Journal: Science Advances

Article Title: A potent CBP/p300-Snail interaction inhibitor suppresses tumor growth and metastasis in wild-type p53-expressing cancer

doi: 10.1126/sciadv.aaw8500

Figure Lengend Snippet: ( A ) Immunoblot analysis of p53 expression in wild-type (left) and mutant (right) p53-expressing cells that were treated with vehicle or CYD19 for 48 hours. ( B ) Immunofluorescence staining of Snail and p53 in MMTV-PyMT (left) and HCT116 (right) cells treated with vehicle or 50 nM CYD19 for 48 hours. ( C ) Reverse transcription quantitative polymerase chain reaction (qPCR) analysis of p53 , p21 , and MDM2 expressions in MMTV-PyMT (top) and HCT116 (bottom) cells as described in (B). ( D ) Immunoblot analysis of p53, p21, and MDM2 expressions in MMTV-PyMT and HCT116 cells treated with vehicle or CYD19 for 48 hours. ( E ) Immunoblot analysis of p53 expression in MMTV-PyMT cells treated with vehicle or 50 nM CYD19 and then with CHX (100 μg/ml) for a total of 48 hours. ( F ) Densitometry of p53 protein in cells as described in (E). ( G ) Comparison of ubiquitinated p53 protein in vehicle- and CYD19-treated MMTV-PyMT cells. MG132 (10 μM) was added 4 hours before harvesting. Lysates from vehicle- and CYD19-treated cells loaded at ratios of 2:1 and 1:1 were subjected to IP assay using an anti-p53 antibody. ( H ) Comparison of acetylated p53 protein in vehicle- and CYD19-treated MMTV-PyMT cells as described in (G). ( I ) Comparison of binding interaction of p53 with HDAC1 in vehicle- and CYD19-treated MMTV-PyMT cells as described in (G). ( J ) Comparison of Snail, p53, and p21 expressions in control (left) and Snail-deleted (right) MMTV-PyMT cells that were treated with vehicle or CYD19 for 48 hours. All representative blots and images as shown are from three independent experiments. All data are presented as means ± SD ( n = 3 independent experiments). ** P < 0.01. Differences are tested using unpaired two-tailed Student’s t test (C).

Article Snippet: p3XFLAG-Snail-WT, p3XFLAG-Slug-WT, and pLKO.1-ms.p53-shRNA vectors were generated and used as described previously ( , ). pET23a(+)-His-Snail-WT, His-Snail-R174A, p3XFLAG-Snail-R174A, FLAG-Snail-K147R/K186R, pLKO.1-hu.p53-shRNA (targeting mRNA sequence from ATG, 176 to 196), pLKO.1-Snail-shRNA1 (468 to 486), pLKO.1-Snail-shRNA2 (1515 to 1533), pCDN3.1-GST-Snail-WT-GFP, and pCDN3.1-GST-Snail-R174A-GFP vectors were generated by GenScript Biotech Inc. (Nanjing, China).

Techniques: Western Blot, Expressing, Mutagenesis, Immunofluorescence, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Comparison, Binding Assay, Control, Two Tailed Test

Journal: Science Advances

Article Title: A potent CBP/p300-Snail interaction inhibitor suppresses tumor growth and metastasis in wild-type p53-expressing cancer

doi: 10.1126/sciadv.aaw8500

Figure Lengend Snippet: ( A ) CCK-8 cell proliferation assay for wild-type and mutant p53-expressing cells treated with vehicle or CYD19 for 48 hours. ( B ) Quantification of apoptotic subpopulation in various cell lines treated with vehicle or CYD19 for 48 hours. ( C ) Immunoblot analysis of the indicated protein expressions in MMTV-PyMT (left) and HCT116 (right) cells as described in (B). C-casp9, cleaved caspase 9. ( D ) Immunoblot analysis of the indicated protein expressions in control and Snail-deleted MMTV-PyMT cells treated with vehicle or CYD19 for 48 hours. ( E ) CCK-8 analysis for control and Snail-deleted MMTV-PyMT cells treated with vehicle or CYD19 for 48 hours. ( F ) Quantification of apoptotic subpopulation in control and Snail-silenced HCT116 cells treated with vehicle or CYD19 for 48 hours. ( G ) CCK-8 analysis for control and Snail-silenced HCT116 cells treated with vehicle or CYD19 for 48 hours. ( H ) Quantification of apoptotic subpopulation in control and p53-silenced HCT116 cells treated with vehicle or CYD19 for 48 hours. ( I ) CCK-8 analysis for control and p53-silenced HCT116 cells treated with vehicle or CYD19 for 48 hours. ( J ) CCK-8 analysis for MMTV-PyMT and HCT116 cells that were treated with vehicle or taxol in combination with vehicle or 25 nM CYD19 for 48 hours. All representative blots as shown are from three independent experiments. All data are presented as means ± SD ( n = 3 independent experiments). ** P < 0.01. Differences are tested using one-way ANOVA with Tukey’s post hoc test (B, F, and H).

Article Snippet: p3XFLAG-Snail-WT, p3XFLAG-Slug-WT, and pLKO.1-ms.p53-shRNA vectors were generated and used as described previously ( , ). pET23a(+)-His-Snail-WT, His-Snail-R174A, p3XFLAG-Snail-R174A, FLAG-Snail-K147R/K186R, pLKO.1-hu.p53-shRNA (targeting mRNA sequence from ATG, 176 to 196), pLKO.1-Snail-shRNA1 (468 to 486), pLKO.1-Snail-shRNA2 (1515 to 1533), pCDN3.1-GST-Snail-WT-GFP, and pCDN3.1-GST-Snail-R174A-GFP vectors were generated by GenScript Biotech Inc. (Nanjing, China).

Techniques: CCK-8 Assay, Proliferation Assay, Mutagenesis, Expressing, Western Blot, Control

Journal: Science Advances

Article Title: A potent CBP/p300-Snail interaction inhibitor suppresses tumor growth and metastasis in wild-type p53-expressing cancer

doi: 10.1126/sciadv.aaw8500

Figure Lengend Snippet: ( A and B ) Primary tumor volumes (A) and weights (B) were measured in MMTV-PyMT mice that were intraperitoneally treated with vehicle or CYD19 (30 mg/kg) for 25 consecutive days ( n = 6 mice, each). ( C ) Immunohistochemical staining of Ki67 (top) and cleaved caspase 3 (bottom) in primary tumors of vehicle- and CYD19-treated mice ( n = 6 mice, each). ( D ) Quantification of Ki67-positive (Ki67 + ; top) and cleaved caspase 3–positive (C-casp3 + ; bottom) cells in tumors as described in (C). ( E ) Immunoblot analysis of Snail and p53 expressions in tumor lysates of vehicle- and CYD19-treated mice ( n = 3 pools from six mice, each). ( F ) Hematoxylin and eosin (H&E) staining for primary tumors as described in (C) ( n = 6 mice, each). Magnified areas of boxed sections are shown in the bottom panels. ( G ) Immunofluorescence staining of E-cadherin and vimentin in primary tumors as described in (C) ( n = 6 mice, each). ( H ) Quantification of staining intensity in primary tumors as described in (G). ( I and J ) Representative histogram (I) and quantification (J) of ALDH + subpopulation in primary tumors as described in (C) ( n = 6 mice, each). DEAB, diethylaminobenzaldehyde. ( K ) Immunofluorescence staining of F4/80 and CD31 in primary tumors as described in (C) ( n = 6 mice, each). ( L ) Quantification of staining intensity in primary tumors as described in (K). ( M ) H&E staining for vehicle- and CYD19-treated lungs ( n = 6 mice, each). ( N ) Magnified areas of boxed sections in (M) are shown. ( O ) Quantification of nodules in vehicle- and CYD19-treated lungs as described in (M). All data are presented as means ± SD ( n = 6 independent experiments). * P < 0.05 and ** P < 0.01. Differences are tested using Mann-Whitney U test.

Article Snippet: p3XFLAG-Snail-WT, p3XFLAG-Slug-WT, and pLKO.1-ms.p53-shRNA vectors were generated and used as described previously ( , ). pET23a(+)-His-Snail-WT, His-Snail-R174A, p3XFLAG-Snail-R174A, FLAG-Snail-K147R/K186R, pLKO.1-hu.p53-shRNA (targeting mRNA sequence from ATG, 176 to 196), pLKO.1-Snail-shRNA1 (468 to 486), pLKO.1-Snail-shRNA2 (1515 to 1533), pCDN3.1-GST-Snail-WT-GFP, and pCDN3.1-GST-Snail-R174A-GFP vectors were generated by GenScript Biotech Inc. (Nanjing, China).

Techniques: Immunohistochemical staining, Staining, Western Blot, Immunofluorescence, MANN-WHITNEY

Journal: Science Advances

Article Title: A potent CBP/p300-Snail interaction inhibitor suppresses tumor growth and metastasis in wild-type p53-expressing cancer

doi: 10.1126/sciadv.aaw8500

Figure Lengend Snippet: ( A and B ) HCT116 xenograft tumor volumes (A) and weights (B) were measured in athymic nude mice that were intraperitoneally treated with vehicle or CYD19 for two consecutive weeks ( n = 6 mice, each). ( C ) Immunohistochemical staining of Ki67 (top) and cleaved caspase 3 (bottom) in xenograft tumors of vehicle- and CYD19-treated mice ( n = 6 mice, each). ( D ) Quantification of Ki67 + (top) and C-casp3 + (bottom) cells in tumors as described in (C). ( E ) Immunoblot analysis of Snail and p53 expressions in tumor lysates of vehicle- and CYD19-treated mice ( n = 3 pools from six mice, each). ( F ) Immunofluorescence staining of E-cadherin and vimentin in xenograft tumors of vehicle- and CYD19-treated mice ( n = 6 mice, each). ( G ) Quantification of staining intensity in xenograft tumors as described in (F). ( H and I ) Representative histogram (H) and quantification (I) of ALDH + subpopulation in xenograft tumors as described in (C) ( n = 6 mice, each). ( J ) Growth of HCT116 xenograft tumors derived from 1 × 10 6 control cells or 2 × 10 6 Snail-silenced cells was monitored in nude mice treated with vehicle or CYD19 for two consecutive weeks ( n = 6 mice, each). ( K ) Immunoblot analysis of Snail and p53 expressions in lysates of xenograft tumors as described in (J). ( L ) Representative phase contrast (top), GFP fluorescence (middle), and H&E (bottom) images of vehicle- and CYD19-treated livers ( n = 6 mice, each). Mice were treated with vehicle or CYD19 for three consecutive weeks starting from the third day after surgery. ( M ) Quantification of fluorescence intensity in livers as described in (L). All data are presented as means ± SD ( n = 6 independent experiments). ** P < 0.01. Differences are tested using Mann-Whitney U test.

Article Snippet: p3XFLAG-Snail-WT, p3XFLAG-Slug-WT, and pLKO.1-ms.p53-shRNA vectors were generated and used as described previously ( , ). pET23a(+)-His-Snail-WT, His-Snail-R174A, p3XFLAG-Snail-R174A, FLAG-Snail-K147R/K186R, pLKO.1-hu.p53-shRNA (targeting mRNA sequence from ATG, 176 to 196), pLKO.1-Snail-shRNA1 (468 to 486), pLKO.1-Snail-shRNA2 (1515 to 1533), pCDN3.1-GST-Snail-WT-GFP, and pCDN3.1-GST-Snail-R174A-GFP vectors were generated by GenScript Biotech Inc. (Nanjing, China).

Techniques: Immunohistochemical staining, Staining, Western Blot, Immunofluorescence, Derivative Assay, Control, Fluorescence, MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Characterization of the SOXB2 expression in the developing dorsal midbrain at E12.5. Coronal sections of SOX21 GFPn midbrains stained with BrdU (A) , MASH1 (B) , LHX2 and SOX14 (C) and GATA3 (D) antibodies. Coronal sections of SOX14 +/− midbrains were stained with BHLHB5 and SOX21 (E) , BHLHB5 and SOX2 (F) , SOX21 and GATA2 (G) , BHLHB5 and GATA2 (H) , BrdU and LHX2 (I) , GATA3 and LHX2 (K) and GAD6 (J) antibodies. The results of this analysis are summarized in the schematic (L) . Red, green and blue arrows indicate the corresponding single stained cells (as shown in the label) and yellow and purple arrows denote co-localization between green and red cells and blue and red, respectively. Scale bar, 150 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Expressing, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Analysis of lineage-determining factors in SOX21 −/− embryos at E12.5. The immunostaining of the lineage-determining factors MASH1 (A,A′) , GATA2 (B,B′) and DBX1 (C,C′) was similar between knockout ( ii ) and wild type embryos ( i ). A significant reduction in the BHLHB5 staining was observed in the Knockout embryos in comparison to wild type (D,D′) . All nuclei were counted dorsally to the ventrolateral midbrain sulcus (indicated by white arrow). These data were confirmed by RT-Q-PCR analysis of embryos at the same age (E) . Data are represented as mean ± SEM, n = 10 (5 pairs of mice). * n = 10 (5 pairs of mice), P = 0.0116, two-tailed Mann-Whitney test. ** n = 6 (3 pairs of mice), p = 0.025, DF = 1, Kruskal Wallis test. *** n = 6 (3 pairs of mice), p = 0.0253, DF = 1, Kruskal Wallis test. Scale bar, 150 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Immunostaining, Knock-Out, Staining, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Lentiviral knock down of SOX21, SOX14 and BHLHB5. GATA3 eGFP embryos were infected through intra amniotic injections at E8.5 with lentiviral shRNA vectors targeting BHLHB5 (PLKO.1-shBHLHB5; ii ), SOX21 (PLKO.1-shSOX21; iii ) and SOX14 (PLKO.1-shSOX14; iv ). The expression of GFP was reduced in the embryos infected with the SOX14 shRNA vector (A,E) . Immunostaining of the embryos with BHLHB5 showed a reduction when embryos were infected with PLKO.1-shBHLHB5 and PLKO.1-shSOX21 (B) . Not surprisingly, SOX21 was only reduced when embryos were infected with PLKO.1-shSOX21 (C) while not noticeable changes were observed in the expression of SOX2 (D) and LHX2 (E) when infected with either vector. These data were confirmed by RT-Q-PCR analysis of embryos at E12.5 (F) . Data are represented as mean ± SEM. * n = 6 (3 pairs of mice), SOX21sh p = 0.0417, DF = 4, Kruskal Wallis test, ** n = 6 (3 pairs of mice), SOX14sh p = 0.0141, DF = 4, Kruskal Wallis test, *** n = 6 (3 pairs of mice), SOX21sh p = 0.016, BHLHB5sh p = 0.0474, DF = 4, Kruskal Wallis test, **** n = 6 (3 pairs of mice), SOX21sh p = 0.0064, SOX14sh p = 0.0021, DF = 4, Kruskal Wallis test. Scale bar, 150 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Knockdown, Infection, shRNA, Expressing, Plasmid Preparation, Immunostaining

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Overexpression of SOX21, SOX14 and BHLHB5 through in utero electroporation. Wild type embryos ( i,ii ) were in utero electroporated at E11 with Plasmids encoding CMV-SOX21-FLAG-IRES-GFP n , CMV-SOX14-FLAG-IRES-GFP n and CMV-BHLHB5-IRES-GFP n . The immunostaining of the embryos with BHLHB5 illustrated that ectopic expression of this gene could be achieved when SOX21 was overexpressed (A) and not with SOX14 (C) . Expression of GATA3 on the other hand was only induced when SOX14 was overexpressed (D) and not with SOX21 (B) . These experiments were repeated on SOX21 −/− embryos ( iii,iv ) providing similar findings (A’,B’,C’,D′) . Interestingly, ectopic expression of BHLHB5 was unable to induce either the GABAergic or Glutamatergic lineage as show by staining of the embryos with GATA3 or LHX2 (E,F) . Scale bar, 75 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Over Expression, In Utero, Electroporation, Immunostaining, Expressing, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Schematic representation of the SOX2B during maturation of GABAergic neurons. SOX21 is expressed both in radial glial and neuronal progenitors at the VZ while been maintained in neuronal precursors at the sub ventricular zone (SVZ) where it appears to govern the expression of BHLHB5. At the MZ SOX14 marks the GABAergic lineage while promoting GATA3 expression.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Expressing