m ) Cynomolgus macaque ( Macaca fascicularis ) fibroblasts on day 25, (m) bovine fetal fibroblasts on day 21, ( o ) porcine fetal fibroblasts on day 21 after nucleofection. p-q , Hierarchical clustering analysis of human ESC and integration-free iPSC lines derived from human newborn foreskin (young, Y) or 56-year-old human dermal (old, O) fibroblasts using episomal OSKML (pCXLE-OCT4+shP53, L-MYC-F2A-LIN28 and SOX-P2A-KLF4 carrying SOX2, SOX AV , or SOX2-17) based on: global gene expression (RNA-seq), TPM ≥1 ( p ) or global methylome (RRBS) ( q ). Clustering was based on Euclidean distance. r , Comparison of the number of genes that lost imprinting in 23 DMRs according to RRBS data. s , A model of the advantageous effects of highly cooperative Sox factors on cell fate reset.

Article Snippet: The pMX-Sox2/Sox17 chimeric TF vectors were based on Addgene ID 13367 and the tet-inducible pHAGE2-tetO-Oct4-P2A-Sox2-17-T2A-Klf4-E2A-cMyc (OS*KM) and pHAGE-tetO-Sox2-17-T2A-Klf4-E2A-cMyc (S*KM) vectors were based on Addgene ID 136551 and 136541, respectively .

Techniques: Transfection, Generated, Titration, Infection, Staining, Derivative Assay, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Enhancing Sox/Oct cooperativity induces higher-grade developmental reset

doi: 10.1101/2022.09.23.509242

Figure Lengend Snippet: a , Spearman correlation of time course ATAC-seq reads for naïve-to-primed differentiation mouse ESC samples . b-c , TOBIAS footprinting analysis of (a) using MEME TF motif database of ESCs versus day one EpiLC samples (b), and day one versus day two EpiLC samples (c). d , EMSAs of whole-cell lysates showing endogenous levels of Sox2 and Oct4 that can bind Nanog promoter DNA element labeled with Cy5. Arrow heads indicate DNA bound by Oct4 (yellow), Sox2 (blue), or the heterodimer (green). All cells were grown without feeders. e , Western blot of lysates used in . f , A supershift assay using anti-Oct4 and anti-Sox2 antibodies to confirm the identity of protein/DNA complexes in whole-cell lysate EMSAs. g , EMSAs of 6 mouse iPSC and ESC lines , of which epi-OKSM1 and epi-OKSM3 were incapable of generating all-iPSC mice (4N-off). The lines were grown in 2iLIF on gelatin-coated plates, then split either in KSR-LIF media (left panel) or again in 2iLIF media (right panel). h , Western blot of lysates used in . i , Immunostaining for naïve pluripotency marker KLF17 of primed-to-naïve converted human iPSCs on day 6 after transduction with constitutive lentiviral vectors (pHAGE2-EF1α). j , EMSAs of integration-free clonal mEpiSC lines primed-to-naïve converted using episomal mCherry-T2A-SOX-KLF4 vectors carrying wild-type SOX2, SOX2 AV , or SOX2-17. All lines were converted and expanded on a feeder layer. The error bars represent SEM, statistical significance was calculated between top three out of six lines converted by each cocktail with Student’s t-test. k , SoxOct model of highgrade developmental reset.

Article Snippet: The pMX-Sox2/Sox17 chimeric TF vectors were based on Addgene ID 13367 and the tet-inducible pHAGE2-tetO-Oct4-P2A-Sox2-17-T2A-Klf4-E2A-cMyc (OS*KM) and pHAGE-tetO-Sox2-17-T2A-Klf4-E2A-cMyc (S*KM) vectors were based on Addgene ID 136551 and 136541, respectively .

Techniques: Footprinting, Labeling, Western Blot, Immunostaining, Marker, Transduction

Journal: bioRxiv

Article Title: Enhancing Sox/Oct cooperativity induces higher-grade developmental reset

doi: 10.1101/2022.09.23.509242

Figure Lengend Snippet: a, FACS of mEpiSCs transfected with episomal mCherry-T2A-SOX-KLF4 vectors using Lipofectamin Stem reagent at day 2. b , Representative phase-contrast/epi-mCherry/Oct4-GFP merged overview images of clonal primed-to-naïve converted mEpiSCs using episomal mCherry-T2A-SOX-P2A-KLF4 vectors grown in KSR-LIF media on a C3H feeder layer, at passage 4, scale=500μm. The same number of cells were plated for each line. At least 100 colonies were quantified from three random overview images of each line, the error bars represent SEM, statistical significance was calculated using Student’s t-test. c , Western blot of lysates used in .

Article Snippet: The pMX-Sox2/Sox17 chimeric TF vectors were based on Addgene ID 13367 and the tet-inducible pHAGE2-tetO-Oct4-P2A-Sox2-17-T2A-Klf4-E2A-cMyc (OS*KM) and pHAGE-tetO-Sox2-17-T2A-Klf4-E2A-cMyc (S*KM) vectors were based on Addgene ID 136551 and 136541, respectively .

Techniques: Transfection, Western Blot

Journal: Oncology Letters

Article Title: SOX2 enhances cell survival and induces resistance to apoptosis under serum starvation conditions through the AKT/GSK-3β signaling pathway in esophageal squamous cell carcinoma

doi: 10.3892/ol.2021.12530

Figure Lengend Snippet: Activation of AKT by adenoviral vector-mediated overexpression of SOX2. (A) KYSE30 and TE4 cells, which are known to express SOX2 at low levels, were infected with Ad-SOX2 or Ad-control. Cells were harvested 48 h after infection and subjected to immunoblot analysis of SOX2, p-AKT (S473), total AKT, p-ERK and total ERK expression. (B) Time course of changes in the levels of SOX2, p-AKT (T308), p-AKT (S473) and total AKT. TE4 cells were infected with Ad-SOX2 and harvested at the indicated time points after infection, then subjected to immunoblot analysis. (C) Effect of MK2206. TE6 cells, which express a high endogenous level of SOX2, were treated for 48 h with MK2206, an AKT inhibitor, at the indicated concentrations for 48 h, then subjected to immunoblot analysis of SOX2, p-AKT (S473) and total AKT levels. (D) TE6 cells were treated with 2 µM MK2206 for different time periods and harvested at the indicated time points, then subjected to immunoblot analysis of SOX2, p-AKT (S473) and total AKT expression. For all experiments, β-actin was detected as a loading control. The numbers presented below the gels represent the expression levels of each protein relative to those of β-actin. Values were normalized so that β-actin expression in each well had a value of 1. The ratio of p-AKT (S473 or T308) to total AKT is shown. Ad, adenovirus vector; p-, phosphorylated.

Article Snippet: Recombinant adenovirus vectors expressing SOX2 or control green fluorescent protein (GFP), which we here refer to as Ad-SOX2 and Ad-control, respectively, were obtained from ViGene.

Techniques: Activation Assay, Plasmid Preparation, Over Expression, Infection, Control, Western Blot, Expressing

Journal: Oncology Letters

Article Title: SOX2 enhances cell survival and induces resistance to apoptosis under serum starvation conditions through the AKT/GSK-3β signaling pathway in esophageal squamous cell carcinoma

doi: 10.3892/ol.2021.12530

Figure Lengend Snippet: Activation of AKT by SOX2 under serum starvation conditions. (A) KYSE30 cells were infected with Ad-SOX2 or Ad-control. After a 24-h incubation, cells were cultured in serum-free medium for 24 h. Subsequently, cells were incubated in growth medium without or with serum (10%) for 15 min (− serum or + serum, respectively), harvested and subjected to immunoblot analysis of SOX2, p-AKT (S473) and total AKT expression. (B) KYSE140 cells were transfected with one of two siRNAs targeting SOX2 (SOX2 siRNA #1 and #2) or with a control siRNA. After a 48-h incubation, cells were cultured in serum-free medium for 12 h. Subsequently, cells were incubated in growth medium without or with serum (10%) for 15 min (− serum or + serum, respectively), harvested and subjected to immunoblot analysis of SOX2, p-AKT (S473) and total AKT expression. For all experiments, β-actin was detected as a loading control. The numbers presented below the gels represent the expression levels of each protein relative to those of β-actin. Values were normalized so that β-actin expression in each well had a value of 1. The ratio of p-AKT (S473) to total AKT is shown. Ad, adenovirus vector; p-, phosphorylated; siRNA, small interfering RNA.

Article Snippet: Recombinant adenovirus vectors expressing SOX2 or control green fluorescent protein (GFP), which we here refer to as Ad-SOX2 and Ad-control, respectively, were obtained from ViGene.

Techniques: Activation Assay, Infection, Control, Incubation, Cell Culture, Western Blot, Expressing, Transfection, Plasmid Preparation, Small Interfering RNA

Journal: Oncology Letters

Article Title: SOX2 enhances cell survival and induces resistance to apoptosis under serum starvation conditions through the AKT/GSK-3β signaling pathway in esophageal squamous cell carcinoma

doi: 10.3892/ol.2021.12530

Figure Lengend Snippet: Promotion of cell survival under serum starvation conditions and resistance to apoptosis through activation of the AKT/GSK-3β signaling pathway by SOX2. (A) TE4 cells were infected with Ad-SOX2 or Ad-control, and then cultured in serum-free medium (− serum) or medium with serum (+ serum). Relative cell viabilities were measured at the indicated time points. (B) TE4 cells were infected with Ad-SOX2 or Ad-control, then treated 24 h later with the indicated concentrations of MK2206 in serum-free medium. Cell viabilities were measured 48 h after MK2206 treatment. (C) TE4 cells were infected with Ad-SOX2 or Ad-control, then treated 24 h later with the indicated concentrations of doxorubicin in serum-free medium. Cell viabilities were measured 48 h after doxorubicin treatment. (D) TE4 cells were infected with Ad-SOX2 or Ad-control, then treated 24 h later with 3 µM doxorubicin and 2 µM MK2206 in serum-free medium. After a 48-h incubation, cells were subjected to cell viability assays. (E) TE4 cells were infected with Ad-SOX2 or Ad-control, then treated 24 h later with or without 3 µM doxorubicin in serum-free medium. Cells were harvested 48 h after doxorubicin treatment and subjected to immunoblot analysis of PARP, cleaved PARP, p-GSK-3β and total GSK-3β expression. β-actin was detected as a loading control. The numbers presented above and below the gel for PARP and cleaved PARP represent the expression levels of PARP and cleaved PARP relative to those of β-actin, respectively. The numbers presented below the gels for p-GSK-3β and total GSK-3β represent the expression levels of p-GSK-3β and total GSK-3β relative to those of β-actin, respectively. Values were normalized so that β-actin expression in each well had a value of 1. The ratio of p-GSK-3β to total GSK-3β is shown. (F) TE4 cells were infected with Ad-SOX2, and treated 24 h later with AR-A014418 or DMSO. The cells were treated 24 h later with or without 3 µM doxorubicin in serum-free medium. Cell viability was measured 72 h after doxorubicin treatment. All data are presented as the mean ± SD (n=3). *P<0.01 analyzed by two-tailed unpaired Student's t-test. OD, optical density; Ad, adenovirus vector; p-, phosphorylated; GSK-3β, glycogen synthase kinase-3β; PARP, poly (ADP-ribose) polymerase.

Article Snippet: Recombinant adenovirus vectors expressing SOX2 or control green fluorescent protein (GFP), which we here refer to as Ad-SOX2 and Ad-control, respectively, were obtained from ViGene.

Techniques: Activation Assay, Infection, Control, Cell Culture, Incubation, Western Blot, Expressing, Two Tailed Test, Plasmid Preparation

Journal: Oncology Letters

Article Title: SOX2 enhances cell survival and induces resistance to apoptosis under serum starvation conditions through the AKT/GSK-3β signaling pathway in esophageal squamous cell carcinoma

doi: 10.3892/ol.2021.12530

Figure Lengend Snippet: PTEN/PI3K/PDK1- and mTORC2-mediated phosphorylation of AKT in SOX2-overexpressing cells. (A) KYSE140 and TE6 cells were treated with the indicated concentrations of LY294002, a PI3K inhibitor, in serum-free medium for 48 h, harvested and subjected to immunoblot analysis of SOX2, p-AKT (S473) and total AKT expression. (B) KYSE70, KYSE140 and TE6 cells were transfected with either SOX2 siRNA#1 or with a control siRNA. After a 48-h incubation in serum-free medium, cells were harvested and subjected to immunoblot analysis of SOX2, PTEN, p-AKT (S473) and total AKT expression. TE4 cells infected with Ad-SOX2 and KYSE140 cells were treated for 1 h in serum-free medium containing the indicated concentrations of (C) GSK2334470, a PDK1 inhibitor, or (D) AZD8055, an mTORC2 inhibitor. Cells were harvested and subjected to immunoblot analysis of SOX2, p-AKT (T308 or S473) and total AKT expression. For all experiments, β-actin was detected as a loading control. The numbers presented below the gels represent the expression levels of each protein relative to those of β-actin. Values were normalized so that β-actin expression in each well had a value of 1. The ratio of p-AKT (S473 or T308) to total AKT is shown. PI3K, phosphatidylinositol 3-kinase; Ad, adenovirus vector; p-, phosphorylated; siRNA, small interfering RNA; PDK1, phosphoinositide-dependent protein kinase 1; mTORC2, mammalian target of rapamycin complex 2.

Article Snippet: Recombinant adenovirus vectors expressing SOX2 or control green fluorescent protein (GFP), which we here refer to as Ad-SOX2 and Ad-control, respectively, were obtained from ViGene.

Techniques: Phospho-proteomics, Western Blot, Expressing, Transfection, Control, Incubation, Infection, Plasmid Preparation, Small Interfering RNA

Journal: Oncotarget

Article Title: Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma

doi: 10.18632/oncotarget.21523

Figure Lengend Snippet: ( A ) Immunoblot analysis of SOX2 in indicated cells. The expression level of β-actin is shown as a control. ( B ) The intensity of SOX2 staining was assigned the following scores: none = -, weak = +, moderate = ++, and strong = +++ expression. Examples of representative immunohistochemistry results are shown. Bars, 50 μm. ( C ) SOX2 expression in primary pulmonary adenocarcinoma samples of 40 patients, lung SCC samples of 40 patients and esophageal SCC samples of 40 patients who underwent surgical tumor resection at the Kawasaki Hospital Attached to Kawasaki Medical School between 2007 and 2012. Percentage values are given in parentheses.

Article Snippet: A plasmid vector expressing sh SOX2 : pBAsi-mU6 sh SOX2 was constructed by ligating a shRNA sequence for SOX2 (GCTCTTGGCTCCATGGGTT) into pBAsi-mU6 Pur (Takara Bio Inc. Otsu.

Techniques: Western Blot, Expressing, Staining, Immunohistochemistry

Journal: Oncotarget

Article Title: Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma

doi: 10.18632/oncotarget.21523

Figure Lengend Snippet: ( A ) Design of Artificial zinc finger protein (AZP) to target a 19 bp sequence (-161: TGCCCCCTCCTCCCCCGGC:-143) in human SOX2 distal promoter region. TSS; transcription start site. ( B ) The Artificial Transcription Factor (ATF) contains the KOX suppressor domain, a nuclear localization signal (NLS), the Artificial zinc finger protein (AZP) and a FLAG tag. We termed this ATF as ATF/ SOX2 . ( C ) Schematic representation of Ad-null, Ad-sh SOX2 and Ad-ATF/ SOX2. The PCR-generated expression cassette of ATF/ SOX2 from pcDNA3.1 ATF/ SOX2 or sh SOX2 from pBAsi-mU6 sh SOX2 (described in Materials and Methods section) were subcloned into the linearized E1 deleted adenovirus type 5 genome. E1; Adenovirus early region 1, E3; Adenovirus early region 3, LITR; Left Inverted Terminal Repeat, RITR; Right Inverted Terminal Repeat.

Article Snippet: A plasmid vector expressing sh SOX2 : pBAsi-mU6 sh SOX2 was constructed by ligating a shRNA sequence for SOX2 (GCTCTTGGCTCCATGGGTT) into pBAsi-mU6 Pur (Takara Bio Inc. Otsu.

Techniques: Sequencing, FLAG-tag, Generated, Expressing

Journal: Oncotarget

Article Title: Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma

doi: 10.18632/oncotarget.21523

Figure Lengend Snippet: ( A ) Schematic representation of SOX2 distal and proximal promoter reporter constructs. TSS; transcription start site. Luc; Luciferase. ( B ) Transient transfection reporter assays in lung SCC cells and esophageal squamous carcinoma cells with the indicated luciferase reporter constructs (2 μg, pGL4), effector constructs (2 μg, pcDNA3.1) and pCMV. β-gal (1 μg). Results are presented as fold induction of relative light units normalized to β-galactosidase activity relative to that observed for control constructs. Statistical analysis was performed using Student’s t test (two-tailed, unpaired). Statistical significance was defined as * p < 0.01 vs pcDNA3.1 transfected group. ( C ) Ad-ATF/ SOX2 dose dependently increased ATF/ SOX2 fused to the FLAG epitope and suppressed SOX2 expression in EBC2 cells, TE1 and TE4 cells 48 hours after infection. SOX2 expression was not changed after Ad-null infection in these kinds of cells.

Article Snippet: A plasmid vector expressing sh SOX2 : pBAsi-mU6 sh SOX2 was constructed by ligating a shRNA sequence for SOX2 (GCTCTTGGCTCCATGGGTT) into pBAsi-mU6 Pur (Takara Bio Inc. Otsu.

Techniques: Construct, Luciferase, Transfection, Activity Assay, Two Tailed Test, FLAG-tag, Expressing, Infection

Journal: Oncotarget

Article Title: Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma

doi: 10.18632/oncotarget.21523

Figure Lengend Snippet: Immunoblot analysis shows Ad-ATF /SOX2 increased CDKN1A expression more than Ad-sh SOX2 48 hours after adenoviral infections in EBC2 lung SCC cells, TE1 and TE4 esophageal SCC cells.

Article Snippet: A plasmid vector expressing sh SOX2 : pBAsi-mU6 sh SOX2 was constructed by ligating a shRNA sequence for SOX2 (GCTCTTGGCTCCATGGGTT) into pBAsi-mU6 Pur (Takara Bio Inc. Otsu.

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma

doi: 10.18632/oncotarget.21523

Figure Lengend Snippet: ( A ) Ad-ATF/ SOX2 more significantly inhibited cell growth of SOX2-expressing EBC2 lung SCC cells and TE1, TE4 esophageal SCC cells than Ad-sh SOX2 . Cell viability was assessed 48 hours after adenoviral infection with a TC20 automated cell counter. Results represent the mean ± SD ( n = 3). Statistical analysis was performed using Student’s t test (two-tailed, unpaired). Statistical significance was defined as $ p < 0.01 vs Ad-sh SOX2 treated group. ( B ) Colony formation of EBC2 lung SCC cells, TE1 and TE4 esophageal SCC cells treated with Ad-null, Ad-sh SOX2 or Ad-ATF/ SOX2 . 7 to 14 days after treatment, cells were fixed and stained with Diff-Quik. Representative pictures of experiments performed in triplicate are shown. ( C ) Mean colony number was derived from quantitation of triplicate dishes for each treatment and was arbitrarily set to 100%. Data are shown relative to the control group. Results represent the mean ± SD ( n = 3). Statistical analysis was performed using Student’s t test (two-tailed, unpaired). Statistical significance was defined as * p < 0.01 vs Ad-null treated group at the same MOI; $ p < 0.01 vs Ad-sh SOX2 treated group at the same MOI.

Article Snippet: A plasmid vector expressing sh SOX2 : pBAsi-mU6 sh SOX2 was constructed by ligating a shRNA sequence for SOX2 (GCTCTTGGCTCCATGGGTT) into pBAsi-mU6 Pur (Takara Bio Inc. Otsu.

Techniques: Expressing, Infection, Two Tailed Test, Staining, Diff-Quik, Derivative Assay, Quantitation Assay

Journal: Oncotarget

Article Title: Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma

doi: 10.18632/oncotarget.21523

Figure Lengend Snippet: ( A ) Volume of the tumors derived from EBC2 lung SCC cells treated with Ad-null, Ad-sh SOX2 or Ad-ATF/ SOX2 at a MOI of 250 is shown. The volume was monitored over time (days) after inoculation of tumor cells. Fifteen mice were studied in each group. Tumor growth is expressed as mean tumor volume; bars represent SD. Statistical analysis was performed using Student’s t test (two-tailed, unpaired). Statistical significance was defined as * p < 0.01. ( B ) Macroscopic appearance of EBC2 lung SCC tumors in xenograft mice at 39 days after inoculation.

Article Snippet: A plasmid vector expressing sh SOX2 : pBAsi-mU6 sh SOX2 was constructed by ligating a shRNA sequence for SOX2 (GCTCTTGGCTCCATGGGTT) into pBAsi-mU6 Pur (Takara Bio Inc. Otsu.

Techniques: Derivative Assay, Two Tailed Test

Journal: eLife

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.7554/eLife.26733

Figure Lengend Snippet: ( A–D ) Pluripotency genes expressions analyzed by qPCR in HCC827 ( A ), PDCL#24 ( B ), A549 ( C ), and H1299 cells ( D ) with NFATc2 knockdown or overexpression. ( E ) Effects of stable NFATc2 knock-down, knockout or overexpression on SOX2 expression in respective lung cancer cells by Western blot analysis. ( F–G ) Pluripotency genes expression analyzed by qPCR in HCC827 ( F ), and PDCL#24 cells ( G ) treated with CSA or FK506, respectively, for 24 hr. ( H–I ) Effects of transient knockdown of PPP3R1 on pluripotency gene expressions analyzed by qPCR in HCC827 ( H ) and PDCL#24 cells ( I ). ( J–K ) Effects of transient knockdown of NFATc1 on pluripotency gene expressions analyzed by qPCR in HCC827 ( J ) and PDCL#24 cells ( K ). *p<0.05, **p<0.01 versus control by t-test. Error bar indicates the mean ±S.D. for at least three independent replicates. DOI: http://dx.doi.org/10.7554/eLife.26733.017

Article Snippet: Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Knockdown, Over Expression, Knock-Out, Expressing, Western Blot, Control

Journal: eLife

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.7554/eLife.26733

Figure Lengend Snippet: ( A ) Computational prediction of NFAT binding sites (marked in red) on 5’ and 3’ SOX2 regulatory regions (Region 1 to 4). TSS: transcription start site. ( B ) Transcriptional activities of the respective SOX2 regions 1–4 of H441 cells analyzed by luciferase reporter assays. *p<0.05, **p<0.01 versus control by Student’s t-test. Error bars indicate the mean ±SD for at least three independent replicates. ( C–D ) Luciferase reporter activities of mutant or wild-type SOX2 reporters of A549 ( C ), or H1299 cells ( D ), with or without NFATc2 stable overexpression. *p<0.05, **p<0.01, comparison with RFP; # p<0.05, ## p<0.01, wild type versus mutant in RFP-NFATc2 cells by t-test. Error bars indicate the mean ±SD for at least three independent replicates. DOI: http://dx.doi.org/10.7554/eLife.26733.021

Article Snippet: Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Binding Assay, Luciferase, Control, Mutagenesis, Over Expression, Comparison

Journal: eLife

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.7554/eLife.26733

Figure Lengend Snippet: ( A ) Genome browser view of NFAT binding sites and H3K27Ac marks (lowest panel) on SOX2 regulatory regions (regions 2 and 3 indicated in ) analyzed in A549 cells. ( B ) Transcriptional activities of sites 1–5 by dual luciferase reporter assays in H441 cells. ( C ) Transcriptional activities of the indicated putative NFAT binding sites by respective luciferase reporters in H441 cells with transient NFATc2 over-expression, with or without CSA treatment. ( D ) Effects of site-directed mutagenesis of the indicated putative NFAT binding sequences by respective luciferase reporter assays in H441 cells with transient NFATc2 overexpression. For B-D, *p<0.05, **p<0.01 versus control by t-test. Error bar indicates the mean ±S.D. for at least three independent replicates. ( E ) Alignment of sites 4 and 5 genomic sequences showing highly homologous regions (gray) in different mammalian species, with putative NFAT binding sites highlighted in red. ( F–G ) ChIP–qPCR assays of NFATc2 binding to the indicated SOX2 sites in A549 cells with or without stable NFATc2 overexpression ( F ), or HCC827 cells with or without NFATc2 knockout ( G ). # p<0.05, ## p<0.01 versus IgG control,m **p<0.01 versus vector control by t-test. Error bar indicates the mean ±S.D. for at least three independent replicates. ( H ) Correlation of immunohistochemical expressions of NFATc2 and SOX2 in 92 moderately to poorly differentiated human lung adenocarcinoma by χ 2 -test. Pearson R, Pearson correlation coefficient. ( I ) Correlation of mRNA levels of SOX2 and NFATc2 in a panel of lung AD cell lines analyzed by q-PCR and Pearson correlation test. ( J ) Expression of NFATc2 and SOX2 in A549 cells with or without NFATc2 overexpression and SOX2 stable knockdown by Western blot. ( K–L ) Effect of SOX2 knockdown on tumorsphere formation ( K ), cell migration and invasion ability ( L ), of A549 cells with NFATc2 overexpression. *p<0.05, **p<0.01 versus control by t-test. Error bars indicate the mean ±SD for at least three independent replicates. ( M ) In vivo tumorigenicity of A549 cells with NFATc2 overexpression and SOX2 knockdown by subcutaneous inoculation of 1 × 10 4 cells in SCID mice. **p<0. 0001 versus control by two-way ANOVA. Error bar indicates the mean ±SD of tumor volumes of six mice. ( N ) Effect of NFATc2 knockdown on SOX2 expression in A549 CR cells analyzed by immunoblot. ( L ) Effect of SOX2 knockdown on cisplatin sensitivity by MTT assay of A549 cells with NFATc2 overexpression. ## p<0.01, versus vector control, **p<0.01 versus RFP-NFATc2_Sh-Ctrl by t-test. Error bar indicates the mean ±SD for at least three independent replicates. DOI: http://dx.doi.org/10.7554/eLife.26733.019 10.7554/eLife.26733.020 Figure 6—source data 1. Statistical analyses for . DOI: http://dx.doi.org/10.7554/eLife.26733.020

Article Snippet: Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Binding Assay, Luciferase, Over Expression, Mutagenesis, Control, Genomic Sequencing, ChIP-qPCR, Knock-Out, Plasmid Preparation, Immunohistochemical staining, Expressing, Knockdown, Western Blot, Migration, In Vivo, MTT Assay

Journal: eLife

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.7554/eLife.26733

Figure Lengend Snippet: ( A–D ) Effects on ALDH + , CD44 + and ALDH + /CD44 + cell populations by flow cytometry analysis of HCC827 with NFATc2 knockdown ( A ), NFATc2 knockout ( B ), or NFATc2 inhibition by CSA or FK506 ( C ), and of A549 cells with NFATc2 overexpression ( D ). ( E–H ) Effects on ALDH1A1 mRNA expression by qPCR analysis of cancer cells with NFATc2 knockdown ( E ), NFATc2 inhibition by CSA or FK506 ( F ), NFATc2 up-regulation ( G ), or of A549 with induced cisplatin resistance ( H ). ( I ) Effects of SOX2 knockdown on ALDH + , CD44 + and ALDH + /CD44 + cell populations by flow cytometry in A549 with NFATc2-overexpression. ( J ) Expression of SOX2 and ALDH1A1 transcripts in A549 cells with NFATc2 overexpression and SOX2 knockdown. ( K ) Representative images of A549 xenografts with or without NFATc2 overexpression immunohistochemically stained for NFATc2, SOX2 and ALDH1A1, respectively. Scale bars, 50 µm. ( L ) Conserved SOX2 binding sequences (ATTCA) at ALDH1A1 enhancer region by ChIP-seq of PDCL#24 cells, aligned with homologous mammalian sequences and H3K27Ac peaks of A549 cells from published databases. ( M ) Detection of endogenous SOX2 binding to ALDH1A1 sites by ChIP–qPCR analysis in PDCL#24 cells. ( N ) Luciferase reporter activities at sites 1 and 2 of ALDH1A enhancer region by dual luciferase reporter assay in A549 cells with SOX2 overexpression. ( O–P ) Effects of transient ALDH1A1 suppression on A549 with upregulated NFATc2 with respect to invasion and migration ( O ), and cisplatin sensitivity. ## p<0.01, versus RFP_Scramble by t-test. ( P ). ( Q ) Correlation between ALDH1A1 and SOX2 expressions by IHC in human lung adenocarcinomas by χ 2 -test. *p<0.05, **p<0.01 versus control by t-test. Error bar indicates the mean ±S.D. for at least three independent replicates. DOI: http://dx.doi.org/10.7554/eLife.26733.022 10.7554/eLife.26733.023 Figure 7—source data 1. Statistical analyses for . DOI: http://dx.doi.org/10.7554/eLife.26733.023

Article Snippet: Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Flow Cytometry, Knockdown, Knock-Out, Inhibition, Over Expression, Expressing, Staining, Binding Assay, ChIP-sequencing, ChIP-qPCR, Luciferase, Reporter Assay, Migration, Control

Journal: eLife

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.7554/eLife.26733

Figure Lengend Snippet: ( A ) ROS levels detected by flow cytometry in A549 and A549 CR cells. ( B–C ) ROS levels in HCC827 cells ( B ) and PDCL#24 cells ( C ) with or without NFATc2 stable knockdown. ( D ) ROS levels in HCC827 cells with or without NFATc2 knockout. ( E–F ) Cisplatin sensitivity expressed as IC 50 by MTT assays of NFATc2-silenced PDCL#24 cells treated with increasing doses of NAC ( E ), or NFATc2-overexpressing A549 cells treated with the oxidizing agent BSO ( F ), respectively. *p<0.05, **p<0.01 versus vector control without REDOX reagents; ## p<0.01 versus the corresponding treatment control by t-tests. Error bar indicates the mean ±S.D. for three independent replicates. ( G–H ) Effects of increasing doses of NAC on tumorsphere formation ability of HCC827 ( G ) cells and PDCL#24 cells ( H ). *p<0.05, **p<0.01 versus corresponding treatment controls, ## p<0.01 versus vector control by t-test. Error bar indicates the mean ±S.D. for three independent replicates. ( I ) Effects of increasing doses of NAC on cell migration and invasion of HCC827 cells with NFATc2 down-regulation by 2 sh-RNA knockdown sequences. ( J–K ) ROS levels in NFATc2-overexpressing A549 cells with stable SOX2 ( J ) or transient ALDH1A1 ( K ) knockdown. *p<0.05, **p<0.01 versus respective control by t-test. Error bar indicates the mean ±S.D. for at least three independent replicates. DOI: http://dx.doi.org/10.7554/eLife.26733.028 10.7554/eLife.26733.029 Figure 8—source data 1. Statistical analyses for . DOI: http://dx.doi.org/10.7554/eLife.26733.029

Article Snippet: Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Flow Cytometry, Knockdown, Knock-Out, Plasmid Preparation, Control, Migration

Journal: eLife

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.7554/eLife.26733

Figure Lengend Snippet: Expressions of total β-catenin, active β-catenin (non-phosphorylated), and phosphorylated β-catenin (p-β-catenin) analyzed by immunoblot in A549 with or without NFATc2 overexpression and SOX2 knockdown. DOI: http://dx.doi.org/10.7554/eLife.26733.027

Article Snippet: Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Western Blot, Over Expression, Knockdown

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: 3q26 genes SOX2 and OPA1 are targeted in glioma. A. Schematic illustration of chromosome segment 3q26 with indication of relative position and orientation of PIK3CA, MFN1, SOX2 and OPA1 genes. B. Frequency of genomic amplification (red) and deletion (blue) of 3q26 genes as determined by quantitative PCR of DNA from 129 glioma biopsies. C. Protein levels of SOX2 and OPA1 determined by mass spectrometry in six representative glioma biopsies (T) compared with corresponding non‐tumorous white matter (N). The glioma biopsies were genotyped to be either IDH wild type or mutant, or carrying SOX2 amplification (SOX2 > 2n) or OPA1 deletion (OPA1 < 2n). “Others” stands for glioma with neither SOX2 amplification nor OPA1 deletion.

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: Amplification, Real-time Polymerase Chain Reaction, Mass Spectrometry, Mutagenesis

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: SOX2 amplification and OPA1 deletion are associated with larger necrotic volumes in the glioma mass. A. Neuroimaging (left) and color modelization (right) of the tumor compartments of the same glioma. B. Neuroimaging of representative glioma cases with neither SOX2 amplification nor OPA1 deletion (left) with SOX2 amplification (center) and OPA1 deletion (right). C. Boxplots visualizing the distribution of the log‐transformed volume of necrotic compartment of gliomas across different statuses of SOX2 and OPA1. The boxes display the median and the interquartile range. Coefficients estimates for a linear model with log‐transformed necrosis volume and adjusted for age and ratio of postoperative to preoperative volume are shown below the boxplots. D. Comparative invasion kinetics of LN319 cells in the presence of autologous necrotic LN319 cells. Parental LN319 (left), LN319 overexpressing cherry‐SOX2 under the control of 30 ng/ml doxycycline (middle) and L319 with OPA1 knocked down by shRNA (right). Invasiveness in absence of necrotic cells was set to 100% (reference score). Significance cutoff: *P < 0.05.

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: Amplification, Transformation Assay, shRNA

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: SOX2 and OPA1 modulate glioma cell invasion in vitro and in vivo. A. Inducible SOX2 overexpression in LN319 cells. LN319 stably transfected with an mCherry‐SOX2 fusion construct under control of a doxycycline‐inducible promoter. Western blot documenting dose‐dependent induction of the fusion protein (anti‐SOX2 staining, two exposure times, left). Fluorescence microscopy verifying effective nuclear localization of mCherry‐SOX2 24 h past induction (middle). SOX2‐dependent modulation of cell invasiveness as investigated by 24 h Boyden chamber assay (right). B. shRNA‐driven OPA1 knock‐down in LN319 cells. Western blot of transfected LN319 cells (left). Verification of knock‐down phenotype by mitochondria morphology in transfected LN319 (middle). Comparative cell invasion in response by 24 h Boyden chamber assay (right).

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: In Vitro, In Vivo, Over Expression, Stable Transfection, Transfection, Construct, Western Blot, Staining, Fluorescence, Microscopy, Boyden Chamber Assay, shRNA

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: A. OPA1 knock‐down increases invasiveness of LN319 cells in vivo. Left: Confocal pictures of zebrafish embryos effectively xenotransplanted with LN319 control cells (shRNA non‐coding, left) or LN319 shOPA1 knock‐down cells (right). LN319 cells (Cell Tracker, red) either established stable cell masses near the site of injection (aggregate growth, top), dispersed throughout the yolk sack (invasive growth, center) or disseminated to the caudal hematopoietic tissue (CHT) of the tail fin (bottom). Inlays indicate absolute animal numbers per condition. Right: Relative quantification of experimental outcomes documenting aggravated invasiveness and dissemination of OPA1 knock‐down vs. respective control cells. B. SOX2 overexpression (OE) increases invasiveness of LN319 cells in vivo. Experimental setup as above, except that TetON (control, left) and TetON mCherry‐SOX2 (SOX2 OE, right) cells had been pretreated with doxycycline (1 µg/ml for 24 h) to selectively induce SOX2 protein formation prior to transplantation. Right: Assay quantification verifying increased invasiveness and dissemination to CHT (by trend) also for SOX2 OE cells when compared to equally treated controls. Significance cut‐off‐: *P < 0.05.

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: In Vivo, shRNA, Stable Transfection, Injection, Over Expression, Transplantation Assay

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: Modulation of SOX2 protein stability by canonical PI3K/AKT signaling. A. Increased PI3K activity fosters SOX2 protein expression in LN319 cells. Cell were stably transfected with Doxycycline‐inducible variants of PIK3CA encoding the catalytic subunit of PI3 kinase p110. From left to right: no insert (control), PIK3CA wild type, and the constitutively active mutant alleles H1047R or E545K. Note elevated SOX2 protein expression in response to aggravated PI3K activity, as indicated by increased pAKT (Ser473) and pS6 (Ser235,236) signatures. Actin staining is shown for loading control. B. Effects of 48 h‐exposure to PI3K/AKT/TOR pathway inhibitors on SOX2 protein levels as assessed by Western blot. SOX2 expression is strongly impaired by pan PI3K‐inhibitor (BYL‐719) and AKT‐inhibitors (AKTi1/2, MK‐2206), but not by further downstream inhibitor rapamycin (anti mTORC1). C. Fluorescence microcopy to document nucleo‐cytoplasmatic displacement and reduction of SOX2 protein expression by PI3K inhibitor MK‐2206. D. Impaired invasiveness of MK‐2206‐treated, SOX2‐depleted LN319 cells as assayed by Boyden chamber experiments. E. Schematic illustration of SOX2 protein expression and turnover in dependence of PI3K/AKT signaling.

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: Activity Assay, Expressing, Stable Transfection, Transfection, Mutagenesis, Staining, Western Blot, Fluorescence

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: SOX2 trans‐activates 3q26 genes PIK3CA and OPA1 . A. Theoretical SOX2‐binding sequences found upstream of PIK3CA, MFN1 and OPA1 transcription initiation sites. B. ChIP of PIK3CA, MFN1 and OPA1 promoter regions containing the potential SOX2‐binding sites (LN319 cells). C. Luciferase assays of PIK3CA, MFN1 and OPA1 promoter regions containing the potential SOX2‐binding sites (HEK293 cells). Co‐transfection of SOX2 expressing vector and comparison between wild‐type and mutated sites.

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: Binding Assay, Luciferase, Cotransfection, Expressing, Plasmid Preparation

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: 3q26 gene products modulate glioma cell invasion in various entangled ways. SOX2 balances glioma cell invasion through intrinsically antagonistic mechanisms: While PI3K/AKT/mTOR 41 and PI3K/AKT‐SOX2 loops promote glioma invasion 9, 20, SOX2 trans‐activates OPA1, which results in an effective inhibition of invasion. OPA1 knock‐down mutations resolve this functional antagonism and lead to an effective deregulation of glioma cell invasion.

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: Inhibition, Functional Assay

Journal: bioRxiv

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.1101/131987

Figure Lengend Snippet: (A-B) Pluripotency genes expressions analyzed by qPCR indicated cells. ( C) Effects of stable NFATc2 knock-down, overexpression, or knockout on SOX2 expression in lung cancer cells by Western blot analysis. (D) Genome browser view of NFAT binding sites (site 1 to 5 shown as black curve) on SOX2 regulatory region 2 and 3 with H3K27Ac mark in A549 cells (shown in black). (E) Promoter activities of site 1-5 by dual luciferase reporter assays in H441 cells. (F) Effect of transient NFATc2 over-expression on transcriptional activities of indicated reporters in H441 cells. (G) Site-directed mutagenesis of NFAT binding sequences in indicated NFAT reporters was performed. Reporter activity of wild type and the corresponding mutant reporters were studied in H441 cells with transient NFATc2 overexpression. (H) Sequence alignment of site 4 and site 5 sequences in different species. Putative NFAT binding sites are highlighted in rectangle. Identical sequences were highlighted in gray. (I) Confirmation of NFATc2 binding to SOX2 sites by ChIP–qPCR analysis in A549 cells with stable NFATc2 overexpression. (J) Correlation of immunohistochemical expressions of NFATc2 and SOX2 in 92 moderately to poorly differentiated human lung adenocarcinoma by χ 2 -test. Pearson r: Pearson correlation coefficient. (L) In vivo tumorigenicity of A549 cells with NFATc2 overexpression and SOX2 knockdown was assessed by subcutaneous inoculation of 1×10 4 cells into SCID. Xenograft formation was monitored by tumor growth curves and tumor sizes. **p<0.0001 versus control vehicle by two-way ANOVA. Error bar indicates the mean ± SD of tumor volumes of six mice. (M) Effect of NFATc2 knockdown on SOX2 expression in A549 CR cells analyzed by immunoblot. (N) MTT assay of cisplatin sensitivity for NFATc2-overexpressing A549 cells with stable knockdown of SOX2. For A, B, E-G, I, N *p<0.05, **p<0.01 versus control by t-test. Error bar indicates the mean ± S.D. for at least three independent replicates. The following figure supplements and source data are available for : : Expression of pluripotency factors in tumorspheres. : NFATc2 regulated SOX2 expression : NFATc2 regulated SOX2 expression through binding to 3’ regulatory regions. : NFATc2 regulated tumor function through SOX2. Figure 4-source data 1: Statistical analyses for figure 4K.

Article Snippet: GFP-VIVIT (11106), pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3H b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Knockdown, Over Expression, Knock-Out, Expressing, Western Blot, Binding Assay, Luciferase, Mutagenesis, Activity Assay, Sequencing, ChIP-qPCR, Immunohistochemical staining, In Vivo, Control, MTT Assay

Journal: bioRxiv

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.1101/131987

Figure Lengend Snippet: (A-B) Expressions of SOX2 , OCT4 , NANOG analyzed by qPCR in PDCL#24 cells with NFATc2 knockdown (A), or H1299 cells with NFATc2 overexpression (B). *p<0.05, **p<0.01 versus control by t-test. Error bar indicates the mean ± S.D. for three replicates . (C) Effects of stable NFATc2 knockdown or upregulation on SOX2 expression in lung cancer cells by Western blot analysis.

Article Snippet: GFP-VIVIT (11106), pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3H b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Knockdown, Over Expression, Control, Expressing, Western Blot

Journal: bioRxiv

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.1101/131987

Figure Lengend Snippet: (A) Computational prediction of NFAT binding sites (marked as red curve) on 5’ and 3’ SOX2 regulatory regions (Regions 1 to 4). TSS: transcription start site. (B) Transcriptional activity of region 1-4 were studied in H441 cells using luciferase reporter assay. (C-D) Luciferase reporter activities of mutant or wild-type SOX2 reporters were analyzed in A549 (C) or H1299 (D) cells with or without NFATc2 stable overexpression. *p<0.05, **p<0.01, comparison with RFP; # p<0.05, ## p<0.01, wild type versus mutant in RFP-NFATc2 cells by t-test. Error bars indicate the mean ± SD for at least three independent replicates. (E) Confirmation of NFATc2 binding to candidate SOX2 sites by ChIP-qPCR analysis in HCC827 cells. For B and E, *p<0.05, **p<0.01 versus control by Student’s t-test. Error bars indicate the mean ± SD for at least three independent replicates.

Article Snippet: GFP-VIVIT (11106), pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3H b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Binding Assay, Activity Assay, Luciferase, Reporter Assay, Mutagenesis, Over Expression, Comparison, ChIP-qPCR, Control

Journal: bioRxiv

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.1101/131987

Figure Lengend Snippet: (A) Expression of NFATc2 and SOX2 analyzed by Western blot in A549 cells with or without NFATc2 overexpression and SOX2 stable knockdown. (B-C) Effect of SOX2 knockdown on tumorsphere formation (B) and cell migration and invasion ability (C) of NFATc2 overexpressing A549 cells. *p<0.05, **p<0.01 versus control by t-test. Error bars indicate the mean ± SD for at least three independent replicates.

Article Snippet: GFP-VIVIT (11106), pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3H b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Expressing, Western Blot, Over Expression, Knockdown, Migration, Control

Journal: bioRxiv

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.1101/131987

Figure Lengend Snippet: (A) Expression of NFATc2 and its target FASL analyzed by qPCR in ALDH + /CD44 + TIC population compared to ALDH - /CD44 - population. ( B-C) Flow cytometry analysis of TIC proportions by ALDH + /CD44 + markers, in HCC827 cells with NFATc2 knockdown (B), or knockout (C). (D-F) mRNA levels of ALDH1A1 analyzed by qPCR in cells with NFATc2 up-(D) or down-regulation (E), or A549 CR cells (F). (G) Effects of SOX2 knockdown in NFATc2-overexpressing cells on ALDH/CD44 distribution by flow cytometry. (H) Expression of SOX2 and ALDH1A1 in A549 cells with NFATc2 overexpression and SOX2 knockdown. (I) Representative image of immunohistochemical expression of NFATc2, SOX2 and ALDH1A1 in xenografts derived from indicated A549 cells. Scale bars, 50 µM. (J) ChIP-seq genome browser view of SOX2 peak with SOX2 binding motif (curved arrow) on ALDH1A1 enhancer. The SOX2 peaks were co-localized with mammalian conservation peaks (blue) and H3K27Ac peak in A549 cells (black). ( K) Confirmation of SOX2 binding to ALDH1A1 sites by ChIP–qPCR analysis in PDCL#24 cells. (L) Site 1 and 2 reporters were co-transfected with SOX2 overexpressing vectors and reporter activity was analyzed by dual luciferase reporter assay in A549 cells. (M-N) Effects of transient ALDH1A1 suppression on invasion and migration abilities (M), and cisplatin sensitivity (N) of indicated cells. (O) Correlation between ALDH1A1 and SOX2 expressions by IHC in human lung adenocarcinomas by χ 2 -test. *P<0.05, **p<0.01 versus control by t-test. Error bar indicates the mean ± S.D. for at least three independent replicates. The following figure supplements and source data are available for : : NFATc2 regulated the ALDH+ population. : NFATc2 regulated ALDH1A1 expression. : Effect of siALDH1A1 on ALDH1A1 expression. : Effect of NFATc2/SOX2 on β-catenin activity. Figure 5-source data 1: Statistical analyses for figure 5B, C and G.

Article Snippet: GFP-VIVIT (11106), pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3H b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Expressing, Flow Cytometry, Knockdown, Knock-Out, Over Expression, Immunohistochemical staining, Derivative Assay, ChIP-sequencing, Binding Assay, ChIP-qPCR, Transfection, Activity Assay, Luciferase, Reporter Assay, Migration, Control

Journal: bioRxiv

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.1101/131987

Figure Lengend Snippet: (A) ALDH + proportions analyzed by flow cytometry in A549 cells with NFATc2 overexpression and transient ALDH1A1 knockdown. (B) mRNA level of ALDH1A1 analyzed by qPCR in HCC827 cells with NFATc2 knockout. (C) Representative images of immunohistochemical expression of NFATc2, SOX2 and ALDH1A1 in xenografts derived from PDCL#24 cells with or without NFATc2 knockdown. Scale bars, 50 µM. (D) Luciferase reporter assay for site1 and 2 in A549 cells with or without NFATc2 overexpression. **p<0.01 versus control by t-test. Error bars indicate the mean ± SD for at least three independent replicates.

Article Snippet: GFP-VIVIT (11106), pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3H b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Flow Cytometry, Over Expression, Knockdown, Knock-Out, Immunohistochemical staining, Expressing, Derivative Assay, Luciferase, Reporter Assay, Control

Journal: bioRxiv

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.1101/131987

Figure Lengend Snippet: (A) ROS levels detected by flow cytometry in A549 and A549 CR cells. (B-C) ROS levels in HCC827 cells (B) and PDCL#24 cells (C) with or without NFATc2 stable knockdown. (D) ROS levels in HCC827 cells with or without NFATc2 knockout. (E-F) Cisplatin sensitivity expressed as IC 50 by MTT assays of NFATc2-silenced PDCL#24 cells treated with increasing doses of NAC (E), or NFATc2-overexpressing A549 cells treated with the oxidizing agent (BSO) (F), respectively. * p<0.05, ** p<0.01 versus vector control without REDOX reagents; ## p<0.01 versus the corresponding treatment control; t-test. Error bar indicates the mean ± S.D. for three independent replicates. (G-H) Effects of increasing doses of NAC on tumorsphere formation ability of HCC827 (G) cells and PDCL#24 cells (H). * p<0.05, ** p<0.01 versus corresponding treatment controls, ## p<0.01 versus vector control, by t-test. Error bar indicates the mean ± S.D. for three independent replicates. (I) Effects of increasing doses of NAC on cell migration and invasion ability of HCC827 cells with NFATc2 knockdown. (J-K) ROS levels in NFATc2 overexpressing A549 cells with stable SOX2 (J) or transient ALDH1A1 (K) knockdown. For A-D, I-K *p<0.05, **p<0.01 versus respective control by t-test. Error bar indicates the mean ± S.D. for at least three independent replicates. Figure 6-source data 1: Statistical analyses for figure 5A-D, J and K.

Article Snippet: GFP-VIVIT (11106), pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3H b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Flow Cytometry, Knockdown, Knock-Out, Plasmid Preparation, Control, Migration

Journal: bioRxiv

Article Title: NFATc2 enhances tumor-initiating phenotypes through the NFATc2/SOX2/ALDH axis in lung adenocarcinoma

doi: 10.1101/131987

Figure Lengend Snippet: Expressions of total β-catenin, active β-catenin (non-phosphorylated), and phosphorylated β-catenin (p-β-catenin) analyzed by immunoblot in A549 with or without NFATc2 overexpression and SOX2 knockdown.

Article Snippet: GFP-VIVIT (11106), pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3H b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org ).

Techniques: Western Blot, Over Expression, Knockdown

Journal: PLoS ONE

Article Title: Efficient Generation of Virus-Free iPS Cells Using Liposomal Magnetofection

doi: 10.1371/journal.pone.0045812

Figure Lengend Snippet: (A) Alkaline phosphatase (AP) staining. Seven LMF-iPS cell lines examined expressed high levels of AP, similar to D3 ES cells. (B) Immunofluorescence staining of the ES cell-specific markers Oct4 and stage-specific embryonic antigen 1 (SSEA1). Scale bars: 200 µm. (C) mRNA expression of ES cell-specific markers (Nanog, Tert, Zfp, Oct4, and Sox2) and exogenous genes (Kl4 and c-Myc). G3PDH was used as a loading control.

Article Snippet: The pCX-OKS-2A (OKS, plasmid vector expressing Oct4, Klf4, and Sox2) and pCX–cMyc (C, plasmid vector expressing cMyc) were purchased from Addgene.

Techniques: Staining, Immunofluorescence, Expressing

Journal: PLoS ONE

Article Title: Efficient Generation of Virus-Free iPS Cells Using Liposomal Magnetofection

doi: 10.1371/journal.pone.0045812

Figure Lengend Snippet: (A) Southern blot analysis of D3 ES cells, seven LMF-iPS cell lines, and MEF cells using probes against Oct4, Sox2, Klf4, and c-Myc. Genomic DNA (15 µg) was digested with EcoRI. The arrows indicate bands derived from the transgenes. Closed and open arrowheads indicate bands derived from endogenous genes or nonspecific bands integrated into the genomic DNA, respectively. (B) Detection of plasmid integration by PCR. Genomic DNA (100 ng) from D3 ES cells, LMF-iPS cells, and MEFs was amplified by PCR using plasmid-specific primers (amplified regions: O, Oct4; S, Sox2; K, Klf4; C, cMyc; pA, polyadenylation signal and backbone; Amp, ampicillin resistant gene; and CAG, CAG promoter). In the K and C PCR results, closed arrowheads indicate bands derived from endogenous genes and open arrowheads indicate integrated exogenous genes.

Article Snippet: The pCX-OKS-2A (OKS, plasmid vector expressing Oct4, Klf4, and Sox2) and pCX–cMyc (C, plasmid vector expressing cMyc) were purchased from Addgene.

Techniques: Southern Blot, Derivative Assay, Plasmid Preparation, Amplification

Journal: PLoS ONE

Article Title: Efficient Generation of Virus-Free iPS Cells Using Liposomal Magnetofection

doi: 10.1371/journal.pone.0045812

Figure Lengend Snippet: (A) Phase-contrast images of the two LMF-iPS cell lines, according to spontaneous differentiation for 20 days. (B) RT-PCR analysis of the spontaneous differentiation of the two LMF-iPS cell lines into three germ-like layers expressing undifferentiated ES cell markers (Oct4 and Nanog), as well as endodermal (α-fetoprotein and α-amylase), mesodermal (β-enolase and renin), and ectodermal (Map2 and β-tubulin) genes. (C) Immunocytochemistry of neuronal cell differentiation in the two LMF-iPS cell lines into Map2-, Tuj1-, and GFAP-positive cells. (D and E) RT-PCR and immunostaining results showing cardiac (TnI) and endothelial (Tie2) cell differentiation of two LMF-iPS cell lines. Blue nuclear staining is by DAPI.

Article Snippet: The pCX-OKS-2A (OKS, plasmid vector expressing Oct4, Klf4, and Sox2) and pCX–cMyc (C, plasmid vector expressing cMyc) were purchased from Addgene.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunocytochemistry, Cell Differentiation, Immunostaining, Staining