Journal:

Article Title: Compartmentalizing VEGF-Induced ERK2/1 Signaling in Placental Artery Endothelial Cell Caveolae: A Paradoxical Role of Caveolin-1 in Placental Angiogenesis in Vitro

doi: 10.1210/me.2008-0475

Figure Lengend Snippet: Down-regulation of caveolin-1 suppresses VEGF-induced angiogenic responses via ERK2/1 pathway. A, Establishment of stable oFPAE cells with down-regulated caveolin-1 expression. oFPAE cells transfected with caveolin-1 shRNAs (see Materials and Methods), selected with 600 μg/ml G418 in complete MCDB 131 medium. G418-resistant colonies were picked up and passaged. Down-regulation of caveolin-1 protein was verified by Western blot analysis. Note the clone from no. 1 shRNA transfection has greatest reduction of caveolin-1 protein (∼90%). Electron microscopic analysis showed that caveolin-1 down-regulation resulted in the loss of caveolae (arrows). B–D, Down-regulation of caveolin-1 inhibits VEGF-stimulated ERK2/1 activation (B), proliferation (C), and tube formation (D). Stable oFPAE cell lines harboring control and caveolin-1 shRNA were treated with VEGF for immunoblotting of total and phosphorylated ERK/2/1 (B), for proliferation (C), and for tube formation (D) assays, as described in Fig. 5​5.. Bars (mean ± sd, n = 4) with different letters differ significantly (P < 0.05). Cav1, Caveolin-1; Ctl, control.

Article Snippet: Establishment of stable caveolin-1 knockdown oFPAE cells Primary oFPAE cells (passage 3) were transfected with Sure Silencing shRNA plasmid (SuperArray Bioscience Corp., Washington, DC) carrying ovine caveolin-1 RNA interference sequences: no. 1: 5′-GGCAGTTGTACCATGCA TTAA-3′, no. 2: 5′-CACCACCTTCACTGTGAC GAA; no. 3: 5′-CGCTGACG TGGTCAAGATT GA-3′; or a negative control sequence: 5′-GGAATCTCATTCGATGCATAC-3′; using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Western Blot, shRNA, Activation Assay, Control

Journal: Oncotarget

Article Title: ATR suppresses the pro-tumorigenic functions of breast stromal fibroblasts

doi: 10.18632/oncotarget.26159

Figure Lengend Snippet: TCF-64 cells were transfected with specific ATR-shRNA (N64-sh) or a scrambled sequence (N64C). (A and B) Whole cell lysates were prepared from the indicated cells and were used for immunoblotting analysis using antibodies against the indicated proteins. (C) Total RNA was extracted from the indicated cells, and the mRNA levels of the indicated genes were assessed by qRT–PCR using specific primers. Error bars represent mean ± S.D. * P < 4x10 −6 . (D) Total RNA was extracted from the indicated cells, and the mRNA levels of the indicated genes were assessed by qRT–PCR using specific primers. Error bars represent mean ± S.D. * P < 4x10 −6 . (E) N64-sh and N64C cells were seeded separately over 3D networks Biotech in 24 well plate, and then were allowed to grow under observation. After 10 days, total RNA was extracted, and the mRNA levels of the indicated genes were assessed by qRT-PCR. Error bars represent mean ± S.D. * P < 0.031. (F and G) SFCM were collected from the indicated cells and the levels of the indicated proteins were determined by ELISA. Error bars represent mean ± S.D (n=3). * P < 0.017811 and * P < 0.003, respectively. (H) Cells (10 5 ) were seeded in SFM onto the upper compartment of the migration and invasion plates and incubated for 24 h in the presence of CpM in the lower chambers of 24-well BD BioCoat plates. The numbers of migrated/invaded cells were calculated and represented in histograms. Error bars represent mean ± SD (n=3). * P < 3.1x10 −5 .

Article Snippet: ATR -shRNA (KHD1318) expressed in sure silencing shRNA plasmid and the corresponding control plasmid were obtained from GenScript Corporation, and were used to carry out transfection using human dermal fibroblast nucleofector 2000 transfection kit (Invitrogen) following the protocol recommended by the manufacturer.

Techniques: Transfection, shRNA, Sequencing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Migration, Incubation

Journal: Journal of Translational Medicine

Article Title: Circular RNA circCSPP1 promotes the occurrence and development of colon cancer by sponging miR-431 and regulating ROCK1 and ZEB1

doi: 10.1186/s12967-022-03240-x

Figure Lengend Snippet: The information of oligonucleotide sequences

Article Snippet: The miR-431 mimics, miR-control, circCSPP1 pcDNA3.1 overexpression plasmid, circCSPP1 pLVX-IRES-Puro silencing plasmids; Rho associated coiled-coil containing protein kinase 1 (ROCK1) and ZEB1 pLVX-IRES-Puro silencing plasmids were obtained from Shanghai Genepharma Co., Ltd. Phorbol-12-myristate-13-acetate (PMA), IL-4 and IL-13 were purchased from Sigma-Aldrich; Merck KGaA.

Techniques: Sequencing, shRNA

Journal: Journal of Translational Medicine

Article Title: Circular RNA circCSPP1 promotes the occurrence and development of colon cancer by sponging miR-431 and regulating ROCK1 and ZEB1

doi: 10.1186/s12967-022-03240-x

Figure Lengend Snippet: CircCSPP1 is highly expressed in colon cancer tissues. A GEO database data analysis of colon cancer differentially expressed circRNA. B Wayne analysis of commonly differentially expressed circRNA in GSE121895 and GSE126094. C The expression of circRNAs in tumor tissues and in adjacent normal tissue was detected with RT-qPCR. D The expression of circCSPP1 in tumor tissues and in adjacent normal tissue was detected with RT-qPCR. E The expression of circCSPP1 in cell lines was tested with RT-qPCR. F The circRNA circularization mechanism is formed by splicing of exons 8 and 9, and sequencing confirmed the sequence is correct. G The localization of circCSPP1 in colon cancer cells was detected with FISH. H The expression of circCSPP1 in the nucleus and cytoplasm was detected by RT-qPCR. I The half-live of linear and circRNA were detected by RT-qPCR after actinomycin treatment. J The expression of linear and circRNA linear was detected with RT-qPCR after RNase R treatment. *p < 0.05 vs normal, **p < 0.01 vs normal, HFC, mock groups; n = 3

Article Snippet: The miR-431 mimics, miR-control, circCSPP1 pcDNA3.1 overexpression plasmid, circCSPP1 pLVX-IRES-Puro silencing plasmids; Rho associated coiled-coil containing protein kinase 1 (ROCK1) and ZEB1 pLVX-IRES-Puro silencing plasmids were obtained from Shanghai Genepharma Co., Ltd. Phorbol-12-myristate-13-acetate (PMA), IL-4 and IL-13 were purchased from Sigma-Aldrich; Merck KGaA.

Techniques: Expressing, Quantitative RT-PCR, Sequencing

Journal: Journal of Translational Medicine

Article Title: Circular RNA circCSPP1 promotes the occurrence and development of colon cancer by sponging miR-431 and regulating ROCK1 and ZEB1

doi: 10.1186/s12967-022-03240-x

Figure Lengend Snippet: Knockdown of circCSPP1 significantly inhibited the tumorgenesis of colon cancer. Colon cancer cells were treated with circCSPP1 shRNAs or negative control (sh-nc) for 24 h. A The expression of circCSPP1 was detected with RT-qPCR. B CCK8 was used to detect the viability of colon cancer cells. C Cell clone formation was used to assess the proliferation of clone cancer cells. D , E The cell migration and invasion ability was detected with transwell assay. **p < 0.01 vs sh-nc; n = 3

Article Snippet: The miR-431 mimics, miR-control, circCSPP1 pcDNA3.1 overexpression plasmid, circCSPP1 pLVX-IRES-Puro silencing plasmids; Rho associated coiled-coil containing protein kinase 1 (ROCK1) and ZEB1 pLVX-IRES-Puro silencing plasmids were obtained from Shanghai Genepharma Co., Ltd. Phorbol-12-myristate-13-acetate (PMA), IL-4 and IL-13 were purchased from Sigma-Aldrich; Merck KGaA.

Techniques: Negative Control, Expressing, Quantitative RT-PCR, Migration, Transwell Assay

Journal: Journal of Translational Medicine

Article Title: Circular RNA circCSPP1 promotes the occurrence and development of colon cancer by sponging miR-431 and regulating ROCK1 and ZEB1

doi: 10.1186/s12967-022-03240-x

Figure Lengend Snippet: CircCSPP1 promotes colon cancer tumor growth and metastasis in vivo. A Tumor volume in each group was imaged in the end of animal study. B The tumors in mice was isolated and weighted. C , D The tumor volumes of SW620 or LOVO tumor-bearing mice were monitored every two days. E , F Liver metastasis of colon cancer in the nude mice in each group was monitored and quantified. G The expression of circCSPP1 in tumor tissues was detected with RT-qPCR. **p < 0.01; n = 6. PcDNA3.1 control (scramble)

Article Snippet: The miR-431 mimics, miR-control, circCSPP1 pcDNA3.1 overexpression plasmid, circCSPP1 pLVX-IRES-Puro silencing plasmids; Rho associated coiled-coil containing protein kinase 1 (ROCK1) and ZEB1 pLVX-IRES-Puro silencing plasmids were obtained from Shanghai Genepharma Co., Ltd. Phorbol-12-myristate-13-acetate (PMA), IL-4 and IL-13 were purchased from Sigma-Aldrich; Merck KGaA.

Techniques: In Vivo, Isolation, Expressing, Quantitative RT-PCR

Journal: Journal of Translational Medicine

Article Title: Circular RNA circCSPP1 promotes the occurrence and development of colon cancer by sponging miR-431 and regulating ROCK1 and ZEB1

doi: 10.1186/s12967-022-03240-x

Figure Lengend Snippet: MiR-431 targets ZEB1 in colon cancer cells. A The binding site between miR-431 and ROCK1 was predicted. B Luciferase reporter experiment was performed to detect the interaction between miR-431 and ZEB1. C and D colon cancer cells were treated with miR-431 mimics or miR-431 inhibitor for 24 h, the gene and protein level of ZEB1 was detected with RT-qPCR and WB, respectively. E RIP assay was performed to verify the binding between ZEB1 and miR-431. Colon cancer cells were treated with pcDNA3.1-circCSPP1, pcDNA3.1-circCSPP1 plus miR-431 mimics or pcDNA3.1-circCSPP1 plus ZEB1 shRNA. F . RT-qPCR was used to detect the expression of ZEB1. G and H Transwell assay was performed to assess the migration and invasion of colon cancer cells. **p < 0.01; n = 3

Article Snippet: The miR-431 mimics, miR-control, circCSPP1 pcDNA3.1 overexpression plasmid, circCSPP1 pLVX-IRES-Puro silencing plasmids; Rho associated coiled-coil containing protein kinase 1 (ROCK1) and ZEB1 pLVX-IRES-Puro silencing plasmids were obtained from Shanghai Genepharma Co., Ltd. Phorbol-12-myristate-13-acetate (PMA), IL-4 and IL-13 were purchased from Sigma-Aldrich; Merck KGaA.

Techniques: Binding Assay, Luciferase, Quantitative RT-PCR, shRNA, Expressing, Transwell Assay, Migration

Journal: Journal of Translational Medicine

Article Title: Circular RNA circCSPP1 promotes the occurrence and development of colon cancer by sponging miR-431 and regulating ROCK1 and ZEB1

doi: 10.1186/s12967-022-03240-x

Figure Lengend Snippet: CircCSPP1 upregulates cyclin D1/CDK4/RB signaling pathway in colon cancer. Colon cancer cells were treated with pcDNA3.1-circCSPP1, pcDNA3.1-circCSPP1 plus miR-431 mimics or pcDNA3.1-circCSPP1 plus ROCK1 shRNA. A , B Western blot was used to detect the expressions of cell cycle-related proteins (cyclin D1, CDK4, Rb) and EMT related protein (Snail, E-cadherin) in each group. C The potential mechanism by which circCSPP1 regulated the progression of colon cancer was presented. N = 3

Article Snippet: The miR-431 mimics, miR-control, circCSPP1 pcDNA3.1 overexpression plasmid, circCSPP1 pLVX-IRES-Puro silencing plasmids; Rho associated coiled-coil containing protein kinase 1 (ROCK1) and ZEB1 pLVX-IRES-Puro silencing plasmids were obtained from Shanghai Genepharma Co., Ltd. Phorbol-12-myristate-13-acetate (PMA), IL-4 and IL-13 were purchased from Sigma-Aldrich; Merck KGaA.

Techniques: shRNA, Western Blot

Journal: iScience

Article Title: JUN mediates glucocorticoid resistance by stabilizing HIF1a in T cell acute lymphoblastic leukemia

doi: 10.1016/j.isci.2023.108242

Figure Lengend Snippet: JUN plays a critical role in dexamethasone resistance in T-ALL cells (A) Heatmap depicting 727 upregulated genes in a T-ALL cohort (GEO, GSE5820) (left). Upregulated genes were selected with p < 0.05 and log2 fold change >0.5 (left). Fragments per kilobase million (FPKM) of AP-1 TF family members ( Jun , Fos , Fosl1 , Fosl2 , FosB ) in a T-ALL cohort with 16 GC-resistant T-ALL patient samples and 13 GC-sensitive T-ALL patient samples (GEO, GSE5820) (right). (B) Event-free survival of T-ALL patients with higher or lower expression of c-Jun , data from TARGET, phs000464. (C) The relative cell viability of T-ALL cell lines. Cell counting was performed at 48 h after treatment with different concentration gradients of dexamethasone, and the IC50 was calculated as the concentration of dexamethasone that caused 50% cell death. (D) Relative mRNA expression of c-Jun in T-ALL cell lines through qPCR (left) and the correlation between the IC50 values and the relative mRNA expression of c-Jun in T-ALL cell lines (right). (E) The knockdown efficiency of JUN in Jurkat cells. Cells were harvested 72 h after transfection using lentivirus vectors, and a nontarget lentivirus vector (control) was used as the negative control (left). Cells were treated with different concentrations of dexamethasone, and cell counting was performed 48 h after treatment. (F) Overexpression efficiency of JUN in CCRF-CEM cells. Cells were harvested 72 h after transfection using lentivirus vectors. A nontarget lentivirus vector (control) was used as the negative control (left). Cells were treated with different concentrations of dexamethasone, and cell counting was performed 48 h after treatment (right). Data are represented as mean ± SD (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ns = no significant).

Article Snippet: Then, Jurkat and CCRF-CEM(Dex) cells were infected with lentivirus packaged by the silencing plasmid (pLKO.1-puro), which includes a puromycin resistance region, and an shRNA construct to target HIF1α and JUN or a non-specific shRNA control (shRNA C: 5′-CAACACAGATGATAGAGCACCAATTGGTGCTCTATCATCTGTGTTGTTTTT-3′).

Techniques: Expressing, Cell Counting, Concentration Assay, Knockdown, Transfection, Plasmid Preparation, Control, Negative Control, Over Expression

Journal: iScience

Article Title: JUN mediates glucocorticoid resistance by stabilizing HIF1a in T cell acute lymphoblastic leukemia

doi: 10.1016/j.isci.2023.108242

Figure Lengend Snippet: HIF1α is essential for dexamethasone resistance in T-ALL cells (A) The relative cell viability of T-ALL cell lines. Cell counting was performed at 48 h after treatment with different concentration gradients of the HIF1a inhibitors KC7F2 (left) and BAY-872243 (right). (B) Gene set enrichment analysis (GSEA) of upregulated genes in the CCRF-CEM(Dex) group. (C) Relative mRNA expression of HIF1a in T-ALL cell lines through qPCR (left). The correlation between the IC50 values and the relative mRNA expression of HIF1a in T-ALL cell lines (right). (D) Protein levels of HIF1a in T-ALL cell lines by western blot. GAPDH was used as an internal control (left), and the correlation between the IC50 values and the protein expression of HIF1a in T-ALL cell lines (right). (E) The protein levels of HIF1a and JUN, CCRF-CEM(Dex) cells were harvested at 72 h after transfection with shHIF1a-1, shHIF1a-2, or nontarget lentivirus vectors (shC), β-tubulin was used as the internal control, CCRF-CEM(Dex) cells were treated with different concentrations of dexamethasone, and cell counting was performed at 48 h after treatment (right). (F–I) CCRF-CEM(Dex) cell-derived xenograft (CDX) experiment in NCG mice, 5×10 6 CCRF-CEM(Dex) cells were injected through the tail vein, dexamethasone (15 mg/kg/2 days), 2-methoxyestradiol (30 mg/kg/2 days) or a combination of these two inhibitors was intraperitoneally administered from day 6 to 20, and the control group was treated with PBS. (F) Schematic outline of cell-derived xenograft (CDX) generation. (G) Kaplan‒Meier survival curves of CCRF-CEM(Dex) cell-derived xenograft mice (CDX) in different treatment groups. (H) Analysis of bone marrow invasion using anti-human CD7 antibody through flow cytometry. (I) Quantification of peripheral blood and bone marrow invasion after drug treatment. (J) The protein levels of HIF1a and JUN after JUN silencing in CCRF-CEM(Dex) and CCRF-CEM(Dex) cells were harvested 72 h after transfection with shJUN-1, shJUN-2, or nontarget lentivirus vectors (shC). GAPDH was used as the internal control. (K) Relative mRNA expression of c-Jun or HIF1a in T-ALL cell lines through qPCR. CCRF-CEM(Dex) cells were harvested 72 h after transfection with shJUN-1, shJUN-2, shHIF1a-1, shHIF1a-2, or nontarget lentivirus vectors (shC). (L and M) Verification of the interaction between JUN and HIF1a. (L) Co-IP of JUN and HIF1a from the lysates of transfected cells. HEK293T cells were cotransfected with the plasmid combination pcDNA3.1-JUN-Flag/pcDNA3.1-HIF1a-HA and measured by western blotting. (M) Co-IP of JUN and HIF1a from the lysates of CCRF-CEM(Dex) cells. Cells were lysed, and co-IP assays were carried out and analyzed by western blotting. (N) Cycloheximide (CHX) chase assays showing that HIF1a retarded protein degradation in CCRF-CEM(Dex) cells (left), quantification rate of degradation (right). Data are represented as mean ± SD (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001).

Article Snippet: Then, Jurkat and CCRF-CEM(Dex) cells were infected with lentivirus packaged by the silencing plasmid (pLKO.1-puro), which includes a puromycin resistance region, and an shRNA construct to target HIF1α and JUN or a non-specific shRNA control (shRNA C: 5′-CAACACAGATGATAGAGCACCAATTGGTGCTCTATCATCTGTGTTGTTTTT-3′).

Techniques: Cell Counting, Concentration Assay, Expressing, Western Blot, Control, Transfection, Derivative Assay, Injection, Flow Cytometry, Co-Immunoprecipitation Assay, Plasmid Preparation

Journal: iScience

Article Title: JUN mediates glucocorticoid resistance by stabilizing HIF1a in T cell acute lymphoblastic leukemia

doi: 10.1016/j.isci.2023.108242

Figure Lengend Snippet:

Article Snippet: Then, Jurkat and CCRF-CEM(Dex) cells were infected with lentivirus packaged by the silencing plasmid (pLKO.1-puro), which includes a puromycin resistance region, and an shRNA construct to target HIF1α and JUN or a non-specific shRNA control (shRNA C: 5′-CAACACAGATGATAGAGCACCAATTGGTGCTCTATCATCTGTGTTGTTTTT-3′).

Techniques: Virus, Recombinant, SYBR Green Assay, Drug discovery, CCK-8 Assay, RNA Extraction, Sequencing, Software, Flow Cytometry