Journal: Autophagy

Article Title: Long-lived mice with reduced growth hormone signaling have a constitutive upregulation of hepatic chaperone-mediated autophagy

doi: 10.1080/15548627.2020.1725378

Figure Lengend Snippet: Liver lysosomes of fed Snell mice show more uptake of CMA substrates than lysosomes from littermate controls. (A) Representative western blots of liver lysosomes from control (C) and Snell (S) mice 2 h after treatments with leupeptin (100 mg/kg body weight) or PBS vehicle control. (B-D) Quantifications of the abundance of CMA substrates GAPDH, ENO1, and ACADL in lysosomes shown in (A). (E) Quantification of PPID, endosomal microautophagy substrate. (F) Quantification of HSPA8, CMA chaperone. (G) Representative western blots for binding and uptake assays for CMA substrate GAPDH, quantified in (H). (I) Representative western blots for binding and uptake assays for CMA substrate MAPT, quantified in (J). For (B-F), n = 9 of each treatment group, 2-way ANOVA results are reported below each bar graph. Comparisons plotted on the bar graphs are results of Student’s t-test. For (H & J), n = 6 of each treatment group, the results of Student’s t-test are shown on the graphs. Error bars are S.E.M

Article Snippet: Recombinant GAPDH (Sino Biological, 51,221-M07E), MAPT (Sino Biological, 10,058-H07E), and/or protease inhibitors (Sigma, 11,836,153,001) were added, as indicated. qPCR and other reagents For mRNA quantification, RNA was isolated from powdered tissue (flash frozen in liquid nitrogen upon dissection), using Qiagen RNeasy kits (Qiagen, 74,106), with QIAshredder (Qiagen, 79,654) tissue homogenizers. cDNA was synthesized using iScript cDNA synthesis reagents (BioRad, L010174A), according to the manufacturer’s instructions. qPCR was conducted in an Applied Biosystems Step One Plus Real-Time PCR System, using Fast SYBR Green Master Mix (Thermo, 4,385,612) as a detection reagent.

Techniques: Western Blot, Binding Assay

Journal: Autophagy

Article Title: Long-lived mice with reduced growth hormone signaling have a constitutive upregulation of hepatic chaperone-mediated autophagy

doi: 10.1080/15548627.2020.1725378

Figure Lengend Snippet: Liver lysosomes of fed ghr KO mice show more uptake of CMA substrates than lysosomes from littermate controls. (A) Representative western blots of liver lysosomes from control (C) and ghr KO (KO) mice 2 h after treatments with leupeptin (100 mg/kg body weight) or PBS vehicle control. (B-D) Quantifications of the abundance of CMA substrates GAPDH, ENO1, and ACADL in lysosomes shown in (A). (E) Quantification of PPID, endosomal microautophagy substrate. (F) Quantification of HSPA8, CMA chaperone. (G) Representative western blots for binding and uptake assays for CMA substrate MAPT, quantified in (H). For (B-F), n = 6 of each treatment group, 2-way ANOVA results are reported below each bar graph. Comparisons plotted on the bar graphs are results of Student’s t-test. For (H), n = 6 of each treatment group; the results of Student’s t-test are shown on the graphs. Error bars are S.E.M

Article Snippet: Recombinant GAPDH (Sino Biological, 51,221-M07E), MAPT (Sino Biological, 10,058-H07E), and/or protease inhibitors (Sigma, 11,836,153,001) were added, as indicated. qPCR and other reagents For mRNA quantification, RNA was isolated from powdered tissue (flash frozen in liquid nitrogen upon dissection), using Qiagen RNeasy kits (Qiagen, 74,106), with QIAshredder (Qiagen, 79,654) tissue homogenizers. cDNA was synthesized using iScript cDNA synthesis reagents (BioRad, L010174A), according to the manufacturer’s instructions. qPCR was conducted in an Applied Biosystems Step One Plus Real-Time PCR System, using Fast SYBR Green Master Mix (Thermo, 4,385,612) as a detection reagent.

Techniques: Western Blot, Binding Assay

Journal: PLoS ONE

Article Title: Spermatogenesis Associated 4 Promotes Sertoli Cell Proliferation Modulated Negatively by Regulatory Factor X1

doi: 10.1371/journal.pone.0075933

Figure Lengend Snippet: After transient transfection of pcDNA3.1-RFX1 for 36 h, the overexpressed RFX1 protein was detected by immunocytochemistry staining and Western blot ( A ), overexpression of RFX1 was shown to down-regulate the promoter activity ( B ) and Spata4 mRNA expression ( C ) and in a dose-dependent manner. The Spata4 mRNA expression was analyzed by quantitative PCR and normalized to GAPDH. ** P <0.01. *** P <0.001.

Article Snippet: The membranes were then incubated with primary antibodies specific for RFX1 (1∶500; Santa Cruz), Spata4 (1∶1000; prepared by our laboratory), GAPDH (1∶1000; Cwbiotech, Beijing, China) or β-tublin (1∶1000; Cwbiotech) at 4°C overnight, followed by an incubation with horseradish peroxidase-conjugated anti-goat IgG (1∶5000; Santa Cruz) or anti-rabbit IgG (1∶10000) at room temperature for 1 h. The chemiluminescence reaction was performed using ECL reagent (Thermo Scientific).

Techniques: Transfection, Immunocytochemistry, Staining, Western Blot, Over Expression, Activity Assay, Expressing, Real-time Polymerase Chain Reaction

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: JNK regulated JUN in dexamethasone-resistant T-ALL cells (A) Schematic outline of drug-resistant stable cell line (CCRF-CEM(Dex)) generation, RNA-seq and ATAC-seq. (B) The relative cell viability of CCRF-CEM and CCRF-CEM(Dex) cells. Cell counting was performed 48 h after treatment with different concentration gradients of dexamethasone. (C) Protein levels of JUN in T-ALL cell lines determined by western blotting. GAPDH was used as an internal control. (D) Correlation between the IC50 values and the protein expression of JUN in T-ALL cell lines. (E) Heatmap depicting the differentially expressed genes (DEGs) between CCRF-CEM and CCRF-CEM(Dex) cells based on RNA-seq data. The color indicates the Z score of the expression of different genes. Genes were selected with p < 0.05 and log2 fold change >0.5. (F) Top 70 significantly activated transcription factors (TFs) in each group based on RNA-seq data. The color indicates the Z -scaled average TF activity score. (G) Rank of motifs enriched in the promoter region based on ATAC-seq. (H) Venn diagram depicting the number of common genes in different groups. “Motifs” was the group of motifs enriched in the promoter, “RNA-seq” was the upregulated genes based on RNA-seq data, and “TF activity (Top 70)” was the group of the top 70 differentially activated TFs based on RNA-seq data. (I) KEGG pathway analysis of upregulated genes in the CCRF-CEM(Dex) groups. (J) Relative mRNA expression of JNK, ERK, and p38 MAPK in CCRF-CEM and CCRF-CEM(Dex) cells. (K) Protein levels of JNK, ERK, p38 MAPK, phosphorylation level of ERK, phosphorylation level of JNK, and phosphorylation level of p38 MAPK in CCRF-CEM and CCRF-CEM(Dex) cells. GAPDH was used as the internal control. (L) The relative cell viability of CCRF-CEM or CCRF-CEM(Dex) cells. Cell counting was performed at 48 h after treatment with different concentration gradients of aristolochic acid, SP600125, SB203580, and SCH772984. Data are represented as mean ± SD (∗p < 0.05, ∗∗p < 0.01, ns = no significant).

Article Snippet: GAPDH was used as the internal control. (L) The relative cell viability of CCRF-CEM or CCRF-CEM(Dex) cells.

Techniques: Stable Transfection, RNA Sequencing, Cell Counting, Concentration Assay, Western Blot, Control, Expressing, Activity Assay, Phospho-proteomics

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: GAPDH was used as the internal control. (L) The relative cell viability of CCRF-CEM or CCRF-CEM(Dex) cells.

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: T-ALL patient samples with glucocorticoid resistance are sensitive to HIF1a inhibition (A) TPM of HIF1a in the resistant and sensitive groups based on a T-ALL cohort (GEO, GSE5820) (left), TPM of HIF1a in different groups based on a T-ALL cohort including ETP-ALL and non-ETP-ALL patient samples (right). (B) Event-free survival of T-ALL patients with higher or lower expression of HIF1a (TARGET, phs000464). (C) The relative cell viability of blast cells from 12 T-ALL patients (T1-T12) treated with different concentration gradients of dexamethasone. (D) Protein levels of HIF1a and JUN in T-ALL patient samples determined by western blotting. GAPDH was used as an internal control. (E) The correlation between the IC50 values and the protein expression of JUN. (F) The correlation between the IC50 values and the protein expression of HIF1α. (G) Synergistic inhibition of Dex and BAY872243 in patient samples. Cell counting was performed at 48 h. (H–K) T5 patient sample cell-derived xenograft (CDX) experiment in NCG mice, 5×10 5 luciferase-labeled T5 patient sample cells were injected through the tail vein, dexamethasone or BAY872243 or a combination of these two inhibitors from day 10 to day 30, and the control group was treated with PBS. (H) Schematic outline of patient-derived xenografts (PDXs). (I) Bioluminescent imaging of T5-derived xenograft mice in different treatment groups. (J) Kaplan‒Meier survival curves of xenograft mice (PDX) in different treatment groups. (K) Analysis of bone marrow invasion using anti-human CD7 antibody through flow cytometry. Data are represented as mean ± SD.

Article Snippet: GAPDH was used as the internal control. (L) The relative cell viability of CCRF-CEM or CCRF-CEM(Dex) cells.

Techniques: Inhibition, Expressing, Concentration Assay, Western Blot, Control, Cell Counting, Derivative Assay, Luciferase, Labeling, Injection, Imaging, Flow Cytometry