Journal: Translational Lung Cancer Research

Article Title: Exosomal ITGB6 from dormant lung adenocarcinoma cells activates cancer-associated fibroblasts by KLF10 positive feedback loop and the TGF-β pathway

doi: 10.21037/tlcr-23-707

Figure Lengend Snippet: KLF10-Smad2 positive feedback loop induces the activation of CAFs by activating the TGF-β pathway. (A) Protein expression of the TGF-β pathway were measured by western blot analysis. MRC-5 cells were treated with DORexo compared to exo. GAPDH was used as the internal control. (B) Protein expression of KLF10 and TGF-β pathway were measured by western blot analysis. MRC-5 cells incubated with DORexo were transfected with si-KLF10. Further, MRC-5 cells were transfected with KLF10 overexpression plasmid. GAPDH was used as the internal control. (C) Protein expression of KLF10 and TGF-β pathway were measured by western blot analysis. MRC-5 cells incubated with DORexo were transfected with si-Smad2. Further, MRC-5 cells were transfected with Smad2 overexpression plasmid. GAPDH was used as the internal control. NC, negative control; exo, A549-secreted exosomes; DORexo, dormant A549-secreted exosomes; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAFs, cancer-associated fibroblasts; TGF-β, transforming growth factor β.

Article Snippet: KLF10 overexpression plasmid, Smad2 overexpression plasmid, ITGB6 overexpression plasmid, and empty vector plasmid were synthesized by GenScript (Nanjing, China).

Techniques: Activation Assay, Expressing, Western Blot, Control, Incubation, Transfection, Over Expression, Plasmid Preparation, Negative Control

Journal: Translational Lung Cancer Research

Article Title: Exosomal ITGB6 from dormant lung adenocarcinoma cells activates cancer-associated fibroblasts by KLF10 positive feedback loop and the TGF-β pathway

doi: 10.21037/tlcr-23-707

Figure Lengend Snippet: KLF10 is the key molecule for activation of CAFs. (A) The RNA expression profile of MRC-5 incubated with exosomes was examined. The heatmap of differentially expressed molecules was presented. (B) Venn diagram showing that the overlapping part between the 3 datasets contained 509 molecules, including our dataset and two independent datasets (GSE116679 and GSE61797). (C) The volcano plot for 509 molecules showed that KLF10 was one of the most representative differentially expressed RNA molecules. (D) The expression of KLF10 was measured by qRT-PCR and western blotting. (E,G) The invasion ability of fibroblast was accessed by transwell assays with crystal violet staining. Magnification: ×40; scale bar =200 µm. (F,H) The contractile ability of fibroblast was accessed by ECM-remodeling assay. Data are presented as the mean ± SD, and analyzed with Student’s t -test. **, P<0.01; ****, P<0.0001. NC, negative control; exo, A549-secreted exosomes; DORexo, dormant A549-secreted exosomes; CAFs, cancer-associated fibroblasts; qRT-PCR, quantitative real-time polymerase chain reaction; ECM, extracellular matrix; SD, standard deviation.

Article Snippet: KLF10 overexpression plasmid, Smad2 overexpression plasmid, ITGB6 overexpression plasmid, and empty vector plasmid were synthesized by GenScript (Nanjing, China).

Techniques: Activation Assay, RNA Expression, Incubation, Expressing, Quantitative RT-PCR, Western Blot, Staining, Negative Control, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Translational Lung Cancer Research

Article Title: Exosomal ITGB6 from dormant lung adenocarcinoma cells activates cancer-associated fibroblasts by KLF10 positive feedback loop and the TGF-β pathway

doi: 10.21037/tlcr-23-707

Figure Lengend Snippet: KLF10-Smad2 positive feedback loop induces the activation of CAFs by activating the TGF-β pathway. (A) Protein expression of the TGF-β pathway were measured by western blot analysis. MRC-5 cells were treated with DORexo compared to exo. GAPDH was used as the internal control. (B) Protein expression of KLF10 and TGF-β pathway were measured by western blot analysis. MRC-5 cells incubated with DORexo were transfected with si-KLF10. Further, MRC-5 cells were transfected with KLF10 overexpression plasmid. GAPDH was used as the internal control. (C) Protein expression of KLF10 and TGF-β pathway were measured by western blot analysis. MRC-5 cells incubated with DORexo were transfected with si-Smad2. Further, MRC-5 cells were transfected with Smad2 overexpression plasmid. GAPDH was used as the internal control. NC, negative control; exo, A549-secreted exosomes; DORexo, dormant A549-secreted exosomes; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAFs, cancer-associated fibroblasts; TGF-β, transforming growth factor β.

Article Snippet: KLF10 overexpression plasmid, Smad2 overexpression plasmid, ITGB6 overexpression plasmid, and empty vector plasmid were synthesized by GenScript (Nanjing, China).

Techniques: Activation Assay, Expressing, Western Blot, Control, Incubation, Transfection, Over Expression, Plasmid Preparation, Negative Control

Journal: Translational Lung Cancer Research

Article Title: Exosomal ITGB6 from dormant lung adenocarcinoma cells activates cancer-associated fibroblasts by KLF10 positive feedback loop and the TGF-β pathway

doi: 10.21037/tlcr-23-707

Figure Lengend Snippet: Exosomal ITGB6 from dormant LUAD cells induces the activation of CAFs by activating TGF-β pathway. (A) The Venn diagram showed that the overlapping part between the two datasets contained 12 molecules, including proteomics and TGF-β associated datasets (GO:0007179). ITGB6 was selected as the key molecule. (B) Proteomics showed that ITGB6 is up-regulation in DORexo. **, P<0.01. (C) ITGB6 was measured in DORexo by western blot analysis. (D) After brief processing (6 hours) with exosomes, the protein level of ITGB6 in MRC-5 cells was detected by western blot analysis. (E) Protein expression of ITGB6, KLF10, and the TGF-β pathway were measured by western blot analysis. MRC-5 cells were transfected with ITGB6 overexpression plasmid. (F,G) The invasion and contractile ability of fibroblast were accessed by transwell assays with crystal violet staining and ECM-remodeling assay. MRC-5 cells were transfected with ITGB6 overexpression plasmid. Magnification: ×40; scale bar =200 µm. **, P<0.01; ****, P<0.0001. (H) Immunohistochemical analysis evaluated the correlation between the expression of ITGB6 and progress state in one case of LUAD after chemotherapy. exo, A549-secreted exosomes; DORexo, dormant A549-secreted exosomes; NC, negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LUAD, lung adenocarcinoma; CAFs, cancer-associated fibroblasts; TGF-β, transforming growth factor β; GO, Gene Ontology; ECM, extracellular matrix.

Article Snippet: KLF10 overexpression plasmid, Smad2 overexpression plasmid, ITGB6 overexpression plasmid, and empty vector plasmid were synthesized by GenScript (Nanjing, China).

Techniques: Activation Assay, Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, Staining, Immunohistochemical staining, Negative Control

Journal: Translational Lung Cancer Research

Article Title: Exosomal ITGB6 from dormant lung adenocarcinoma cells activates cancer-associated fibroblasts by KLF10 positive feedback loop and the TGF-β pathway

doi: 10.21037/tlcr-23-707

Figure Lengend Snippet: ITGB6 was associated with TGF-β pathway and ECM remodeling. (A) The correlation between ITGB6 and TGF-β pathway associated molecules was showed in LUAD patients by scatter diagram (TIMER2.0 database). (B) The correlation between ITGB6 and ECM-associated molecules in LUAD patients was shown by scatter diagram (TIMER2.0 database). (C) A proposed working model for exosomal ITGB6 derived from dormant LUAD cells was transferred into fibroblasts and induced the activation of a KLF10 positive feedback loop and the TGF-β pathway. The pattern diagram was drawn by Figdraw. TPM, transcripts per million; TGF-β, transforming growth factor β; ECM, extracellular matrix; CAFs, cancer-associated fibroblasts; α-SMA, alpha smooth muscle actin; IL, interleukin; LUAD, lung adenocarcinoma; TIMER2.0, Tumor Immune Estimation Resource.

Article Snippet: KLF10 overexpression plasmid, Smad2 overexpression plasmid, ITGB6 overexpression plasmid, and empty vector plasmid were synthesized by GenScript (Nanjing, China).

Techniques: Derivative Assay, Activation Assay

Journal: Atherosclerosis plus

Article Title: Smad2 inhibition of MET transcription potentiates human vascular smooth muscle cell apoptosis

doi: 10.1016/j.athplu.2021.08.005

Figure Lengend Snippet: Met is anti-apoptotic in AoSMCs and negatively regulated by Smad2. A and B. Smad2 loss-of-function increases MET expression at protein and mRNA levels (Western blots in A and qRT-PCR analyses in B). C and D. Met gain- and loss-of-function respectively inhibits and enhances AoSMC apoptosis (FACS assay). Black bars represent averaged values based on three independent repeat experiments (mean ± SD, n = 3). The apoptotic cells include Q2 and Q4, late- and early-phase apoptotic populations, respectively. The Flow Jo software was used. E and F. Met gain- and loss-of-function respectively inhibits and enhances AoSMC apoptosis (cleaved caspase3 assay). Human primary aortic SMCs (AoSMCs) were transfected with scrambled siRNA (Scr), Smad2-specific siRNA, empty vector (VEC), or overexpression plasmid (OE) for 12 h in basal medium (no FBS). The cells were cultured for another 12 h in fresh basal medium (no transfection reagents) to recover, and then treated with solvent or 10 ng/ml TGFβ1 or 50 ng/ml PDGF-BB for 20 h before harvest for Western blot and qRT-PCR analyses. Quantification: Densitometry of Western blots (similar ECL exposure) from independent repeat experiments was normalized (to GAPDH) and then averaged to calculate mean ± SEM (n = 3 independent experiments). Readings of triplicate qRT-PCR reactions were normalized (to GAPDH) and averaged to calculate mean ± SD (n = 3 repeats). a.u., arbitrary unit. Statistics: One-way ANOVA followed by Bonferroni post-hoc test in A and B; Student’s t-test in C–F; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: In brief, AoSMCs were transfected with empty vector (VEC) or smad2 overexpression plasmid (Addgene, 11734), or AoSMCs were starved and treated with Nutlin 3a for 24 h. Post transfected or treated AoSMCs were lysed on ice for 30min in IP Lysis/Wash Buffer (included in kit) containing Halt protease inhibitor cocktail (Thermo Fisher Scientific, 87785).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Software, Caspase-3 Assay, Transfection, Plasmid Preparation, Over Expression, Cell Culture, Solvent

Journal: Atherosclerosis plus

Article Title: Smad2 inhibition of MET transcription potentiates human vascular smooth muscle cell apoptosis

doi: 10.1016/j.athplu.2021.08.005

Figure Lengend Snippet: Smad2 positively regulates AoSMC expression of FAS. Human primary aortic SMCs (AoSMCs) were transfected with scrambled siRNA (Scr), Smad2-specific siRNA, empty vector (VEC), or Smad2 overexpression plasmid (Smad2-OE) for 12 h in basal medium (no FBS). The cells were cultured for another 12 h in fresh basal medium (no Lipofectamine) to recover, and then treated with solvent or 10 ng/ml TGFβ1 or 50 ng/ml PDGF-BB for 20 h before harvest for Western blot and qRT-PCR analyses. A and B. Smad2 loss-of-function reduces FAS expression at protein and mRNA levels (Western and qRT-PCR analyses). C. Smad2 gain-of-function enhances FAS promoter activity (luciferase assay). D. Smad2 enrichment at FAS promoter regions (ChIP-qPCR). Quantification: Densitometry of Western blots (similar ECL exposure) from independent repeat experiments was normalized (to GAPDH) and then averaged to calculate mean ± SEM, n = 3 independent experiments. Readings of triplicate qRT-PCR reactions were normalized (to GAPDH) and averaged to calculate mean ± SD (n = 3 repeats). a.u., arbitrary unit. Statistics: One-way ANOVA/Bonferroni post-hoc test; Student’s t-test was performed in C; **P < 0.01, ***P < 0.001. E. Schematic working model of Smad2 regulations of MET and FAS that promote SMC apoptosis. While Smad2 partnering with P53 represses the transcription of MET, it also activates the transcription of FAS. The sum of decreased Met (anti-apoptotic) and increased Fas (pro-apoptotic) prompts SMC apoptosis.

Article Snippet: In brief, AoSMCs were transfected with empty vector (VEC) or smad2 overexpression plasmid (Addgene, 11734), or AoSMCs were starved and treated with Nutlin 3a for 24 h. Post transfected or treated AoSMCs were lysed on ice for 30min in IP Lysis/Wash Buffer (included in kit) containing Halt protease inhibitor cocktail (Thermo Fisher Scientific, 87785).

Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression, Cell Culture, Solvent, Western Blot, Quantitative RT-PCR, Activity Assay, Luciferase, ChIP-qPCR

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: The expression of TGIF2 and SOX2 in human PC and adjacent pancreas. A. TGIF2 and SOX2 expression in PC and paired pancreas specimens by IHC. B and C. TGIF2, SOX2, EGFR and E-cad expression in two PC specimens (#21 and #37). D. The mRNA level of TGIF2 in 20 cases of human PC and adjacent pancreas by qRT-PCR (T: PC; N: paired pancreas) and SOX2. E. The mRNA level of SOX2 in our cohort. F and G. High and low expression of TGIF2 and SOX2 against prognosis. H. Combination of TGIF2 and SOX2 against prognosis.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: The relationship among TGIF2, SOX2, EGFR and E-cad expression in 88 cases of clinical PC samples

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Expressing

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: Univariate and Multivariate analysis in survival time

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: Coordination of TGIF2/SOX2 promotes EMT of PC in vitro . A and B. TGIF2 and SOX2 protein (A) and mRNA (B) level in 4 PC cell lines. C. The protein expression of TGIF2, SOX2 and EMT signaling in Scramble, TGIF2-OE, siSOX2 and TGIF2-OE/siSOX2 groups in PANC-1 cells. D. The protein expression of TGIF2, SOX2 and EMT signaling in Scramble, siTGIF2, SOX2-OE, and siTGIF2/SOX2-OE groups in BxPC-3 cells. E. The EMT phenotype in Scramble, TGIF2-OE, siSOX2 and TGIF2-OE/siSOX2 groups in PANC-1 cells. F. The EMT phenotype in Scramble, SOX2-OE, siTGIF2 and siTGIF2/SOX2-OE groups in BxPC-3 cells. G and H. Cell invasion (G) and migration (H) in Scramble, TGIF2-OE, siSOX2 and TGIF2-OE/siSOX2 groups in PANC-1 cells. I and J. Cell invasion (I) and migration (J) in Scramble, siTGIF2, SOX2-OE and siTGIF2/SOX2-OE groups Scramble, SOX2-OE, siTGIF2 and siTGIF2/SOX2-OE in BxPC-3 cells. Bars indicate ± S.E.*, P <0.05; **, P <0.01 compared with the control.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: In Vitro, Expressing, Migration, Control

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: Coordination of TGIF2/SOX2 promotes CSCs and drug resistance of PC in vitro . A and B. Sphere formation in PANC-1 cells (A) and BxPC-3 (B) cells following the activation of TGIF2, SOX2, CD133, CD44 and EGFR/MAPK signaling. C and D. The sphere number (C) and the protein expression of TGIF2, SOX2, CD133, CD44 and EGFR/MAPK signaling (D) in Scramble, TGIF2-OE, siSOX2 and TGIF2-OE/siSOX2 groups in PANC-1 cells. E and F The sphere number (E) and the protein expression of TGIF2, SOX2, CD133, CD44 and EGFR/MAPK signaling (F) in Scramble, siTGIF2, SOX2-OE and siTGIF2/SOX2-OE groups in BxPC-3 cells. ( G-H ) the spheroids number of PC in wildtype (treated with EGF to activate EGFR/ERK signaling) and Kras-mutant (constant activation of EGFR/ERK signaling) mice. ( I ) The overexpression of TGIF2, SOX2, CD133 and p-ERK were in pancreatic intraepithelial neoplasia (PanIN) of KC mice. J. Cell growth in Scramble, TGIF2-OE, siSOX2, TGIF2-OE/siSOX2 and TGIF2-OE/Erlotinib groups of PANC-1 cells and in Scramble, siTGIF2, SOX2-OE and siTGIF2/SOX2-OE of BxPC-3 cells by MTT. K. Under various concentration of Gemcitabine treatment, Cell proliferation rate in Scramble, TGIF2-OE, siSOX2, TGIF2-OE/siSOX2 and TGIF2-OE/Erlotinib groups of PANC-1 cells and in Scramble, siTGIF2, SOX2-OE and siTGIF2/SOX2-OE of BxPC-3 cells by MTT. Bars indicate ± S.E.*, P <0.05; **, P <0.01 compared with the control.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: In Vitro, Activation Assay, Expressing, Mutagenesis, Over Expression, Concentration Assay, Control

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: TGIF2 is a transactivation factor of SOX2 and interacts with Smad2 to co-regulate SOX2. A. The mRNA level of TGIF2 and SOX2 in TGIF2 overexpressing PANC-1 and TGIF2 silencing BxPC-3 cells, respectively. B. The binding site of TGIF2 in its zinc fingers domain was obtained from the JASPAR database. C. The predicted potential binding site of TGIF2 to SOX2 promoter, as well as wild-type/mutant SOX2 promoter plasmids (SOX2-WT or SOX2-Mut) designed accordingly. D. Chip assays in PANC-1 and BxPC-3 cells. E. Luciferase assay in 293 T cells co-transfected with SOX2-WT or SOX2-Mut promoter plasmid and TGIF2 overexpression plasmid. F. The protein interaction network of TGIF2 via String database. G. Co-IP was performed in PANC-1 and BxPC-3 cells. H and I. The protein and mRNA level of Smad2 and SOX2 in Smad2 silencing BxPC-3 cells. J. The binding site of Smad2 in its zinc fingers domain was obtained from the JASPAR database. K. The predicted potential binding site of Smad2 to SOX2 promoter, as well as wild-type/mutant SOX2 promoter plasmids (SOX2-WT or SOX2-Mut2) designed accordingly. L. Chip assays in BxPC-3 cells. M. Luciferase assay in 293 T cells co-transfected with SOX2-WT or SOX2-Mut2 promoter plasmid and Smad2 overexpression plasmid (or empty vector scramble). N. The protein level of Smad2 and SOX2 in Scramble, TGIF2-OE, si2-Smad2 and TGIF2-OE/si2-Smad2 groups in PANC-1 cells. Bars indicate ± S.E.*, P <0.05; **, P <0.01 in contrast with the control.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Binding Assay, Zinc-Fingers, Mutagenesis, Luciferase, Transfection, Plasmid Preparation, Over Expression, Co-Immunoprecipitation Assay, Control

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: SOX2 is a transactivation factor of EGFR and Slug. A. The mRNA level of SOX2, EGFR and Slug in SOX2 overexpressing PANC-1 and SOX2 silencing BxPC-3 cells, respectively. B. The DNA binding site of SOX2 was obtained from the JASPAR database. C. The predicted potential binding site of SOX2 to Slug promoter. D and E. Chip assays in PANC-1 (D) and BxPC-3 (E) cells. F. The predicted potential binding site of SOX2 to EGFR promoter, as well as wild-type/mutant EGFR promoter plasmids designed accordingly. G. Chip assays in PANC-1 and BxPC-3 cells. H. Luciferase assay in 293 T cells co-transfected with EGFR-WT (or EGFR-Mut) promoter plasmid and SOX2 overexpression plasmid (or empty vector corresponding to scramble group). I. The roles of SOX2-OE and Erlotinib treatment to TGIF2 nuclear translocation. J. IF staining of TGIF2 and SOX2 in Scramble, SOX2-OE and SOX2-OE plus Erlotinib groups of PANC-1. Bars indicate ± S.E.*, P <0.05; **, P <0.01 in contrast with the control.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Binding Assay, Mutagenesis, Luciferase, Transfection, Plasmid Preparation, Over Expression, Translocation Assay, Staining, Control

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: Coordination of TGIF2/SOX2 promotes subcutaneous tumor size in vivo . A. Tumor volumes in Scramble, TGIF2-OE, TGIF2-OE/shSOX2, and TGIF2-OE plus Erlotinib groups implanted with PANC-1 cells. B. Tumor growth curve in above group. C. HE staining of harvested tumor. D. The statistical data of IHC assays. E. The different expression of TGIF2, SOX2, E-cad, EGFR and Vimentin in Scramble, TGIF2-OE, and TGIF2-OE/shSOX2 groups by IHC. Bars indicate ± S.E. *, P<0.05; **, P<0.01 compared with control.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: In Vivo, Staining, Expressing, Control

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: Coordination of TGIF2/SOX2 promotes liver metastasis in vivo . A . Liver metastasis in Scramble, shTGIF2 and shTGIF2/SOX2-OE groups. B-C. The Liver body ratio and the number of liver metastases in above groups. D. The different expression of TGIF2, SOX2, E-cad, EGFR and Vimentin in Scramble, shTGIF2 and shTGIF2/SOX2-OE groups by IHC. E. The statistical data of IHC assays. Bars indicate ± S.E. *, P<0.05; **, P<0.01 compared with control.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: In Vivo, Expressing, Control

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: Smad2 cooperating with TGIF2 contributes to CSC and EMT via co-targeting SOX2. TGIF2 activates SOX2 promoter via interacting with Smad2, which stimulates EMT and EGFR/MAPK signaling by transactivating Slug and EGFR, and promoting EMT and CSCs function. Moreover, the stimulation of EGFR/MAPK signaling by SOX2 promotes TGIF2 nuclear translocation, forming a positive feedback loop.

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Translocation Assay

Journal: International Journal of Biological Sciences

Article Title: Smad2 Cooperating with TGIF2 Contributes to EMT and Cancer Stem Cells Properties in Pancreatic Cancer via Co-Targeting SOX2

doi: 10.7150/ijbs.102381

Figure Lengend Snippet: The clinicopathological significance of TGIF2 and SOX2 expression in 88 cases of clinical PC samples

Article Snippet: The siRNA and shRNA sequences for TGIF2 and SOX2 were summarized in , which were synthesized by GenePharma Co., Ltd. (Shanghai, China). shRNA-mediated TGIF2 and SOX2 silencing and lentivirus vector (GV492 and CV186)-mediated TGIF2, SOX2, and Smad2 overexpression (TGIF2-OE, SOX2-OE, and Smad2-OE, respectively) were purchased from GeneChem (Shanghai, China). siRNAs and plasmids were mixed with Oligofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: lncRNA XIST knockdown suppresses cell proliferation and promotes apoptosis in diabetic cataracts through the miR-34a/SMAD2 axis

doi: 10.3892/mmr.2021.12523

Figure Lengend Snippet: XIST is upregulated and miR-34a is downregulated in DC tissues and cells. (A and B) RT-qPCR showed the relative XIST and miR-34a expression in DC tissues and normal samples. *P<0.05. (C) Pearson's correlation analysis was used to determine the correlation between the miR-34a expression and XIST in DC tissues. (D and E) RT-qPCR showed the relative XIST and miR-34a expression in SRA01/04 cells treated with HG. n=3. *P<0.05 vs. NG. XIST, X-inactive specific transcript; miR, microRNA; DC, diabetic cataracts; RT-qPCR, reverse transcription-quantitative PCR; NG, normal glucose; HG, high glucose.

Article Snippet: Small hairpin (sh)RNA against XIST (shXIST, 5′-GUGCGUACAGUGCUGUACAGCAU-3′) and its negative control (NC; shNC, 5′-UACGCUCAGCAUGUGUCACUC-3′), miR-34a mimics (5′-UCGUUCGUGAGCACUUGCGACG-3′), NC mimics (5′-UCGUCGGAUCGACUGAGAUCU-3′), miR-34a inhibitors (5′-AGCCUUGCUGCAGGUGCGCAU-3′) and NC inhibitors (5′-UGCCUUACUGACGGUCGGAGA-3′) were obtained from Shanghai GenePharma Co., Ltd. pcDNA3.1 vector (Thermo Fisher Scientific, Inc.) was used to construct a XIST and SMAD2 overexpression vector.

Techniques: Quantitative RT-PCR, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: lncRNA XIST knockdown suppresses cell proliferation and promotes apoptosis in diabetic cataracts through the miR-34a/SMAD2 axis

doi: 10.3892/mmr.2021.12523

Figure Lengend Snippet: XIST depletion inhibits diabetic cataracts development in HG-induced lens epithelial cells. (A) Reverse transcription-quantitative PCR was used to determine the relative XIST expression in SRA01/04 transfected with shXIST. *P<0.05 vs. shNC. (B) MTT, (C) wound healing and (D) Transwell assays were used to analyze the proliferation, migration and invasion of SRA01/04 cells transfected with shXIST or shNC after treatment of HG. (E) TUNEL assay was used to determine the apoptosis in SRA01/04 cells transfected with shXIST or shNC after treatment of HG. n=3. *P<0.05. XIST, X-inactive specific transcript; sh, short hairpin RNA; NG, normal glucose; HG, high glucose; NC, negative control.

Article Snippet: Small hairpin (sh)RNA against XIST (shXIST, 5′-GUGCGUACAGUGCUGUACAGCAU-3′) and its negative control (NC; shNC, 5′-UACGCUCAGCAUGUGUCACUC-3′), miR-34a mimics (5′-UCGUUCGUGAGCACUUGCGACG-3′), NC mimics (5′-UCGUCGGAUCGACUGAGAUCU-3′), miR-34a inhibitors (5′-AGCCUUGCUGCAGGUGCGCAU-3′) and NC inhibitors (5′-UGCCUUACUGACGGUCGGAGA-3′) were obtained from Shanghai GenePharma Co., Ltd. pcDNA3.1 vector (Thermo Fisher Scientific, Inc.) was used to construct a XIST and SMAD2 overexpression vector.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Transfection, Migration, TUNEL Assay, shRNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: lncRNA XIST knockdown suppresses cell proliferation and promotes apoptosis in diabetic cataracts through the miR-34a/SMAD2 axis

doi: 10.3892/mmr.2021.12523

Figure Lengend Snippet: XIST knockdown modulates diabetic cataracts progression by targeting miR-34a in HG-induced lens epithelial cells. (A) StarBase website was used to predict the binding site between XIST and miR-34a. (B) Luciferase reporter assay showed luciferase activity of XIST-wt or XIST-mut in SRA01/04 cells transfected with NC mimics or miR-34a mimics. (C) RIP assay was performed to determine the enrichment of XIST and miR-34a in Anti-IgG and Anti-Ago2. (D) Reverse transcription-quantitative PCR showed the relative miR-34a expression in SRA01/04 cells transfected with pcDNA3.1, pcDNA3.1/XIST, shNC or shXIST. (E) MTT, (F) wound healing and (G) Transwell assays were used to analyze the proliferation, migration and invasion of SRA01/04 cells transfected with shNC, shXIST or shXIST + miR-34a inhibitor after treatment of HG. (H) TUNEL assay determined the apoptosis in SRA01/04 cells transfected with shNC, shXIST or shXIST + miR-34a inhibitor after treatment of HG. n=3. *P<0.05. XIST, X-inactive specific transcript; miR, microRNA; wt, wild-type; mut, mutant; sh, short hairpin RNA; NG, normal glucose; HG, high glucose; NC, negative control.

Article Snippet: Small hairpin (sh)RNA against XIST (shXIST, 5′-GUGCGUACAGUGCUGUACAGCAU-3′) and its negative control (NC; shNC, 5′-UACGCUCAGCAUGUGUCACUC-3′), miR-34a mimics (5′-UCGUUCGUGAGCACUUGCGACG-3′), NC mimics (5′-UCGUCGGAUCGACUGAGAUCU-3′), miR-34a inhibitors (5′-AGCCUUGCUGCAGGUGCGCAU-3′) and NC inhibitors (5′-UGCCUUACUGACGGUCGGAGA-3′) were obtained from Shanghai GenePharma Co., Ltd. pcDNA3.1 vector (Thermo Fisher Scientific, Inc.) was used to construct a XIST and SMAD2 overexpression vector.

Techniques: Knockdown, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Migration, TUNEL Assay, Mutagenesis, shRNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: lncRNA XIST knockdown suppresses cell proliferation and promotes apoptosis in diabetic cataracts through the miR-34a/SMAD2 axis

doi: 10.3892/mmr.2021.12523

Figure Lengend Snippet: SMAD2 is a target of miR-34a. (A) StarBase website was used to predict the binding site between miR-34a and SMAD2. (B) Luciferase reporter assay showed luciferase activity of SMAD2-wt or SMAD2-mut in SRA01/04 cells transfected with NC mimics or miR-34a mimics. *P<0.05. (C) RT-qPCR showed the relative SMAD2 expression in DC tissues and normal samples. *P<0.05. (D) Pearson's correlation analysis showed the correlation between XIST and SMAD2 expression in DC tissues. (E) RT-qPCR showed the relative SMAD2 expression in SRA01/04 cells treated with HG. *P<0.05 vs. NG group. (F) RT-qPCR showed the expression of miR-34a in SRA01/04 cells transfected with NC inhibitor and miR-34a inhibitor. *P<0.05 vs. NC inhibitor. (G and H) RT-qPCR and western blot assays showed the relative SMAD2 expression in SRA01/04 cells transfected with NC mimics or miR-34a mimics and NC inhibitor or miR-34a inhibitor. n=3. *P<0.05. SMAD2, SMAD family member 2; miR, microRNA; wt, wild-type; mut, mutant; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; DC, diabetic cataracts; XIST, X-inactive specific transcript; NG, normal glucose; HG, high glucose.

Article Snippet: Small hairpin (sh)RNA against XIST (shXIST, 5′-GUGCGUACAGUGCUGUACAGCAU-3′) and its negative control (NC; shNC, 5′-UACGCUCAGCAUGUGUCACUC-3′), miR-34a mimics (5′-UCGUUCGUGAGCACUUGCGACG-3′), NC mimics (5′-UCGUCGGAUCGACUGAGAUCU-3′), miR-34a inhibitors (5′-AGCCUUGCUGCAGGUGCGCAU-3′) and NC inhibitors (5′-UGCCUUACUGACGGUCGGAGA-3′) were obtained from Shanghai GenePharma Co., Ltd. pcDNA3.1 vector (Thermo Fisher Scientific, Inc.) was used to construct a XIST and SMAD2 overexpression vector.

Techniques: Binding Assay, Luciferase, Reporter Assay, Activity Assay, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Mutagenesis, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: lncRNA XIST knockdown suppresses cell proliferation and promotes apoptosis in diabetic cataracts through the miR-34a/SMAD2 axis

doi: 10.3892/mmr.2021.12523

Figure Lengend Snippet: XIST accelerates diabetic cataracts progression through SMAD2 by sponging miR-34a in HG-treated lens epithelial cells. (A) RT-qPCR showed the expression of XIST in SRA01/04 cells transfected with pcDNA3.1 and pcDNA3.1/XIST. (B) RT-qPCR showed the expression of SMAD2 in SRA01/04 cells transfected pcDNA3.1 and pcDNA3.1/SMAD2. (C and D) RT-qPCR and western blot assays determined SMAD2 expression in SRA01/04 cells transfected with NC mimics, miR-34a mimics, miR-34a mimics + pcDNA3.1 and miR-34a mimics + pcDNA3.1/XIST. (E) MTT, (F) wound healing and (G) Transwell assays were used to analyze the proliferation, migration and invasion of SRA01/04 cells transfected with shNC, shXIST and shXIST + pcDNA3.1/SMAD2 after treatment of HG. (H) TUNEL assay determined the apoptosis in SRA01/04 cells transfected with shNC, shXIST and shXIST + pcDNA3.1/SMAD2 after treatment of HG. n=3. *P<0.05. XIST, X-inactive specific transcript; SMAD2, SMAD family member 2; miR, microRNA; HG, high glucose; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; sh, short hairpin RNA.

Article Snippet: Small hairpin (sh)RNA against XIST (shXIST, 5′-GUGCGUACAGUGCUGUACAGCAU-3′) and its negative control (NC; shNC, 5′-UACGCUCAGCAUGUGUCACUC-3′), miR-34a mimics (5′-UCGUUCGUGAGCACUUGCGACG-3′), NC mimics (5′-UCGUCGGAUCGACUGAGAUCU-3′), miR-34a inhibitors (5′-AGCCUUGCUGCAGGUGCGCAU-3′) and NC inhibitors (5′-UGCCUUACUGACGGUCGGAGA-3′) were obtained from Shanghai GenePharma Co., Ltd. pcDNA3.1 vector (Thermo Fisher Scientific, Inc.) was used to construct a XIST and SMAD2 overexpression vector.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Migration, TUNEL Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, shRNA