Journal: Current issues in molecular biology

Article Title: Identification and Characterization of Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma Using the SORE6 Reporter.

doi: 10.3390/cimb43020041

Figure Lengend Snippet: Figure 3. Experimental manipulation of Sox2 and Oct4 in SupM2 SORE6 clones. (A) FACS analysis showing the effect on SORE6 reporter activity of overexpressing Sox2 in single-cell clone 5-4 (SupM2 SORE6−). (B) FACS analysis showing the effect on SORE6 reporter activity of overexpressing shSox2 in single-cell clone 6-5 (SupM2 SORE6+). (C) FACS analysis showing the effect of Oct4 downregulation on SORE6 reporter activity in clone 5-4 (SupM2 SORE6−). (D) FACS analysis showing the effect of shOct4 transfection on SORE6 reporter activity in single-cell clone 6-5 (SupM2 SORE6+). All results shown are representative of three independent experiments and were also repeated in a second batch of single-cell clones. Side panels show the fold change of GFP in log scale (mean ± SEM). Bottom panels of each figure are the Western blots showing the effect of transient transfection with Sox2, Oct4, shSox2, and shOct4 plasmids. Empty vector (EV) was included as a negative control. ** p < 0.01, *** p < 0.001.

Article Snippet: The Oct4 expression vector (pLVEF1a-hOCT4-IRES-Neo) was purchased from BiOSETTIA (San Diego, CA, USA). shRNA for Sox2 (#26353) and the Sox2 expression vector (#16577) were purchased from Addgene (Watertown, MA, USA).

Techniques: Clone Assay, Activity Assay, Transfection, Western Blot, Plasmid Preparation, Negative Control

Journal: Current issues in molecular biology

Article Title: Identification and Characterization of Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma Using the SORE6 Reporter.

doi: 10.3390/cimb43020041

Figure Lengend Snippet: Figure 6. SORE6−and SORE6+ clones are biochemically distinct. (A) The subcellular localization of Sox2, Oct4, and c-Myc in SORE6−and SORE6+ cells derived from SupM2, assessed by the nuclear cytoplasmic fractionation assay. (B) The DNA pull-down assay was performed to assess Sox2, Oct4, and c-Myc transcriptional activity in SORE6−and SORE6+ cells using a biotin-labeled SORE6 probe.

Article Snippet: The Oct4 expression vector (pLVEF1a-hOCT4-IRES-Neo) was purchased from BiOSETTIA (San Diego, CA, USA). shRNA for Sox2 (#26353) and the Sox2 expression vector (#16577) were purchased from Addgene (Watertown, MA, USA).

Techniques: Clone Assay, Derivative Assay, Fractionation, Pull Down Assay, Activity Assay, Labeling

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