Journal: Oncogene

Article Title: Genome-wide CRISPR screen identifies LGALS2 as an oxidative stress-responsive gene with an inhibitory function on colon tumor growth

doi: 10.1038/s41388-020-01523-5

Figure Lengend Snippet: A Western blotting showing the expression of p-STAT3 and STAT3 in healthy colon or colon tumors. B Densitometry quantification of p-STAT3/total STAT3 for colons or colorectal tumors. *** p < 0.001 (one-way ANOVA). C Representative images of p-STAT3 immunostaining of colons or colon tumors. Scale bar: 50 μm. D Quantification of p-STAT3 + cells in colon tumors of WT and Gal2-KO mice. *** p < 0.001 ( t -test).

Article Snippet: Primary antibodies including the goat polyclonal anti-Gal2 (STJ24400, 1:500, St John’s Laboratory Ltd, London, UK), anti-STAT3 (#9139, 1:1000), anti-p-STAT3 (#9145, 1:1000), and anti-GAPDH (MAB374, 1:2000) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Expressing, Immunostaining

Journal: Oncogene

Article Title: Genome-wide CRISPR screen identifies LGALS2 as an oxidative stress-responsive gene with an inhibitory function on colon tumor growth

doi: 10.1038/s41388-020-01523-5

Figure Lengend Snippet: A Western blotting showing the expression of Gal2 in stable Gal2-overexpressing HCT116 cells. The red arrow indicates the un-cleaved GFP-2A-Gal2 fusion protein. B The CCK8 proliferation assay of HCT116 with or without Gal2 overexpression. C The survival assay of HCT116 with or without Gal2 overexpression in the presence of 0.6 mM H 2 O 2 . ** p < 0.01, *** p < 0.001 ( t -test). D Western blotting showing the expression of p-STAT3 and STAT3 in HCT116 cells with or without Gal2 overexpression. E Densitometry quantification of p-STAT3/total STAT3 in HCT116 cells with or without Gal2 overexpression. ** p < 0.01 (one-way ANOVA).

Article Snippet: Primary antibodies including the goat polyclonal anti-Gal2 (STJ24400, 1:500, St John’s Laboratory Ltd, London, UK), anti-STAT3 (#9139, 1:1000), anti-p-STAT3 (#9145, 1:1000), and anti-GAPDH (MAB374, 1:2000) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Expressing, Proliferation Assay, Over Expression, Clonogenic Cell Survival Assay

Journal: Oncotarget

Article Title: Addiction of pancreatic cancer cells to zinc-finger transcription factor ZIC2

doi:

Figure Lengend Snippet: A and B , The proliferation curves and cell cycle analysis of PDAC cells transfected either ZIC2 -specific (siZIC2-1, siZIC2-2) or control (siControl) siRNAs. The data represent the mean ± SD from three independent experiments. C, immunoblot analysis of PDAC cells transfected with siRNAs.

Article Snippet: The antibodies used were as follows: anti-ZIC2 antibody (ARP35821, Aviva Systems Biology); anti-GLI2 antibody (sc-271786; Santa Cruz Biotechnology), anti-p-FGFR-3 (Tyr724) antibody (sc-33041, Santa Cruz Biotechnology); anti-GLI1 L42B10 antibody, anti-FGFR3 C51F2 antibody, anti-p44/42 MAPK 137F5 antibody, anti-phospho-p44/42 MAPK 20G11 antibody, anti-cleaved PARP D64E10 antibody (Cell Signaling); anti-FLAG M2 antibody, anti-β-actin AC-74 antibody (SIGMA); and anti-ANXA8 antibody (GTX103853, GenTex).

Techniques: Cell Cycle Assay, Transfection, Control, Western Blot

Journal: Oncotarget

Article Title: Addiction of pancreatic cancer cells to zinc-finger transcription factor ZIC2

doi:

Figure Lengend Snippet: A , Comparative presentation of three sets of the expression microarray analysis. Tet/empty, the genes of PANC-1 Tet/empty cells not changed by the DOX elimination; Tet/ZIC2, the genes of PANC-1 Tet/ZIC2 cells changed by the DOX elimination; siZIC2-1, the genes of PANC-1 cells changed by siZIC2-1-treanfection, which was normalized by siControl transfection. The numbers are on the log2 scale. B , RT-PCR and immunoblot analyses of PDAC cells transiently transfected with either a FLAG-tagged ZIC2 expression vector or an empty (Mock) vector. C , Schematic of the transgenes ( upper panel ) and RT-PCR / immunoblot analysis ( lower panel ) of KP-4 cells, which were transiently transfected with either ZIC2ER or a control ER vector. CHX , cycloheximide; E2 , β-estradiol.

Article Snippet: The antibodies used were as follows: anti-ZIC2 antibody (ARP35821, Aviva Systems Biology); anti-GLI2 antibody (sc-271786; Santa Cruz Biotechnology), anti-p-FGFR-3 (Tyr724) antibody (sc-33041, Santa Cruz Biotechnology); anti-GLI1 L42B10 antibody, anti-FGFR3 C51F2 antibody, anti-p44/42 MAPK 137F5 antibody, anti-phospho-p44/42 MAPK 20G11 antibody, anti-cleaved PARP D64E10 antibody (Cell Signaling); anti-FLAG M2 antibody, anti-β-actin AC-74 antibody (SIGMA); and anti-ANXA8 antibody (GTX103853, GenTex).

Techniques: Expressing, Microarray, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Control

Journal: Oncotarget

Article Title: Addiction of pancreatic cancer cells to zinc-finger transcription factor ZIC2

doi:

Figure Lengend Snippet: A , Immunoblot ( left ) and qRT-PCR ( right ) analyses of GLI low-expressing PDAC cells (PA-TU-8988T and PA-TU-8902) transiently transfected with indicated siRNAs. The immunoblot of GLI high-expressing KP-4 cells is also shown as a comparison. The RT-PCR data were normalized to that of siControl transfectants and are shown on the log2 scale. Columns , the mean values of three independent experiments; bars , SD. B , qRT-PCR analysis of GLI low-expressing PDAC cells transiently transfected with either a FLAG-ZIC2 expression vector or an empty (Mock) vector. The data were normalized to that of mock vector transfectants and are shown on the log2 scale. Columns , the mean values of three independent experiments; bars , SD. C , Immunoblot ( left ) and qRT-PCR ( right ) analyses of GLI high-expressing PDAC cells (PANC-1 and KP-4) transiently transfected with either siRNAs for a GLI1/2-double knockdown (siGLI1&siGLI2) or a control siRNA (siControl) in conjunction with FLAG-ZIC2 (+) or a mock control vector (−). In qRT-PCR analysis, the data were normalized to that of siControl plus a mock control expression vector transfectants and are shown on the log2 scale. Columns , the mean values of three independent experiments; bars , SD.

Article Snippet: The antibodies used were as follows: anti-ZIC2 antibody (ARP35821, Aviva Systems Biology); anti-GLI2 antibody (sc-271786; Santa Cruz Biotechnology), anti-p-FGFR-3 (Tyr724) antibody (sc-33041, Santa Cruz Biotechnology); anti-GLI1 L42B10 antibody, anti-FGFR3 C51F2 antibody, anti-p44/42 MAPK 137F5 antibody, anti-phospho-p44/42 MAPK 20G11 antibody, anti-cleaved PARP D64E10 antibody (Cell Signaling); anti-FLAG M2 antibody, anti-β-actin AC-74 antibody (SIGMA); and anti-ANXA8 antibody (GTX103853, GenTex).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Transfection, Comparison, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Knockdown, Control

Journal: Oncotarget

Article Title: Addiction of pancreatic cancer cells to zinc-finger transcription factor ZIC2

doi:

Figure Lengend Snippet: A , Immunoblot analysis of PDAC cells transiently transfected with either an FGFR3-IIIb variant of the human FGFR3 expression vector (FGFR3) or an empty (Mock) control vector. The ratio of phosphorylated ERK versus ERK protein was semi-quantified using NIH Image software. B , Immunoblot analysis of PDAC cells transiently transfected with either FGFR3 -specific siRNAs (siFGFR3-1, siFGFR3-2) or a control siRNA (siControl) in conjunction with either FLAG-ZIC2 (+) or an empty (Mock) vector (−). C , immunoblot analysis of PDAC cells transiently transfected with either FGFR3 -specific , ANXA8 -specific (siANXA8-1, siANXA8-2) or control siRNAs. D , Immunoblot analysis of PDAC cells transfected with either siZIC2-1 or siControl in conjunction with the indicated expression vectors (EGFP, FGFR3 and ANXA8). The DNA amounts of transfected expression vectors were equalized using the EGFP expression vector.

Article Snippet: The antibodies used were as follows: anti-ZIC2 antibody (ARP35821, Aviva Systems Biology); anti-GLI2 antibody (sc-271786; Santa Cruz Biotechnology), anti-p-FGFR-3 (Tyr724) antibody (sc-33041, Santa Cruz Biotechnology); anti-GLI1 L42B10 antibody, anti-FGFR3 C51F2 antibody, anti-p44/42 MAPK 137F5 antibody, anti-phospho-p44/42 MAPK 20G11 antibody, anti-cleaved PARP D64E10 antibody (Cell Signaling); anti-FLAG M2 antibody, anti-β-actin AC-74 antibody (SIGMA); and anti-ANXA8 antibody (GTX103853, GenTex).

Techniques: Western Blot, Transfection, Variant Assay, Expressing, Plasmid Preparation, Control, Software

Journal: Oncotarget

Article Title: Addiction of pancreatic cancer cells to zinc-finger transcription factor ZIC2

doi:

Figure Lengend Snippet: A , H&E and immunohistochemical staining of precancerous pancreatic intraepithelial lesion (PanIN) and PDAC. Staining intensities of ZIC2, ANXA8 and FGFR3 were semi-quantitatively scored as negative, 0; weak, 1; strong, 2. Labeling index of Ki-67 staining was calculated by counting over 100 cells per lesion. Bar , 20μm. Statistical analyses are provided in . B and C , Correlation between ZIC2 and ANXA8 (B) or FGFR3 (C) expression. D, Correlation between ZIC2 expression and the Ki-67 labeling index.

Article Snippet: The antibodies used were as follows: anti-ZIC2 antibody (ARP35821, Aviva Systems Biology); anti-GLI2 antibody (sc-271786; Santa Cruz Biotechnology), anti-p-FGFR-3 (Tyr724) antibody (sc-33041, Santa Cruz Biotechnology); anti-GLI1 L42B10 antibody, anti-FGFR3 C51F2 antibody, anti-p44/42 MAPK 137F5 antibody, anti-phospho-p44/42 MAPK 20G11 antibody, anti-cleaved PARP D64E10 antibody (Cell Signaling); anti-FLAG M2 antibody, anti-β-actin AC-74 antibody (SIGMA); and anti-ANXA8 antibody (GTX103853, GenTex).

Techniques: Immunohistochemical staining, Staining, Labeling, Expressing