flag atf2  (OriGene)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATF2 predicts poor prognosis and promotes malignant phenotypes in renal cell carcinoma

doi: 10.1186/s13046-016-0383-2

Figure Lengend Snippet: ATF2 is highly expressed in RCC samples. a qRT-PCR analysis of ATF2 in RCC cells versus human normal renal cells (293 T and HK-2). b qRT-PCR analysis of ATF2 in RCC tissues and adjacent non-tumor tissues from 17 RCC patients. c Immunohistochemistry analysis of ATF2 in RCC tissues ( right ) and adjacent non-tumor tissues ( left ). Scale bar = 50 μm. d Immunohistochemical comparison of ATF2 expression in human RCC tissues versus adjacent non-tumor tissues. The horizontal lines in the box plots represent the median, the boxes represent the interquartile range, and the whiskers represent the 2.5th and 97.5th percentiles. e qRT-PCR analysis of ATF2 in primary RCC tissues and tumor thrombus tissues from 9 patients. Results are presented as mean ± SEM from three independent experiments. * p < 0.05 and ** p <0.01

Article Snippet: Human full-length cDNA of ATF2 was cloned into expression plasmid pCMV3-ATF2-Flag (Sino Biological lnc.).

Techniques: Quantitative RT-PCR, Immunohistochemistry, Immunohistochemical staining, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATF2 predicts poor prognosis and promotes malignant phenotypes in renal cell carcinoma

doi: 10.1186/s13046-016-0383-2

Figure Lengend Snippet: ATF2 promotes RCC cells proliferation in vitro. a CCK8 assay of ATF2 knockdown and control RCC cells at indicated times. b Plate colony formation assay of ATF2 knockdown and control ACHN cells in 10 cm dish for 3 weeks ( n = 3). Average number of colonies ( upper ) and representative images ( lower ) were shown. c Flow cytometry analysis of cell cycle in ATF2 knockdown and control RCC cells. Representative cell cycle distributions were shown ( left ) and the histogram columns represent the average percentages of G0/G1, S and G2/M phases ( right ). d Flow cytometry analysis of apoptotic cells in ATF2 knockdown and control RCC cells ( left ) and the percentage of cells at the different apoptosis phases ( right ). The bar charts showed the increases in the early and late apoptotic indexes of RCC cells transfected with shATF2. e CCK8 assay of ATF2 overexpression and control RCC cells at indicated times. f Plate colony formation assay of ATF2 overexpression and control ACHN cells in 10 cm dish for 3 weeks ( n = 3). Average number of colonies ( upper ) and representative images ( lower ) were shown. g Flow cytometry analysis of cell cycle in ATF2 overexpression and control RCC cells. Representative cell cycle distributions were shown ( left ) and the histogram columns represent the average percentages of G0/G1, S and G2/M phases ( right ). h & i Western blotting analysis of Cyclin B1 and Cyclin D1 in ATF2 knockdown ( h ), ATF2 overexpression ( i ) and control ACHN cells. β-Actin was used as an internal standard. j ChIP assay analysis of the enrichment of ATF2 at the proximal promoter region of Cyclin B1 and Cyclin D1 in the context of ATF2 overexpression. The enrichment of ATF2 ( upper ) on Cyclin B1 or Cyclin D1 promoter relative to input in 786-O cells and gel electrophoresis of PCR products from ChIP assay ( lower ). Results are presented as mean ± SEM from three independent experiments. * p < 0.05, ** p <0.01 and *** p < 0.001

Article Snippet: Human full-length cDNA of ATF2 was cloned into expression plasmid pCMV3-ATF2-Flag (Sino Biological lnc.).

Techniques: In Vitro, CCK-8 Assay, Colony Assay, Flow Cytometry, Transfection, Over Expression, Western Blot, Nucleic Acid Electrophoresis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATF2 predicts poor prognosis and promotes malignant phenotypes in renal cell carcinoma

doi: 10.1186/s13046-016-0383-2

Figure Lengend Snippet: ATF2 facilitates RCC cell migration and invasion in vitro. a Left: representative images of the wound-healing assay of ATF2 knockdown and control RCC cells photographed at 0, 6 and 12 h after scratching. Scale bar = 200 μm. Right: the relative migration rate was calculated by dividing the change in the distance between the scratch edges by the initial distance. b & c Transwell assays were performed to evaluate cell migration following ATF2 knockdown ( b ), ATF2 overexpression ( c ) and control RCC cells. The statistical graph indicates the means ± SEM of the number of cells from 8 random high power fields (magnification, ×200) counted from three independent experiments. Scale bar = 2 mm. d & e Transwell assays were performed to evaluate the invasion of ATF2 knockdown ( d ), ATF2 overexpression ( e ) and control RCC cells. The statistical graph indicates the means ± SEM of the number of cells from 8 random high power fields (magnification, ×200) counted from three independent experiments. Scale bar = 2 mm. f & g Western blotting analysis of Snail, Vimentin and E-Cadherin in ATF2 knockdown ( f ), ATF2 overexpression ( g ) and control 786-O cells. β-Actin was used as an internal standard. h ChIP assay analysis of the enrichment of ATF2 at the proximal promoter region of Snail and Vimentin in the context of ATF2 overexpression. The enrichment of ATF2 on Snail and Vimentin promoter relative to input in 786-O cells (upper). The gel electrophoresis of PCR products from ChIP assay (lower). Results are presented as mean ± SEM from three independent experiments. * p < 0.05, ** p <0.01, and *** p < 0.001

Article Snippet: Human full-length cDNA of ATF2 was cloned into expression plasmid pCMV3-ATF2-Flag (Sino Biological lnc.).

Techniques: Migration, In Vitro, Wound Healing Assay, Over Expression, Western Blot, Nucleic Acid Electrophoresis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATF2 predicts poor prognosis and promotes malignant phenotypes in renal cell carcinoma

doi: 10.1186/s13046-016-0383-2

Figure Lengend Snippet: ATF2 knockdown suppresses tumor growth in vivo. a A representative nude mice showing the morphology of the tumors derived from ATF2 knockdown and control ACHN cells (upper). The tumors were dissected and photographed (lower). b The growth curve of the ATF2 knockdown versus the control ACHN tumors. c The average weight of ATF2 knockdown versus the control ACHN tumors. d Ki-67 staining of ATF2 knockdown versus the control ACHN tumors. Scale bar = 50 μm. e TUNEL assay analysis of the cell apoptosis in ATF2 knockdown versus the control ACHN tumors (left). Apoptosis cells numbers per view were shown (right). Scale bar = 50 μm. f qRT-PCR analysis of ATF2, Cyclin B1 and Cyclin D1 in ATF2 knockdown versus the control ACHN tumors. g Left: representative images of HE staining of metastatic nodules in the lungs of nude mice. The metastatic nodules are indicated by yellow arrows. Scale bar = 100 μm. Right: the numbers of nude mice with metastatic nodules in the lungs were calculated and compared ( p = 0.016). Results are presented as means ± SEM for each group ( n = 6). * p < 0.05, ** p <0.01, and *** p < 0.001

Article Snippet: Human full-length cDNA of ATF2 was cloned into expression plasmid pCMV3-ATF2-Flag (Sino Biological lnc.).

Techniques: In Vivo, Derivative Assay, Staining, TUNEL Assay, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ATF2 predicts poor prognosis and promotes malignant phenotypes in renal cell carcinoma

doi: 10.1186/s13046-016-0383-2

Figure Lengend Snippet: High levels of ATF2 predicts poor prognosis of RCC patients. a & b & c Comparison of ATF2 expression in human RCC tissues with diameter > 4 cm or ≤ 4 cm ( a ), RCC tissues with or without tumor thrombus ( b ), RCC tissues with or without tumor metastasis ( c ) determined by immunohistochemistry. The horizontal lines in the box plots represent the median, the boxes represent the interquartile range, and the whiskers represent the 2.5th and 97.5th percentiles. d Patients in comparative ATF2 high group ( n = 102) had lower overall survival time than those in comparative ATF2 low group ( n = 103) ( P = 0.017). e Patients in comparative ATF2 high group ( n = 102) had lower disease-free survival time than those in comparative ATF2 low group ( n = 103) ( P = 0.031)

Article Snippet: Human full-length cDNA of ATF2 was cloned into expression plasmid pCMV3-ATF2-Flag (Sino Biological lnc.).

Techniques: Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: TurboID-based proximity labeling discovers ABCF2 as an adhesion receptor for the zoonotic pathogen Pasteurella multocida

doi: 10.1101/2024.12.03.626657

Figure Lengend Snippet: (A, B) P. multocida infection induced increased levels of ABCF2 in NPTr and A549 cells evaluated by qPCR (A) and western-blotting (B) assays. (C) Western-blotting results showing the expression of ABCF2 in NPTr cells induced by P. multocida wild type strain (WT), the plpE -deleted strain (ΔPlpE), and plpE -complementary strains (CPlpE). (D) Results from western-blotting assays quantified by the image J software. (E) Western-blotting results showing the expression of ABCF2 and P65 phosphorylation (P-P65) in NPTr cells at 0 (mock), 1, 2, 6, and 12 hours post P. multocida inoculation. (F) Results from western-blotting assays quantified by the image J software. (G) Western-blotting results showing the expression of ABCF2 and P65 phosphorylation (P-P65) in NPTr cells treated with (+) or without (-) the NF-κB inhibitor (BAY11-7082), followed by P. multocida inoculation. (H) Results from western-blotting assays quantified by the image J software. (I) qPCR detecting the transcriptional levels of atf2 , stat1 , and abcf2 in NPTr cells at 12 hours post P. multocida inoculation. (J) Western-blotting results showing the P38 phosphorylation (P-P38) in NPTr cells at 0, 4, 8, 12, 16, and 20 hours post P. multocida inoculation. (K) Western-blotting results showing the expression of ABCF2 and P38 phosphorylation (P-P38) in NPTr cells treated with (+) or without (-) p38 inhibitor (BIRB796), followed by P. multocida inoculation. (L) P38 phosphorylation (P-P38) from western-blotting assays quantified by the image J software. (M) ABCF2 expression from western-blotting assays quantified by the image J software. (N) Dual luciferase assays demonstrating the regulation of atf2 on the expression of abcf2 . (O) qPCR assays verifying ATF2 overexpression (ATF2-OE) in NPTr cells with or without P. multocida infection. (P) qPCR assays showing ATF2 overexpression (ATF2-OE) contribute to ABCF2 expression after P. multocida infection. (Q) Western-blotting revealing ATF2 overexpression (ATF2-OE) contribute to ABCF2 expression after P. multocida infection. (R) ABCF2 expression from western-blotting assays quantified by the image J software. (S) Western-blotting showing inhibition of p38 MAPK signaling using the inhibitor BIRB796 decreases P65 phosphorylation (P-P65) after P. multocida infection. (T) The level of P65 phosphorylation (P-P65) from western-blotting assays quantified by the image J software. In all column charts, data were presented as mean ± standard deviation (SD). The significance level was set at P > 0.05 (no significance [ns]), P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***).

Article Snippet: Proteins were transferred to PVDF membranes (Bio-Rad, Hercules, USA), blocked in PBST (containing 5%BSA) for 3 h at room temperature, and probed with specific antibodies overnight at 4 °C to examine the expression of ABCF2 (ABCF2 polyclonal antibody [1:200, MyBioSource, San Diego, USA]), Flag-ATF2 (DYKDDDDK tag polyclonal antibody [1:5,000, Proteintech, San Diego, USA]), p53 (p53 monoclonal antibody [1:10000, Proteintech, San Diego, USA]), P-p53 (phospho-p53 (Ser15) monoclonal antibody [1:2000, Proteintech, San Diego, USA]), Bcl-2 (Bcl-2 polyclonal antibody [1:2000, Proteintech, San Diego, USA]), BAX (BAX monoclonal antibody [1:10000, Proteintech, San Diego, USA]), Caspase3 (Caspase 3/p17/p19 polyclonal antibody [Proteintech, San Diego, USA]), Caspase9 (Caspase 9/p35/p10 polyclonal antibody [Proteintech, San Diego, USA]), and/or GAPDH (GAPDH polyclonal antibody [1:5000, Proteintech, San Diego, USA]).

Techniques: Infection, Western Blot, Expressing, Software, Luciferase, Over Expression, Inhibition, Standard Deviation