Journal: The Journal of Biological Chemistry

Article Title: Heat shock proteins stimulate APOBEC-3–mediated cytidine deamination in the hepatitis B virus

doi: 10.1074/jbc.M116.760637

Figure Lengend Snippet: Effect of Hsp90 or Hsp70 inhibitor on A3G and A3B activity. Hsp inhibitors were used to evaluate the Hsp90 or Hsp70 effect on A3G and A3B. APOBEC-3s with or without Hsp90β were co-transfected with HBV into HepG2 cells in the presence or absence of Hsp90 inhibitor 17-AAG or Hsp70 inhibitor PES. After 2 days of treatment, HBV DNA mutation rates were determined by primer extension with the 88 °C 3D-PCR for A3G or 94 °C PCR for A3B. A, effect of Hsp90 inhibitor 17-AAG on A3G + Hsp90β mutation activity by pe1664; B, effect of Hsp90 inhibitor 17-AAG on A3B mutation activity by pe1674; C, effect of Hsp70 inhibitor PES on A3B mutational activity by pe1674. The data are presented graphically on the right. Each bar represents the average of triplicates for each treatment. Vector, background control with mock vector co-transfection; asterisks, statistically significant differences comparing treatments with controls: **, 0.01 < p < 0.001; *, 0.05 < p < 0.01. The one-factor ANOVA statistical test was used.

Article Snippet: Hsp40 (DNAJB1), Hsp70 (HSPA1A), Hsp90α, Hsp90β, Hsp104, and Hsp60 genes were purchased from Origene or Addgene.

Techniques: Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Cotransfection

Journal: The Journal of Biological Chemistry

Article Title: Heat shock proteins stimulate APOBEC-3–mediated cytidine deamination in the hepatitis B virus

doi: 10.1074/jbc.M116.760637

Figure Lengend Snippet: Effect of Hsp knockdown through siRNA on HBV mutation frequency. Each Hsp siRNA was transfected into HepG2 cells by a reverse transfection method with Lipofectamine RNAiMax. After a 24-h siRNA treatment, the cells were transfected with an HBV viral genome-encoding plasmid with or without A3G. The HepG2 cells were harvested for RNA or HBV extraction 24 h after HBV transfection. A, Hsp mRNA levels after siRNA treatment by quantitative RT-PCR determination. The relative mRNA levels for each siRNA treatment were determined by quantitative RT-PCR, using GAPDH as an internal reference. The mRNA levels relative to control were calculated and are represented graphically as percentages with the control as 100%. Each bar represents the average of triplicates for each treatment. Blank, background control by a scrambled negative siRNA without A3G co-transfection. Control, another control by the scrambled negative siRNA with A3G co-transfection. B, Hsp protein expression level analyses after siRNA treatment. Total cellular proteins were extracted from HepG2 cells after siRNA treatment, and Hsp protein levels in the cell lysates were analyzed by Western blotting with antibodies against endogenous Hsp90β, Hsp90α, Hsp70, and Hsp40. GAPDH was analyzed as the protein loading reference. C, HBV DNA mutation analyses. HBV DNAs were extracted from the cell lysates after a 24-h HBV transfection, and the resultant HBV DNA mutations were determined by pe1453 using an 88 °C 3D-PCR. The data are presented graphically on the right. Each bar represents the average of triplicates for each treatment. Asterisks, statistically significant differences comparing treatments with their corresponding control: **, 0.01 < p < 0.001; *, 0.05 < p < 0.01. The one-factor ANOVA statistical test was used.

Article Snippet: Hsp40 (DNAJB1), Hsp70 (HSPA1A), Hsp90α, Hsp90β, Hsp104, and Hsp60 genes were purchased from Origene or Addgene.

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Quantitative RT-PCR, Cotransfection, Expressing, Western Blot

Journal: Cell Reports Medicine

Article Title: Venetoclax acts as an immunometabolic modulator to potentiate adoptive NK cell immunotherapy against leukemia

doi: 10.1016/j.xcrm.2024.101580

Figure Lengend Snippet:

Article Snippet: Lisaftoclax , MedChemExpress , Cat# HY-129179.

Techniques: Recombinant, Staining, Blocking Assay, CCK-8 Assay, Western Blot, Cell Isolation, Software

Journal: Frontiers in Pharmacology

Article Title: MDM2 inhibitor APG-115 synergizes with ABT-199 to induce cell apoptosis in chronic lymphocytic leukemia

doi: 10.3389/fphar.2024.1441383

Figure Lengend Snippet: APG-115 restores p53 expression and activity. mRNA expression of MDM2 , TP53 , and CDKN1A/p21 were detected in (A,B) CLL patient primary cells and (C,D) EHEB cells treated with different concentrations APG-115 or different times of APG-115 (10 μM). Relative gene expression was calculated based on the threshold cycle (Ct) values and normalized to β-actin internal control using the 2 −ΔΔCt method. Experiments were performed in triplicate and repeated at least three times. The results showed representative primary cells from three CLL patients. (E) The protein expressions of MDM2, p53, and p21 in EHEB cells were detected by WB. (F) The quantification analysis of protein bands in panel E was performed using ImageJ software; the values of protein bands were divided by the internal control β-actin. The data shown are representative images of three independent experiments.

Article Snippet: APG-115 was obtained from Suzhou Ascentage Pharma Co., Ltd. ABT-199 was a product of MCE.

Techniques: Expressing, Activity Assay, Gene Expression, Control, Software

Journal: Frontiers in Pharmacology

Article Title: MDM2 inhibitor APG-115 synergizes with ABT-199 to induce cell apoptosis in chronic lymphocytic leukemia

doi: 10.3389/fphar.2024.1441383

Figure Lengend Snippet: APG-115 inhibits cellular viability and induces apoptosis. (A) EHEB cells were treated with different concentrations of APG-115 for 48 h, cell viability was assessed by MTS assay; (B) cell apoptosis was detected by flow cytometry. Experiments were performed in triplicate and repeated at least three times. (C) Cell apoptosis was detected on CLL patient primary cells (P) (n = 8) cultured with 10 ng CD40L and IL-4 and treated with APG-115 for 48 h. Data was expressed as mean ± standard deviation. Statistical significance was determined using the Student unpaired t -test, with Welch’s correction. *, compared to the control group (no APG-115 treatment) * p < 0.05, ** p < 0.01, *** p < 0.001. Non-significant results are denoted by ns. (D) Protein expression of caspase-3 and PARP was detected by WB in EHEB cells after 12-h treatment with APG-115. β-actin was a control protein. (E) Quantification analysis of the protein bands in D using ImageJ software; the values were divided by the internal control β-actin. The data shown are representative images of two independent experiments. (F) Statistical histogram of cell cycle in EHEB cells exposed to increasing concentrations of APG-115 for 48 h.

Article Snippet: APG-115 was obtained from Suzhou Ascentage Pharma Co., Ltd. ABT-199 was a product of MCE.

Techniques: MTS Assay, Flow Cytometry, Cell Culture, Standard Deviation, Control, Expressing, Software

Journal: Frontiers in Pharmacology

Article Title: MDM2 inhibitor APG-115 synergizes with ABT-199 to induce cell apoptosis in chronic lymphocytic leukemia

doi: 10.3389/fphar.2024.1441383

Figure Lengend Snippet: APG-115 inhibits the expression of BCL-2, BCL-xL, and MCL-1. (A) EHEB cells and (B) CLL patient primary cells were treated with different concentrations of APG-115 for 12 h. The protein expression of BCL-2, BCL-xL, MCL-1, and BAX were detected by WB, and β-actin was the internal control protein. (C,D) The quantification analysis of protein bands in (A,B) by ImageJ software, a statistical graph showing the value divided by the internal control and compared with the control group without treatment.

Article Snippet: APG-115 was obtained from Suzhou Ascentage Pharma Co., Ltd. ABT-199 was a product of MCE.

Techniques: Expressing, Control, Software

Journal: Frontiers in Pharmacology

Article Title: MDM2 inhibitor APG-115 synergizes with ABT-199 to induce cell apoptosis in chronic lymphocytic leukemia

doi: 10.3389/fphar.2024.1441383

Figure Lengend Snippet: APG-115 suppresses the activation of AKT and ERK signaling pathways (A) The protein expression of AKT, p-AKT, ERK, and p-ERK were detected by WB in EHEB cells and (B) CLL patient primary cells treated with different concentrations of APG-115 for 12 h. β-actin was the internal reference protein. The results were representatives of primary cells from three CLL patients. (C,D) The quantification analysis of protein bands in (A,B) by ImageJ software, a statistical graph showing the value divided by the internal reference protein β-actin, and compared with the control group.

Article Snippet: APG-115 was obtained from Suzhou Ascentage Pharma Co., Ltd. ABT-199 was a product of MCE.

Techniques: Activation Assay, Protein-Protein interactions, Expressing, Software, Control

Journal: Frontiers in Pharmacology

Article Title: MDM2 inhibitor APG-115 synergizes with ABT-199 to induce cell apoptosis in chronic lymphocytic leukemia

doi: 10.3389/fphar.2024.1441383

Figure Lengend Snippet: APG-115 synergizes ABT-199 to induce cell apoptosis in CLL (A,B) Cell apoptosis was detected by flow cytometry in CLL patient primary cells, and (C) EHEB cells that were treated with described concentrations of APG-115 and ABT-199 for 48 h. The figure shows the representatives of three independent experiments, and the results are expressed as mean ± standard deviation. (D) The synergic effect was evaluated using the Compusyn software. The criteria for the Combination index (CI) value are as follows: <0.1, Very strong synergism; 0.1–0.3, Strong synergism; 0.3–0.7, Synergism; 0.7–0.85, Moderate synergism; 0.85–0.9, Slight synergism; 0.90–1.1, Nearly additive; >1.1 antagonism. In green, shows synergistic combinations. In red, shows additive combinations. In blue, shows average synergy in all combinations. All concentrations are in μM. (E) The effect of combination of APG-115 and ABT-119 on BCL-2, BCL-xL, and MCL-1 expression in EHEB cells. The data shown are representative images of three independent experiments. (F) The quantification analysis of protein bands in (E) by ImageJ software, the graph shows the value divided by the internal reference β-actin and compared with the control group without treatment. Statistical significance was determined using two-way ANOVA analysis with the Geisser-Greenhouse correction. A p -value of < 0.05 was considered statistically significant.* p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: APG-115 was obtained from Suzhou Ascentage Pharma Co., Ltd. ABT-199 was a product of MCE.

Techniques: Flow Cytometry, Standard Deviation, Software, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: MDM2 inhibitor APG-115 synergizes with ABT-199 to induce cell apoptosis in chronic lymphocytic leukemia

doi: 10.3389/fphar.2024.1441383

Figure Lengend Snippet: The proposed pro-apoptotic mechanism of APG-115 in CLL.

Article Snippet: APG-115 was obtained from Suzhou Ascentage Pharma Co., Ltd. ABT-199 was a product of MCE.

Techniques:

Journal: RNA

Article Title: Establishment of 5′–3′ interactions in mRNA independent of a continuous ribose-phosphate backbone

doi: 10.1261/rna.073759.119

Figure Lengend Snippet: Assembly of chimeric RNA constructs. ( A ) Outline of the procedure. Regulatory RNAs contained a 5′ biotin, two SREs (SRE + = WT or SRE − , that is, with an inactivating point mutation in each SRE) and a poly(A) sequence, which was protected against deadenylation by an N 40 sequence at the 3′ end. Alternatively, regulatory RNA was used that carried a poly(A) sequence but no SREs (not shown). The regulatory RNA was hybridized to immobilized oligo(dT), then divalent streptavidin and the capped and 3′-biotinylated nLuc reporter RNA were added in a stepwise manner. The chimeric construct was eluted in low-salt buffer. The scheme shows the assembly of forward constructs. Flipped constructs were assembled in the same manner, but 3′-biotinylated regulatory RNAs were used. For a full description of the procedure, see Materials and Methods. ( B ) Assembly of chimeric RNAs assayed by native gel electrophoresis. Chimeras [forward-SRE ± p(A)] were assembled as described in panel A and Materials and Methods. The figure shows the analysis of input and product RNAs by electrophoresis through a 5% nondenaturing polyacrylamide gel. Lanes 1 – 3 display the input RNAs as indicated. Lanes 4 – 6 demonstrate the retardation of input RNAs upon mixing with divalent streptavidin (STV). Two retarded bands in one lane are presumably due to binding of one or two RNAs to one molecule of streptavidin. Lanes 7 , 8 show the flow-through of the oligo(dT) matrix after loading with regulatory RNAs; binding was essentially complete. Lanes 9 and 10 show the flow-through of excess nLuc RNA in the last assembly step. “nLuc + ”′ and “nLuc − ” refer to the nLuc RNAs from the assembly reactions with SRE + and SRE − regulatory RNAs, respectively. Lanes 11 and 12 show the eluate in formamide-containing loading buffer without heat denaturation. The main band (arrowhead) is a novel species that we interpret as the desired chimeric RNA. Upon heat denaturation (lanes 13 , 14 ), the input RNAs are restored. Residual amounts of input RNAs can be detected: Based on a comparison to other lanes in this gel, numbered bands are (1) a complex between one regulatory RNA and streptavidin, (2) bare nLuc RNA, and (3) a complex between two regulatory RNAs and streptavidin. The broken line indicates the upper end of the gel.

Article Snippet: After 1 h at 37°C, 0.15 mM GTP was again added, and the reactions were further incubated for 3 h. For the synthesis of 3′-biotinylated regulatory RNAs, transcription reactions were as above except that 5′ biotin-ApG was replaced by 7 mM A-cap (Jena Bioscience), and GTP, ATP, and CTP were all used at 3 mM.

Techniques: Construct, Mutagenesis, Sequencing, Nucleic Acid Electrophoresis, Electrophoresis, Binding Assay

Journal: RNA

Article Title: Establishment of 5′–3′ interactions in mRNA independent of a continuous ribose-phosphate backbone

doi: 10.1261/rna.073759.119

Figure Lengend Snippet: Translation of chimeric RNAs is stimulated by poly(A) tails and repressed by SREs. ( A ) Scheme of chimeric constructs. All nLuc RNAs (blue) had a 5′ m7G cap and a 3′ biotin. Regulatory RNAs had either a 5′ biotin combined with an unmodified 3′ end (forward constructs) or a 5′ A cap combined with a 3′ biotin (flipped constructs). The green RNA section in the forward-long-SRE ± p(A) construct represents the firefly luciferase stuffer fragment (see text). Regulatory RNAs attached to the right of the central divalent streptavidin were as described in and the text. All SRE-containing RNAs were synthesized as SRE + and SRE − variants as indicated. “N” indicates the number of independent chimeric RNA preparations tested in translation. The number of independent translation experiments (each carried out as three technical replicates) is represented by “n.” Each type of RNA was tested in at least three batches of embryo extract except the flipped-p(A) construct, which was tested in two batches. ( B ) Translation assays of nLuc and chimeric RNAs containing poly(A) segments in forward and flipped orientations. RNAs were assayed in parallel for translation in Drosophila embryo extract as described in Materials and Methods. The pairwise comparisons used to calculate poly(A)-dependent stimulation are indicated at the top . ( C ) Assays as in B were carried out with the chimeric constructs containing poly(A) segments plus SREs. The pairwise comparisons used to calculate poly(A)-dependent stimulation or SRE-dependent repression, respectively, are indicated at the top . ( D , E ) The same RNA preparations as in B and C were translated in parallel in rabbit reticulocyte lysate (RRL). It was assumed that differences in luciferase activities represented variable qualities of the RNA preparations rather than SRE- or poly(A)-effects. Numbers from such assays were therefore used to correct luciferase yields obtained in embryo extract in , 7, and 8. Panels B through E show the results of single representative experiments with error bars representing the standard deviations of three technical replicates.

Article Snippet: After 1 h at 37°C, 0.15 mM GTP was again added, and the reactions were further incubated for 3 h. For the synthesis of 3′-biotinylated regulatory RNAs, transcription reactions were as above except that 5′ biotin-ApG was replaced by 7 mM A-cap (Jena Bioscience), and GTP, ATP, and CTP were all used at 3 mM.

Techniques: Construct, Luciferase, Synthesized