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

Journal: Journal of Thoracic Disease

Article Title: Autophagic tumor-associated macrophages promote the endothelial mesenchymal transition in lung adenocarcinomas through the FUT4/p-ezrin pathway

doi: 10.21037/jtd-21-1519

Figure Lengend Snippet: FUT4 overexpression promoted EMT of human lung adenocarcinoma cells induced by autophagic TAMs. (A) The relative mRNA levels of FUT4 and TGF-β1 in shATG5-NC-TAMs, and shATG5-FUT4-TAMs as determined by qRT-PCR; (B) the protein levels of FUT4, LC3, and p-ezrin in shATG5-NC-TAMs and shATG5-FUT4-TAMs were assessed by Western blotting; (C) the expression of p-ezrin in shATG5-NC-TAMs and shATG5-FUT4-TAMs was visualized by cell immunofluorescence studies; (D) the expression of TGF-β1 in shATG5-NC-TAMs, and shATG5-FUT4-TAMs was assessed by enzyme-linked immunosorbent assays; (E) the shATG5-FUT4-TAMs were stimulated with rapamycin and then co-cultured with A549 cells. The protein levels of N-cadherin (N-cad), E-cadherin (E-cad), vimentin, Snail, and Slug in the treated A549 cells were determined by Western blot analysis; (F) the cell migration and invasion of the treated A549 cells was assessed by Transwell assays (×100 magnification), in which cells in the lower compartment were stained with crystal violet; (G) the relative wound area of the treated A549 cells was examined by wound healing assays (×40 magnification). **, P<0.01 vs. shATG5-NC group; ***, P<0.001 vs. shATG5-NC group. FUT4, fucosyltransferase IV; p-erzin, phosphorylated erzin; TGF-β1, tumor growth factor β1; TAM, tumor-associated macrophage; ATG5, autophagy related gene 5; sh, short hairpin; NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction; LC3, microtubule-associated protein 1A/1B-light chain 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: ATG5 silencing and overexpression lentiviral vectors, fucosyltransferase IV (FUT4) silencing and overexpression plasmids, ezrin silencing plasmid, and their corresponding negative controls were obtained from GenePharma (Shanghai, China).

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Cell Culture, Migration, Staining, Negative Control, Reverse Transcription, Polymerase Chain Reaction

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