Journal: Pharmaceuticals

Article Title: JTE-013 Alleviates Pulmonary Fibrosis by Affecting the RhoA/YAP Pathway and Mitochondrial Fusion/Fission

doi: 10.3390/ph16101444

Figure Lengend Snippet: JTE-013 alleviated fibrosis by inhibiting the expression of RHOA, YAP, and related pathway proteins in lung tissues. ( A ): Protein–protein-interaction network constructed using the String database; ( B ): Co-expression of proteins retrieved from databases; ( C ): Immunohistochemical staining was used to detect the expression of RHOA, YAP, CTGF, and CYR61 and their OD quantitative analysis chart (scale bar = 50 μm). ( D ): Immunofluorescence staining was used to detect the expression of RHOA and their quantitative analysis chart (scale bar = 200 μm). ( E ): Immunofluorescence staining was used to detect the colocalization of YAP and α-SMA. ( F ): Western blot was used to detect the expression of RHOA, TAZ, CTGF, CYR61, and the phosphorylation of Lats1 and YAP. Their relative levels were analyzed and compared. (** p < 0.01 vs. control group, ## p < 0.01 vs. BLM group, && p < 0.01 vs. BLM + JTE-013 group). Original Western blot available in .

Article Snippet: Rabbit anti-RHOA (#bs-1180R), rabbit anti-Lats1 (#bs-2904R), rabbit anti-P-Lats1 (#bs-3246R), rabbit anti-TAZ (#bs-12367R), rabbit anti-connective tissue growth factor (CTGF) (#bs-0743R), Rabbit anti-Human Cysteine Rich Protein, Angiogenic Inducer 61 (CYR61) (#bs-1290R) were purchased from Bioss (Beijing, China).

Techniques: Expressing, Construct, Immunohistochemical staining, Staining, Immunofluorescence, Western Blot, Control

Journal: Pharmaceuticals

Article Title: JTE-013 Alleviates Pulmonary Fibrosis by Affecting the RhoA/YAP Pathway and Mitochondrial Fusion/Fission

doi: 10.3390/ph16101444

Figure Lengend Snippet: JTE-013 inhibited the expression of the RHOA/YAP pathway proteins and fibrosis marker proteins in alveolar epithelial MLE-12 cells. ( A ): MTT assay was used to detect the viability of MLE-12 cells treated with S1P * p < 0.05 vs. 0 μmol/L, ** p < 0.01 vs. 0 μmol/L). ( B – D ): Immunofluorescence staining was used to detect the expression of RHOA ( B ), CTGF ( C ), and YAP ( D ) (scale bar = 100 μm). ( E ): Immunofluorescence staining was used to detect the colocalization of YAP and Drp1 (scale bar = 20 μm). ( F ): Western blot was used to detect the expression of RHOA, TAZ, CTGF, CYR61, and the phosphorylation of Lats1 and YAP. ( G ): The expressions of α-SMA, COL1A1, and MMP-9 were detected by Western blot. (** p < 0.01 vs. control group, ## p < 0.01 vs. S1P group, && p < 0.01 vs. S1P + JTE-013 group). Original Western blot available in .

Article Snippet: Rabbit anti-RHOA (#bs-1180R), rabbit anti-Lats1 (#bs-2904R), rabbit anti-P-Lats1 (#bs-3246R), rabbit anti-TAZ (#bs-12367R), rabbit anti-connective tissue growth factor (CTGF) (#bs-0743R), Rabbit anti-Human Cysteine Rich Protein, Angiogenic Inducer 61 (CYR61) (#bs-1290R) were purchased from Bioss (Beijing, China).

Techniques: Expressing, Marker, MTT Assay, Immunofluorescence, Staining, Western Blot, Control

Journal: Pharmaceuticals

Article Title: JTE-013 Alleviates Pulmonary Fibrosis by Affecting the RhoA/YAP Pathway and Mitochondrial Fusion/Fission

doi: 10.3390/ph16101444

Figure Lengend Snippet: Schematic representation of the role of JTE-013 in pulmonary fibrosis. JTE-013 or S1PR2 knockdown inhibited the binding of S1P and S1PR2 to regulate the RHOA/YAP pathway. This further affected the downstream CTGF/CYR61 expression and mitochondrial dynamics, promoted mitochondrial fusion and the phenotypic transition of MFN, inhibited mitochondrial fission, down-regulated the phosphorylation level of Fis1 and Drp1, and reduced the production of mitochondrial ROS and ROS, thereby alleviating pulmonary fibrosis.

Article Snippet: Rabbit anti-RHOA (#bs-1180R), rabbit anti-Lats1 (#bs-2904R), rabbit anti-P-Lats1 (#bs-3246R), rabbit anti-TAZ (#bs-12367R), rabbit anti-connective tissue growth factor (CTGF) (#bs-0743R), Rabbit anti-Human Cysteine Rich Protein, Angiogenic Inducer 61 (CYR61) (#bs-1290R) were purchased from Bioss (Beijing, China).

Techniques: Knockdown, Binding Assay, Expressing

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: RNA-seq on placentas from mice exposed to NaAsO 2 . (A–G) All pregnant mice except controls were exposed to NaAsO 2 ( 15 mg / L ) by drinking water throughout pregnancy. All pregnant mice were sacrificed on GD 18. (A) Volcano plot of differentially expressed genes in mouse placentas. A total of 743 genes (red triangle) were up-regulated, and 1,889 genes (green dot) were down-regulated by at least 1.5-fold with a significance level ( p < 0.05 ). (B) KEGG pathway analysis. (C) Top 100 genes were screened based on the p -value ranking of all differential genes and then we screened 40 candidate genes among them, with the average FPKM > 10 of all samples as the filter condition. RNA-seq of mouse placentae from three dams were analyzed per group. (D) Validation of trophoblasts development-related differentially expressed genes by real-time RT-PCR ( n = 6 mice/group). (E) Fold differences in differentially expressed genes between RNA-seq and real-time RT-PCR. (F) Placental CYR61 was detected by immunoblotting ( n = 3 mice/group, repeated three times). (G) Representative pictures of CYR61-positive cells in labyrinth zone and junctional zone. Scale bars, 20 μ m ( n = 6 mice/group). (H–J) HTR-8/SVneo cells were exposed to NaAsO 2 ( 2 μ M ). (H) Cyr61 mRNA was measured by real-time RT-PCR ( n = 6 samples/group). (I) CYR61 protein was detected by immunoblotting ( n = 3 samples/group, repeated three times). (J) CYR61-positive cells were stained with by immunofluorescence ( n = 3 samples/group). All data are expressed as mean ± SEM . The numeric data are shown in Table S8. RNA-seq data are reported in an Excel sheet and uploaded to the National Center for Biotechnology Information (GSE222092). Unpaired two-tailed Student’s t -test was used for D–F, H, I. * p < 0.05 , ** p < 0.01 . Note: As, arsenic; As-H, 15 mg / L NaAsO 2 ; Ceacam 3/13, carcinoembryonic antigen-related cell adhesion molecule 3/13; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; FPKM, fragments per kilobase per million; Not DEG, not differential gene expression; RNA-seq, RNA sequencing; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean; Sparcl1, SPARC-like protein 1; TGF- β 2 , transforming growth factor β 2 ; μ M , μ mol / L .

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Staining, Immunofluorescence, Two Tailed Test, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: The role of CYR61 in induced inhibition of migration and invasion in As-exposed human placental trophoblasts. HTR-8/SVneo cells were exposed to NaAsO 2 ( 2 μ M ). (A–D) HTR-8/SVneo cells were transfected with CYR61 overexpression plasmid and then exposed to NaAsO 2 ( 2 μ M ). (A) The efficiency of CYR61 overexpression was determined by real-time RT-PCR ( n = 6 samples/group). (B) Vimentin, N-cadherin, and MMP2 proteins were measured by immunoblotting ( n = 2 samples/group, repeated three times), and β -actin was used as a loading control. (C) Cell migration was determined by transwell migration assay ( n = 3 samples/group, repeated two times). (D) Cell invasion was measured by transwell invasion assay ( n = 3 samples/group, repeated 2 times). (E–H) HTR-8/SVneo cells were transfected with CYR61 knockdown plasmid and then exposed to NaAsO 2 ( 2 μ M ). (E) The efficiency of CYR61 knockdown was determined by real-time RT-PCR ( n = 6 samples/group). (F) Vimentin, N-cadherin, and MMP2 proteins were measured by immunoblotting ( n = 2 samples/group, repeated three times); β -actin was used as a loading control. (G) Cell migration was determined by transwell migration assay ( n = 3 samples/group, repeated two times). (H) Cell invasion was measured by transwell invasion assay ( n = 3 samples/group, repeated 2 times). Scale bars, 200 μ m . All data were expressed as mean ± SEM . The numeric data are shown in Table S8. Unpaired two-tailed Student’s t -test was used for A and E. When ANOVA showed p < 0.05 , the Student-Neuman-Keuls (SNK) multiple comparisons tests were used for B–D and F–H. * p < 0.05 , ** p < 0.01 . Note: ANOVA, analysis of variance; As, arsenic; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; CYR61 OE, CYR61 overexpression; shCYR61, CYR61 shRNA; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean; μ M , μ mol / L .

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: Inhibition, Migration, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Transwell Migration Assay, Transwell Invasion Assay, Two Tailed Test, shRNA, Reverse Transcription Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: Effect of m 6 A modification on Cyr61 mRNA stability and CYR61 protein in As-exposed human placental trophoblasts. HTR-8/SVneo cells were exposed to NaAsO 2 ( 2 μ M ). (A) HTR8/SVneo cells were treated with actinomycin D ( 5 μ g / mL ), followed by detection of Cyr61 mRNA at indicated times ( n = 3 samples/group, repeated two times). (B) The m 6 A content was measured by EpiQuik m 6 A methylation quantification kit ( n = 5 samples/group, repeated two times). (C) Schematic representation of the position of m 6 A motifs with CYR61 transcript. (D) The m 6 A -modified Cyr61 levels were measured by MeRIP-qPCR ( n = 3 samples/group). (E) Schematic representation of the interaction between Cyr61 mRNA and IGF2BP1/2/3 proteins. (F) The interaction between Cyr61 mRNA and IGF2BP2 proteins was detected by RIP-qPCR ( n = 3 samples/group). (G) The expression of m 6 A methyltransferases and demethylases was measured by real-time RT-PCR ( n = 6 samples/group). (H) The m 6 A methylase activity was measured by Epigenase m 6 A methylase activity/inhibition assay kit ( n = 6 samples/group). (I) A schematic model of the reversible m 6 A methylation. SAM is not only a methyl donor for m 6 A methylation but also a cofactor for catalytic domain of Mettl3. (J) Intracellular SAM content was measured by LC-MS/MS ( n = 6 samples/group). All data were expressed as mean ± SEM . The numeric data are shown in Table S8. Unpaired two-tailed Student’s t -test was used for A, B, D, F, G, H, and J. * p < 0.05 , ** p < 0.01 . Note: ActD, actinomycin D; Alkbh5, a-ketoglutarate-dependent dioxygenase alkB homolog 5; ANOVA, analysis of variance; As, arsenic; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; FTO, fat-mass obesity-associated protein; IGF2BP1/2/3, insulin-like growth factor 2 mRNA-binding protein one-half/3; LC-MS/MS, liquid chromatography–tandem mass spectrometry; m 6 A , N 6 -methyladenosine ; MeRIP-qPCR, methylated RNA immunoprecipitation–qPCR; Mettl3/14, methyltransferase-like 3/14; Rbm15/15b, RNA-binding motif protein 15/15b; RIP-qPCR, RNA-binding protein immunoprecipitation–qPCR; RT-PCR, reverse transcription polymerase chain reaction; SAH, s-adenosyl-l-homocysteine; SAM, s-adenosylmethionine; SEM, standard error of the mean; μ M , μ mol / L ; WTAP, wilms tumor 1-associating protein.

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: Modification, Methylation, Expressing, Quantitative RT-PCR, Activity Assay, Inhibition, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Binding Assay, Liquid Chromatography, Mass Spectrometry, Immunoprecipitation, RNA Binding Assay, Reverse Transcription Polymerase Chain Reaction, Wilms Tumor Assay

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: Effect of As3MT knockdown on Cyr61 m 6 A modification in As-exposed human placental trophoblasts. HTR8/SVneo cells were exposed to 2 μ M NaAsO 2 in presence or absence of As3MT-shRNA (A) The efficiency of As3MT knockdown was determined by real-time RT-PCR ( n = 6 samples/group). (B) Intracellular SAM content was measured by LC-MS/MS ( n = 6 samples/group). (C) The m 6 A methylase activity was measured by Epigenase m 6 A methylase activity/inhibition assay kit ( n = 6 samples/group). (D and E) HTR8/SVneo cells were treated with actinomycin D ( 5 μ g / mL ), followed by detection of Cyr61 mRNA at indicated times ( n = 3 samples/group, repeated two times). (F) CYR61, Vimentin, N-cadherin, and MMP2 proteins were measured by immunoblotting ( n = 3 samples/group). (G) Cell migration was determined by transwell migration assay ( n = 3 samples/group, repeated two times). (H) Cell invasion was measured by transwell invasion assay ( n = 3 samples/group, repeated two times). Scale bars, 200 μ m . All data were expressed as mean ± SEM . The numeric data are shown in Table S8. When ANOVA showed p < 0.05 , the Student-Neuman-Keuls (SNK) multiple comparisons tests were used for A–H. Asterisks indicate comparison with the control group. Hashtags indicate comparison with the As group. * p and # p < 0.05 , ** p and ## p < 0.01 . Note: ActD, actinomycin D; ANOVA, analysis of variance; As, arsenic; As3MT, arsenite methyltransferase; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MMP2, matrix metalloproteinases 2; RT-PCR, reverse transcription polymerase chain reaction; SAM, s-adenosylmethionine; shAs3MT 1#, As3MT shRNA 1#; shAs3MT 2#, As3MT shRNA 2#; SEM, standard error of the mean; μ M , μ mol / L .

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: Modification, shRNA, Quantitative RT-PCR, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Inhibition, Western Blot, Migration, Transwell Migration Assay, Transwell Invasion Assay, Comparison, Liquid Chromatography, Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: Effect of SAM supplementation on induced inhibition of Cyr61 m 6 A modification in As-exposed human placental trophoblasts. HTR8/SVneo cells were incubated with NaAsO 2 ( 2 μ M ) in presence or absence of SAM ( 10 μ M ). (A) Intracellular SAM content was measured by LC-MS/MS ( n = 6 samples/group). (B) The m 6 A methylase activity was measured by Epigenase m 6 A methylase activity/inhibition assay kit ( n = 6 samples/group). (C) HTR8/SVneo cells were treated with actinomycin D ( 5 μ g / mL ), followed by detection of Cyr61 mRNA at indicated times ( n = 3 samples/group, repeated two times). (D and E) CYR61, Vimentin, N-cadherin, and MMP2 proteins were determined by immunoblotting ( n = 3 samples/group, repeated three times). (F) Cell migration was determined by transwell migration assay ( n = 3 samples/group, repeated two times). (G) Cell invasion was measured by transwell invasion assay ( n = 6 samples/group, repeated two times). Scale bars, 200 μ m . All data were expressed as mean ± SEM . The numeric data are shown in Table S8. When ANOVA showed p < 0.05 , the Student-Neuman-Keuls (SNK) multiple comparisons tests were used for A–G. Asterisks indicate comparison with the control group. Hashtags indicate comparison with the As group. * p and # p < 0.05 , ** p and ## p < 0.01 . Note: ANOVA, analysis of variance; As, arsenic; ActD, actinomycin D; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; MMP2, matrix metalloproteinases 2; RT-PCR, reverse transcription polymerase chain reaction; SAM, s-adenosylmethionine; SEM, standard error of the mean; μ M , μ mol / L .

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: Inhibition, Modification, Incubation, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Western Blot, Migration, Transwell Migration Assay, Transwell Invasion Assay, Comparison, Reverse Transcription Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: Effect of FA supplementation on placental development and fetal growth restriction in As-exposed mice. Pregnant mice were divided into four groups: CTRL, FA, As, and As + FA groups. In the As and As + FA groups, pregnant mice drank ultrapure water containing NaAsO 2 ( 15 mg / L ) throughout pregnancy. In FA and As + FA groups, pregnant mice were administered FA ( 150 μ g / kg ) by gavage daily throughout pregnancy. All pregnant mice were sacrificed on GD18. (A) Fetal weight ( n = 8 dams/group, 12–15 fetuses per dam). (B) Crown–rump length ( n = 8 dams/group, 12–15 fetuses per dam). (C) Placental weight ( n = 8 dams/group, 12–15 fetuses per dam). (D) Placental diameter ( n = 8 dams/group, 12–15 fetuses per dam). (E) Placental cross-section was stained with H&E. Scale bars, 500 μ m (left); 200 μ m (right) ( n = 6 mice/group). (F) The percentage of labyrinth zone area in the entire placenta area ( n = 6 mice/group). (G) The ratio of cross-sectional thickness of labyrinth zone to junctional zone ( n = 6 mice/group). (H) Placental SAM content was determined by LC-MS/MS ( n = 6 mice/group). (I) The m 6 A methylase activity was measured by Epigenase m 6 A methylase activity/inhibition assay kit ( n = 6 mice/group). (J) Placental CYR61 and MMP2 were measured by immunoblotting ( n = 3 mice/group, repeated two times). All data were expressed as mean ± SEM . The numeric data are shown in Table S8. When ANOVA showed p < 0.05 , the Student-Neuman-Keuls (SNK) multiple comparisons tests were used for A–D and F–J. * p < 0.05 , ** p < 0.01 . Note: ANOVA, analysis of variance; As, arsenic; CTRL, control; CYR61, cysteine-rich angiogenic inducer 61; FA, folic acid; H&E, hematoxylin and eosin; JZ, junctional zone; LC-MS/MS, liquid chromatography–tandem mass spectrometry; LZ, labyrinth zone; MMP2, matrix metalloproteinases 2; SAM, s-adenosylmethionine; SEM, standard error of the mean.

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: Staining, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Inhibition, Western Blot, Liquid Chromatography, Mass Spectrometry

Journal: Environmental Health Perspectives

Article Title: Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m 6 A

doi: 10.1289/EHP12207

Figure Lengend Snippet: Based on a case–control study: association among maternal urinary As concentration, plasma CYR61 content, and fetal growth restriction. (A) Maternal urinary As concentration in AGA and SGA infants was determined by hydride generation-atomic fluorescence spectrometry ( n = 45 human sample/group). (B) Maternal plasma CYR61 content in AGA and SGA infants was measured by ELISA ( n = 45 human sample/group). (C) The correlation between maternal urinary As concentration and plasma CYR61 content in AGA infants ( n = 45 human sample). (D) The correlation between maternal urinary As concentration and plasma CYR61 content in SGA infants ( n = 45 human sample). (E) Placental CYR61-positive cells in SGA infants and AGA infants were stained by immunohistochemistry ( n = 18 human sample/group). Representative histology images were showed. Blue arrows indicate CYR61-positive cells. Scale bars, 20 μ m . (F) Placental vimentin, MMP2, and MMP9 were detected by immunoblotting ( n = 18 human sample/group). The data in A and B are expressed as mean ± SD . The data in A–D are represented by dots indicating individual values. The data in E and F are expressed as mean ± SEM . The numeric data are shown in Table S8. Two-tailed Student’s t -test was used for A, B, E, and F. The Spearman correlation was used for C and D. * p < 0.05 , ** p < 0.01 . Note: AGA, appropriate for gestational age; As, arsenic; CYR61, cysteine-rich angiogenic inducer 61; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; SEM, standard error of the mean; SGA, small for gestational age.

Article Snippet: The cysteine-rich angiogenic inducer 61 (CYR61) ELISA kit (#CSB-E13884h) was purchased from Cusabio.

Techniques: Concentration Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Western Blot, Two Tailed Test, Standard Deviation

Journal: Cancer Biology & Medicine

Article Title: MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis

doi: 10.20892/j.issn.2095-3941.2020.0296

Figure Lengend Snippet: MIIP negatively regulates CYR61 expression. (A) RNA-seq analyses were performed in triplicate on 786-O-MIIP and 786-O-Vector cells, and the datasets were analyzed by GSEA. Tumor angiogenesis pathway GSEA (hallmark gene sets) of the RNA-seq dataset is shown. (B) Heatmap of expression changes in a portion of angiogenesis genes determined from GSEA analysis. (C) mRNA levels of CYR61 in MIIP-overexpression and MIIP-knockdown cells were detected with qRT-PCR. (D) Protein levels of MIIP and CYR61 in lysates (Lys) or culture medium (Medium) were detected by Western blot. (E) Protein levels of CYR61 secreted in the culture medium were determined by ELISA. In (C) and (E), the data are represented as mean ± SD, n = 3. ** P < 0.01.

Article Snippet: The concentration of the CYR61 protein released in the supernatant was tested with a commercial CYR61 ELISA kit (Boster, China).

Techniques: Expressing, RNA Sequencing Assay, Plasmid Preparation, Over Expression, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cancer Biology & Medicine

Article Title: MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis

doi: 10.20892/j.issn.2095-3941.2020.0296

Figure Lengend Snippet: Restoration of CYR61 reverses the inhibitory role of MIIP in ccRCC cells. Cells stably overexpressing MIIP OS-RC-2 or 786-O, or their control cells were transfected with pCDNA3.1-HA-CYR61 or pCDNA3.1, respectively. (A) Protein levels of MIIP and CYR61 were detected by Western blot. (B) Viability was measured with CCK-8. (C) Cell cycle profiles were analyzed by flow cytometry. (D) Colony formation ability was detected with colony formation assays. (E) Proangiogenic activity was measured with HUVEC tube formation assays, and the HUVECs were cocultured with conditioned medium for 5 h (scale bar: 100 μm). In (B)–(E), the data are represented as mean ± SD, n = 3. * P < 0.05; ** P < 0.01.

Article Snippet: The concentration of the CYR61 protein released in the supernatant was tested with a commercial CYR61 ELISA kit (Boster, China).

Techniques: Stable Transfection, Transfection, Western Blot, CCK-8 Assay, Flow Cytometry, Activity Assay

Journal: Cancer Biology & Medicine

Article Title: MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis

doi: 10.20892/j.issn.2095-3941.2020.0296

Figure Lengend Snippet: MIIP down-regulates the expression of CYR61 by enhancing the degradation of HIF-2α (A) The protein levels of HIF-2α in MIIP-overexpression and MIIP-knockdown cells were detected by Western blot. (B) Cells stably overexpressing MIIP and control cells were transfected with pCDNA3.1-Flag-HIF2A (HIF-2α) or pCDNA3.1 (control), respectively; cells with stable MIIP knockdown and control cells were transfected with siRNA targeting HIF-2α (siHIF-2α 1# or siHIF-2α 2#) or control (siNC), respectively. HIF-2α and CYR61 protein levels were detected by Western blot. (C) mRNA levels of HIF2A in MIIP-overexpressing and MIIP-knockdown cells were detected by qRT-PCR. Data represent mean ± SD, n = 3. (D) MIIP-overexpressing OS-RC-2 and 786-O cells, and control cells were treated with MG132 (10 μM) or mock (DMSO). MIIP and HIF-2α levels were detected with Western blot. (E) MIIP-overexpressing 786-O cells and control cells were treated with 100 μg/mL CHX at the indicated time points. MIIP and HIF-2α levels were detected with Western blot. (F) The 786-O cells were co-transfected with plasmids expressing HA-Ub and MIIP and treated with MG-132. HIF-2α ubiquitination was assessed with in vivo ubiquitination assays, via immunoprecipitation with anti-HIF-2α antibody followed by immunoblotting with anti-HA antibody.

Article Snippet: The concentration of the CYR61 protein released in the supernatant was tested with a commercial CYR61 ELISA kit (Boster, China).

Techniques: Expressing, Over Expression, Western Blot, Stable Transfection, Transfection, Quantitative RT-PCR, In Vivo, Immunoprecipitation

Journal: Cancer Biology & Medicine

Article Title: MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis

doi: 10.20892/j.issn.2095-3941.2020.0296

Figure Lengend Snippet: MIIP suppresses tumor growth and angiogenesis of ccRCC in vivo. OS-RC-2-MIIP, 786-O-shMIIP 1#, and their corresponding control cells were subcutaneously bilaterally injected into the groin in nude mice (five mice per group). (A) Tumor growth was monitored by caculation of tumor volumes with the formula width 2 × length × 0.5 along time. (B) The tumor xenografts were photographed, and their weights were measured at the end of the experiment. (C) MIIP, CD31, Ki67, HIF-2α, and CYR61 levels in xenograft tissues were examined by immunohistochemistry or immunofluorescence staining (scale bar: 50 μm). (D) MIIP, HIF-2α, and CYR61 levels in xenograft tissues were detected by Western blot. In (A, B), the data are represented as mean ± SD, n = 5. * P < 0.05; ** P < 0.01.

Article Snippet: The concentration of the CYR61 protein released in the supernatant was tested with a commercial CYR61 ELISA kit (Boster, China).

Techniques: In Vivo, Injection, Immunohistochemistry, Immunofluorescence, Staining, Western Blot

Journal: Cancer Biology & Medicine

Article Title: MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis

doi: 10.20892/j.issn.2095-3941.2020.0296

Figure Lengend Snippet: MIIP is weakly expressed in RCC and associated with progression, prognosis, and the expression of CYR61 and HIF-2α. (A) MIIP, CYR61, and HIF-2α expression in 13 pairs of RCC tissues vs. adjacent non-tumor tissues was detected by Western blot. C: RCC tissue; N: adjacent non-tumor tissue. (B) MIIP, CYR61, and HIF-2α expression in an RCC tissue microarray was detected with immunohistochemistry (scale bar: 50 μm). (C) MIIP, CYR61, and HIF-2α expression levels were quantified with a scoring system. The staining score was calculated by multiplying the stained area (%) score and the intensity score. Expression group: low, score < 9; high, score ≥ 9. Stage refers to WHO histological grade. * P < 0.05; ** P < 0.01, χ 2 test. (D) The Kaplan-Meier curve depicts the relationship between MIIP expression level and OS of patients with RCC, and the P -value was calculated with a stratified log rank test ( P = 0.026). (E) Schematic representation of the MIIP/HIF-2α/CYR61 axis in ccRCC. In normal cells, MIIP promotes HSP90 acetylation through inhibiting HDAC6 activity, thus impairing HSP90’s chaperone function and its binding to HIF-2α, which in turn causes RACK1 binding and subsequent HIF-2α degradation. Meanwhile, under normoxia, VHL promotes HIF-2α ubiquitination and degradation in an oxygen-dependent manner. In RCC, VHL deficiency and MIIP downregulation together cause HIF-2α accumulation, thereby leading to overexpression of CYR61, which in turn contributes to RCC progression.

Article Snippet: The concentration of the CYR61 protein released in the supernatant was tested with a commercial CYR61 ELISA kit (Boster, China).

Techniques: Expressing, Western Blot, Microarray, Immunohistochemistry, Staining, Activity Assay, Binding Assay, Over Expression

Journal: Cancer Biology & Medicine

Article Title: MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis

doi: 10.20892/j.issn.2095-3941.2020.0296

Figure Lengend Snippet: Correlation of MIIP expression to clinicopathological features and the expression of CYR61 and HIF-2α in RCC

Article Snippet: The concentration of the CYR61 protein released in the supernatant was tested with a commercial CYR61 ELISA kit (Boster, China).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Lamin A and the LINC complex act as potential tumor suppressors in Ewing Sarcoma

doi: 10.1038/s41419-022-04729-5

Figure Lengend Snippet: a Lamin A/C (green) and β3-tubulin (red) localization in non-treated TC-71 cells (CTRL) and in mevinolin treated (5 μM) EWS cells (MEV). DNA was counterstained with DAPI (DAPI). Merge of fluorescence signals are shown (MERGE); magnification 100x, scale bar 10 μm; b Lamin A/C (green) and neurofilament-H (NEF-H) (red) localization in non-treated TC-71 cells (CTRL) and in mevinolin treated (5 μM) EWS cells (MEV). DNA was counterstained with DAPI (DAPI). Merge of fluorescence signals are shown (MERGE); magnification 100x, scale bar 10 μm; c qRT-PCR analysis of LMNA , NEF-H , β3-tubulin, nestin and SOX2 genes in untreated TC-71 cells (CTRL) and in mevinolin treated EWS cells (2.5 μM or 5μM MEV). Data are shown as 2- ΔΔ Ct . GAPDH was used as a housekeeping gene. Data are shown as mean values ± SD of three different experiments. Asterisks indicate statistically significant differences with respect to CTRL cells; one-way ANOVA test, * p < 0.05, ** p < 0.01, *** p < 0.001; d Lamin A/C (green) and YAP (red) localization in non-treated TC-71 cells (CTRL) and in mevinolin treated (5 μM) EWS cells (MEV). DNA was counterstained with DAPI (DAPI). Merge of fluorescence signals are shown (MERGE). Graphs indicate the fluorescence intensity profile along the white arrows. Representative graphs of at least 30 nuclei analyzed for each sample were shown; magnification 100x, scale bar 10 μm; e qRT-PCR analysis of CTGF and CYR61 in non-treated TC-71 cells (CTRL) and in mevinolin treated EWS cells (2.5 μM or 5μM MEV). Data are shown as 2- ΔΔ Ct . GAPDH was used as a housekeeping gene. Data are shown as mean values ± SD of three different experiments. Asterisks indicate statistically significant differences with respect to CTRL cells; one-way ANOVA test, * p < 0.05, ** p < 0.01; f Western blotting analysis of YAP, p(Ser127) YAP, MYC and ROCK2 protein expression in non-treated TC-71 cells (CTRL) and in mevinolin treated EWS cells (2.5 μM or 5μM MEV). GAPDH was used as loading control. Densitometric analysis is shown as mean values ± SD of three different experiments. Asterisks indicate statistically significant differences with respect to CTRL cells; one-way ANOVA test, * p < 0.05, ** p < 0.01.

Article Snippet: Gene expression was assessed using the TaqMan® Gene Expression Master Mix, and predesigned TaqMan probes (Thermo Fisher) LMNA (Hs.PT.58.24496716), nestin gene (Hs.PT.58.1185097), SOX2 (Hs.PT.58.237897.g), Connective Tissue Growth Factor ( CTGF ) (Hs00170014), Cysteine Rich Angiogenic Inducer 61 (CYR61 ) (Hs00155479), Neurofilament H ( NEF-H ) (Hs00606024) and β3-tubulin gene (Hs.PT.58.20385221) were employed, using the 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA).

Techniques: Fluorescence, Quantitative RT-PCR, Western Blot, Expressing