primary antibodies against twist1  (Proteintech)

Journal: Bioengineered

Article Title: CircRAB3IP upregulates twist family BHLH transcription factor (TWIST1) to promote osteosarcoma progression by sponging miR-580-3p.

doi: 10.1080/21655979.2021.1948487

Figure Lengend Snippet: Figure 4. TWIST1 was targeted by miR-580-3p in OS (a) Bioinformatics was used to predict the potential binding site between miR- 580-3p and TWIST1. (b) TWIST1 was a target gene of miR-580-3p. (c, d) miR-580-3p mimics inhibited TWIST1 expression. (e) qRT-PCR indicated that TWIST1 was up-regulated in OS tissues. (f) Pearson’s analysis showed that circRAB3IP expression was positively correlated with TWIST1 expression in OS tissues. OS cells were transfected with sh-NC, sh-circRAB3IP, sh-circRAB3IP+miR-580-3p inhibitor. (g) Luciferase reporter assay was used to determined the activity of TWIST1-Wt reporter. (h) TWIST1 expression was detected by Western blot. *P < 0.05.

Article Snippet: Primary rabbit antihuman antibodies against TWIST1 (1:1000, cell signaling technology, #46702) and GAPDH (1:2000, Abcam, ab9485) were incubated overnight at 4°C.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Reporter Assay, Activity Assay, Western Blot

Journal: Bioengineered

Article Title: CircRAB3IP upregulates twist family BHLH transcription factor (TWIST1) to promote osteosarcoma progression by sponging miR-580-3p.

doi: 10.1080/21655979.2021.1948487

Figure Lengend Snippet: Figure 5. miR-580-3p inhibitor or TWIST1 overexpression reversed effects of sh-circRAB3IP. MG63 cells were transfected with the control sh RNA (sh-NC), sh-circRAB3IP, sh-circRAB3IP+miR-580-3p inhibitor or sh-circRAB3IP+TWIST1 overexpression vector (sh- circRAB3IP+oeTWIST1). (a, b) Cell proliferation was detected by CCK-8 assay and colony formation assay. (c, d) Transwell assay was employed to detect cell migration and invasion of OS cells. *P < 0.05.

Article Snippet: Primary rabbit antihuman antibodies against TWIST1 (1:1000, cell signaling technology, #46702) and GAPDH (1:2000, Abcam, ab9485) were incubated overnight at 4°C.

Techniques: Over Expression, Transfection, Control, Plasmid Preparation, CCK-8 Assay, Colony Assay, Transwell Assay, Migration

Journal: Cancer Cell International

Article Title: Identification of key genes as predictive biomarkers for osteosarcoma metastasis using translational bioinformatics

doi: 10.1186/s12935-021-02308-w

Figure Lengend Snippet: ARHGAP25 inhibited MG63 and U2OS cells’ growth, migration and invasion. a ARHGAP25 expression was determined in vector transfected MG63 and U2OS cells. b Overexpression of ARHGAP25 in MG63 and U2OS cells inhibited cell growth. c Representative images of MG63 and U2OS cells wound healing assay at 0 and 24 h after scratching. Bars indicate: 100 µm. d , e Quantification of the gap size in wound healing assay (n = 3), which showed that overexpression of ARHGAP25 suppressed wound healing. f Representative images of MG63 and U2OS cells stained with crystal violet in the transwell assay. Bars indicate: 100 µm. g Counts of cells invaded through the chambers (n = 3). h Representative images of colony formation assay to analyze cell proliferation in ARHGAP25 overexpressed OS cells. i Quantification of the colony number (n = 3). j Protein expression of Twist1, Vimentin, and E-cadherin was measured in OS cells with ARHGAP25 overexpression. k Quantification of protein expression using ImageJ. *Indicates P < 0.05, **indicates P < 0.01 and ***indicates P < 0.001. Picture c , f are magnified 100 times and h is not magnified

Article Snippet: Primary antibodies against TWIST1 (25465), anti-E-cadherin (20874), anti-vimentin (10366-1), and GAPDH (60004-1) were purchased from Proteintech (NJ, USA).

Techniques: Migration, Expressing, Plasmid Preparation, Transfection, Over Expression, Wound Healing Assay, Staining, Transwell Assay, Colony Assay

Journal: Frontiers in Oncology

Article Title: Bioinformatics Analysis and Functional Verification of ADAMTS9-AS1/AS2 in Lung Adenocarcinoma

doi: 10.3389/fonc.2021.681777

Figure Lengend Snippet: Functional enrichment analysis.

Article Snippet: After blocked with 4% non-fat milk, the membranes were incubated with a 1:1,000 dilution of primary antibodies against vimentin, Snail1, Twist1, ZEB1 and Beta3 (Abclonal, Wuhan, China) at 4°C overnight.

Techniques: Functional Assay, Membrane, Binding Assay, Activity Assay, Transferring, Transmission Assay

Journal: Frontiers in Oncology

Article Title: Bioinformatics Analysis and Functional Verification of ADAMTS9-AS1/AS2 in Lung Adenocarcinoma

doi: 10.3389/fonc.2021.681777

Figure Lengend Snippet: ADAMTS9-AS1/ADAMTS9-AS2 inhibits the LUAD cells proliferation, migration and invasion. (A) The proliferative ability of ADAMTS9-AS1/ADAMTS9-AS2 was detected by CCK-8. (B) The cell healing ability of ADAMTS9-AS1/ADAMTS9-AS2 was detected by a wound healing scratch assay. (C) The migratory ability of ADAMTS9-AS1/ADAMTS9-AS2 was detected by a transwell migration assay. (D) The invasion ability of ADAMTS9-AS1/ADAMTS9-AS2 was detected by a transwell invasion assay. (E) The influence of ADAMTS9-AS1/ADAMTS9-AS2 on Beta3 and EMT signal pathway detected by Western blot assays. *p < 0.05; **p < 0.01; ***p < 0.001. LUAD, lung adenocarcinoma.

Article Snippet: After blocked with 4% non-fat milk, the membranes were incubated with a 1:1,000 dilution of primary antibodies against vimentin, Snail1, Twist1, ZEB1 and Beta3 (Abclonal, Wuhan, China) at 4°C overnight.

Techniques: Migration, CCK-8 Assay, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Thymidine phosphorylase promotes malignant progression in hepatocellular carcinoma through pentose Warburg effect

doi: 10.1038/s41419-018-1282-6

Figure Lengend Snippet: a Heat map of Twist1 co-expression genes downloaded from The Cancer Genome Atlas (TCGA) database. b Venn analysis for identifying Twist1 target genes in human hepatocellular carcinoma (HCC). The blue diagram represents Twist1 co-expression genes from TCGA, and the green diagram represents Twist1 targeting genes by chromatin immunoprecipitation–sequencing (ChIP-seq). c TP expression was significantly upregulated in LIHC samples compared with the normal liver tissues. d Correlation analysis of Twist1 and TP expression in clinical HCC specimens. e Relative TP expression analysis in seven HCC cell lines. The ratio of densitometry value to the corresponding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) value was used to indicate relative protein expression. f Western blot used to analyze TP protein levels influenced by Twist1 in PLC-PRF-5 and Hep3B cells. g Twist1 promoted TP gene transcription as detected by luciferase assay. h Diagram of the ChIP-seq binding peaks related to the TP promoter. Binding site analysis showed two individual conserved motifs within the promoter of TP. i Luciferase assay results of Twist1 to the truncated TP promoter regions demonstrated that Twist1 bound to the TP promoters of region 3/4. j Luciferase assay results of Twist1 to the mutated TP promoter regions demonstrated that Twist1 bound to motif 1/2 in the TP promoter. (mean ± SD; n = 3 in triplicate; ** P < 0.01)

Article Snippet: The membrane was incubated with primary antibodies against Twist1 (Santa Cruz Biotechnology, USA), TP (Abcam, UN), VE–cadherin (Affinity Bioreagents, USA), VEGFR1 (Affinity Bioreagents, USA), VEGFR2 (Affinity Bioreagents, USA), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Affinity Bioreagents, USA), followed by secondary antibody (Santa Cruz Biotechnology, USA).

Techniques: Expressing, ChIP-sequencing, Western Blot, Luciferase, Binding Assay

Journal: Cell Death & Disease

Article Title: Thymidine phosphorylase promotes malignant progression in hepatocellular carcinoma through pentose Warburg effect

doi: 10.1038/s41419-018-1282-6

Figure Lengend Snippet: a Overexpression of Twist1 activated a series of pathways related to glycometabolism and amino acid metabolism. b Enrichment analysis of the gene expression profiles in PLC-PRF-5 cells overexpressing Twist1 detected by gene set enrichment analysis. c Enzyme-linked immunosorbent assay was used to analyze the TP factor levels in the medium. Overexpression of TP promoted the amount of TP secreted in PLC-PRF-5 cells; knocking down of TP reduced the amount of TP secreted in Hep3B cells. d Correlation between Twist1/TP expression levels and secreted TP factor levels in PLC-PRF-5 and Hep3B cells. e Overexpression of TP promoted the enzymatic metabolism of extracellular dT in the medium without glucose. f Enzymatic metabolism of extracellular dT regulated by TP promoted HCC cell proliferation. g Enzymatic metabolism of extracellular dT regulated by TP promoted ATP generation in PLC-PRF-5 cells. h Enzymatic metabolism of extracellular dT regulated by TP affected glycometabolism and amino acid metabolism in glucose-free cultured PLC-PRF-5 cells. NG means “No Glucose.” (mean ± SD; n = 3 in triplicate; * P < 0.05; ** P < 0.01)

Article Snippet: The membrane was incubated with primary antibodies against Twist1 (Santa Cruz Biotechnology, USA), TP (Abcam, UN), VE–cadherin (Affinity Bioreagents, USA), VEGFR1 (Affinity Bioreagents, USA), VEGFR2 (Affinity Bioreagents, USA), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Affinity Bioreagents, USA), followed by secondary antibody (Santa Cruz Biotechnology, USA).

Techniques: Over Expression, Gene Expression, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture

Journal: Cell Death & Disease

Article Title: Thymidine phosphorylase promotes malignant progression in hepatocellular carcinoma through pentose Warburg effect

doi: 10.1038/s41419-018-1282-6

Figure Lengend Snippet: PLC-PRF-5 cells were upregulated with Twist1 and/or TP or upregulated with Twist1 and knocked down of TP. Hep3B cells were knocked down of Twist1 and/or TP or knocked down of Twist1 and upregulated with TP. Cells were cultured in glucose-free medium added with the TP substrate of dT. a Wound healing assay showed a significant difference in the speed of cell migration among different treated groups. b Invasion assay showed a significant difference in the speed of cell invasion among different treated groups. c In vitro assay for VM in three-dimensional culture at 24 h. Upregulation of Twist1 and/or TP promoted the tube formation in PLC-PRF-5 cells, and knocking down of TP attenuated the promotion effect of Twist1. Knocking down of Twist1 and/or TP inhibited the tube formation in Hep3B cells, and upregulating TP significantly reduced the inhibitory effect of knocking down of Twist1. d Morphological observation of different treated PLC-PRF-5 cells in pseudopod and cell rounding. e TP relied on the extracellular dT to promote the tube formation of glucose-free cultured PLC-PRF-5 cells. f Transcriptional regulatory activities of Twist1 and TP on VE–Cad, vascular endothelial growth factor receptor 1 (VEGFR1), and VEGFR2. g Protein expression analysis of VE–Cad, VEGFR1, and VEGFR2 with the HCS systems. NG means “No Glucose” (mean ± SD; n = 3 in triplicate; * P < 0.05; ** P < 0.01)

Article Snippet: The membrane was incubated with primary antibodies against Twist1 (Santa Cruz Biotechnology, USA), TP (Abcam, UN), VE–cadherin (Affinity Bioreagents, USA), VEGFR1 (Affinity Bioreagents, USA), VEGFR2 (Affinity Bioreagents, USA), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Affinity Bioreagents, USA), followed by secondary antibody (Santa Cruz Biotechnology, USA).

Techniques: Cell Culture, Wound Healing Assay, Migration, Invasion Assay, In Vitro, Expressing

Journal: Cell Death & Disease

Article Title: Thymidine phosphorylase promotes malignant progression in hepatocellular carcinoma through pentose Warburg effect

doi: 10.1038/s41419-018-1282-6

Figure Lengend Snippet: HCC samples were divided into four groups of Twist1/TP (−/−), Twist1/TP (−/+), Twist1/TP (+/−), and Twist1/TP (+/+) according to the Twist1/TP expression levels. a Correlation between Twist1/TP expression and HCC characteristics, including metastasis, clinical stage, pathology grade, carcinoembryonic antigen (CEA) level, alpha-fetoprotein (AFP) level, and gender. b Overall survival analysis of Twist1/TP on patients with HCC. c Correlation analysis between vasculogenic mimicry (VM) formation and Twist1 and TP expression in 306 cases of HCC. The VM channel was PAS positive, but it did not express CD31 (red arrow). Endothelial vessels were PAS- and CD31-positive (yellow arrow). d Analysis of the HCC specimens by IHC. VE–Cad, vascular endothelial growth factor receptor 1 (VEGFR1), and VEGFR2 were minimally expressed in the Twist1- and TP-negative expression groups. When Twist1 and TP were individually or both positively expressed, the expression levels of the three marker proteins increased. e Statistical analysis of protein expression in 306 HCC specimens among the four groups (mean ± SD; * P < 0.05; ** P < 0.01)

Article Snippet: The membrane was incubated with primary antibodies against Twist1 (Santa Cruz Biotechnology, USA), TP (Abcam, UN), VE–cadherin (Affinity Bioreagents, USA), VEGFR1 (Affinity Bioreagents, USA), VEGFR2 (Affinity Bioreagents, USA), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Affinity Bioreagents, USA), followed by secondary antibody (Santa Cruz Biotechnology, USA).

Techniques: Expressing, Marker

Journal: Cell Death & Disease

Article Title: Thymidine phosphorylase promotes malignant progression in hepatocellular carcinoma through pentose Warburg effect

doi: 10.1038/s41419-018-1282-6

Figure Lengend Snippet: a Twist1 and TP promoted PLC-PRF-5 xenograft growth, whereas knocking down TP impaired the promotion effects of Twist1. b Number of tumors formed and lung metastasis in different groups. Twist1 and TP promoted lung metastasis of HCC, while knocking down of TP attenuated the promotion effect of Twist1. Images were taken at ×100 magnification. c Statistical analysis of VM formations in different groups. d Analysis of Twist1, TP, VE–Cad, vascular endothelial growth factor receptor 1 (VEGFR1), and VEGFR2 expression levels in xenograft tumors. Images were taken at ×400 magnification. e Statistical analysis of Twist1, TP, VE–Cad, VEGFR1, and VEGFR2 expression levels in different treated xenograft tumors (mean ± SD; * P < 0.05; ** P < 0.01)

Article Snippet: The membrane was incubated with primary antibodies against Twist1 (Santa Cruz Biotechnology, USA), TP (Abcam, UN), VE–cadherin (Affinity Bioreagents, USA), VEGFR1 (Affinity Bioreagents, USA), VEGFR2 (Affinity Bioreagents, USA), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Affinity Bioreagents, USA), followed by secondary antibody (Santa Cruz Biotechnology, USA).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Thymidine phosphorylase promotes malignant progression in hepatocellular carcinoma through pentose Warburg effect

doi: 10.1038/s41419-018-1282-6

Figure Lengend Snippet: Hep3B cells were treated by knocking down TP and/or adding TP enzyme inhibitor tipiracil (TPI). a TPI exhibited good binding activity to TP determined by molecular docking. b Wound healing assay was performed. Quantitative analysis showed a significant difference in the speed of migration among different treated Hep3B cells. c Invasion assay showed a significant difference in the speed of invasion among different treated Hep3B cells. d Knocking down TP and/or adding TPI inhibited tube formation as detected by three-dimensional culture assay. e Effects of knocking down TP and/or adding TPI on Hep3B xenograft tumor growth. f Number of tumor formations and lung metastasis in different groups. Images were taken at ×100 magnification. g Statistical analysis of VM formation in different treated Hep3B xenograft tumors. h Statistical analysis of VE–Cad, vascular endothelial growth factor receptor 1 (VEGFR1), and VEGFR2 expression levels in different treated Hep3B xenograft tumors. i Proposed regulatory mechanism between Twist1–TP and VM in HCC. (mean ± SD; ** P < 0.01)

Article Snippet: The membrane was incubated with primary antibodies against Twist1 (Santa Cruz Biotechnology, USA), TP (Abcam, UN), VE–cadherin (Affinity Bioreagents, USA), VEGFR1 (Affinity Bioreagents, USA), VEGFR2 (Affinity Bioreagents, USA), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Affinity Bioreagents, USA), followed by secondary antibody (Santa Cruz Biotechnology, USA).

Techniques: Binding Assay, Activity Assay, Wound Healing Assay, Migration, Invasion Assay, Expressing