Journal: Oncotarget

Article Title: FOXO4 expression is related to stem cell-like properties and resistance to treatment in diffuse large B-cell lymphoma

doi: 10.18632/oncotarget.13690

Figure Lengend Snippet: ( A ) Study scheme of gene expression analyses using doxorubicin (Dox) and phenylbutyrate (PB)-treated surviving cells: Four different cell lines (BJAB, Raji, Toledo, and Daudi) are treated with doxorubicin (300 nM) or phenylbutyrate (8 mM) for 48 h, and cDNA microarray analysis is done to identify differentially expressed target genes between treatment-surviving cells and parental control cells. ( B ) FOXO4 mRNA level is significantly higher in phenylbutyrate-treated surviving (PB) cells of BJAB, Raji and Daudi than control cells. ( C ) Vorinostat-treated surviving cells show higher mRNA level of FOXO4 than control (Con) cells of BJAB and Raji. ( D ) Primary lymphoma cells from three patients with refractory diffuse large B-cell lymphoma (DLBCL) show increased expression of FOXO4 in phenylbutyrate-treated surviving cells (PB) compared to the corresponding control cells. ( E ) The FOXO4 mRNA level is significantly higher in primary cells from four patients with refractory DLBCL than that of a patient with DLBCL who achieved complete response. Data represent means ± SD from three independent experiments.

Article Snippet: After endogenous peroxidase blocking for 5 min, tissues were incubated with primary polyclonal antibody against FOXO4 (1:1000; GTX50500; GeneTex, Irvine, CA, USA) for 15 min using a BOND-MAX autoimmunostainer (Leica Biosystems, Melbourne, Australia) for 15 min.

Techniques: Gene Expression, Microarray, Control, Expressing

Journal: Oncotarget

Article Title: FOXO4 expression is related to stem cell-like properties and resistance to treatment in diffuse large B-cell lymphoma

doi: 10.18632/oncotarget.13690

Figure Lengend Snippet: ( A, B ) Phenylbutyrate-treated surviving cells (BJAB-PB and Raji-PB) increase the expression of FOXO4 and its transcriptional targets (p21, p27, and SOD) compared to control cells. ( C, D ) BJAB-PB and Raji-PB cells show decreased expression of cyclin D1, CDK4, and cyclin A compared to control cells.

Article Snippet: After endogenous peroxidase blocking for 5 min, tissues were incubated with primary polyclonal antibody against FOXO4 (1:1000; GTX50500; GeneTex, Irvine, CA, USA) for 15 min using a BOND-MAX autoimmunostainer (Leica Biosystems, Melbourne, Australia) for 15 min.

Techniques: Expressing, Control

Journal: Oncotarget

Article Title: FOXO4 expression is related to stem cell-like properties and resistance to treatment in diffuse large B-cell lymphoma

doi: 10.18632/oncotarget.13690

Figure Lengend Snippet: ( A ) Soft agar colony formation in FOXO4-transfected or siFOXO4-transfected BJAB cells shows the amplification of FOXO4 increase colony forming ability and the knockdown of FOXO4 decrease colony formation in BJAB cell line. ( B ) Decrease in mRNA levels of Nanog, Oct-4 and Sox-2 in siFOXO4-transfected (siFOXO4) is noted compared to siControl-transfected (siCon) BJAB cells. ( C ) Phosphorylated AKT level is decreased in BJAB-PB cells with FOXO4 overexpression whereas siFOXO4-transfected BJAB-PB cells show the reverse of phosphorylated AKT. ( D ) The western blot shows the expression of FOXO4 protein in a BJAB clone (c2) overexpressing FOXO4. ( E ) Tumor sphere formation is observed from a BJAB clone (c2) overexpressing FOXO4. ( F ) Immunohistochemical staining for FOXO4 in tumor tissue of DLBCL (× 200). ( G ) Kaplan-Meier curves shows superior progression-free survival and overall survival of FOXO4-high group with diffuse large B cell lymphoma than FOXO4-low group. The P value is calculated using the log-rank test.

Article Snippet: After endogenous peroxidase blocking for 5 min, tissues were incubated with primary polyclonal antibody against FOXO4 (1:1000; GTX50500; GeneTex, Irvine, CA, USA) for 15 min using a BOND-MAX autoimmunostainer (Leica Biosystems, Melbourne, Australia) for 15 min.

Techniques: Transfection, Amplification, Knockdown, Over Expression, Western Blot, Expressing, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: FOXO4 expression is related to stem cell-like properties and resistance to treatment in diffuse large B-cell lymphoma

doi: 10.18632/oncotarget.13690

Figure Lengend Snippet: Characteristics of patients

Article Snippet: After endogenous peroxidase blocking for 5 min, tissues were incubated with primary polyclonal antibody against FOXO4 (1:1000; GTX50500; GeneTex, Irvine, CA, USA) for 15 min using a BOND-MAX autoimmunostainer (Leica Biosystems, Melbourne, Australia) for 15 min.

Techniques:

Journal: Applied and Environmental Microbiology

Article Title: A Novel Small RNA, DsrO, in Deinococcus radiodurans Promotes Methionine Sulfoxide Reductase ( msrA ) Expression for Oxidative Stress Adaptation

doi: 10.1128/aem.00038-22

Figure Lengend Snippet: msrA expression is strongly induced by oxidative stress in D. radiodurans . (A and C) msrA transcriptional level (A) and protein level (C) in D. radiodurans treated with different concentrations of H 2 O 2 for 30 min. (B and D) msrA transcriptional level (B) and protein level (D) in D. radiodurans treated with 80 mM H 2 O 2 for different times. The msrA transcriptional level was analyzed by qRT-PCR with 16S rRNA as the reference gene. GroEL was used as the internal control for Western blotting. Asterisks indicate statistically significant differences of the value compared to that of untreated cells (one-way analysis of variance [ANOVA], Dunnett’s multiple-comparison test; ***, P ≤ 0.001; *, P ≤ 0.05). The experiments were performed at least three times, and the data are presented as means ± standard error of the mean (SEM).

Article Snippet: The primary polyclonal antibody against MsrA was produced by Genscript Biotechnology Company based on the MsrA protein sequence (Genscript, Nanjing, China).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Comparison

Journal: Applied and Environmental Microbiology

Article Title: A Novel Small RNA, DsrO, in Deinococcus radiodurans Promotes Methionine Sulfoxide Reductase ( msrA ) Expression for Oxidative Stress Adaptation

doi: 10.1128/aem.00038-22

Figure Lengend Snippet: The deletion of the msrA gene in D. radiodurans decreases its tolerance to oxidative stress. (A) H 2 O 2 sensitivity assays of different D. radiodurans strains. Spotted agar plates after H 2 O 2 treatment and serial dilution. (Left) 0 mM H 2 O 2 ; (right) 80 mM H 2 O 2 for 30 min; WT, wild-type strain, Δ msrA , msrA -deleted mutant; msrA-pRADZ3 , msrA mutant transformed with pRADZ3 empty plasmid; msrA-com , msrA mutant supplemented with the msrA gene. (B to D) Total antioxidant capacity (B), ROS level (C), and intracellular carbonylation level (D) of different strains after H 2 O 2 treatment. The total level of various antioxidant macromolecules, antioxidant small molecules, and enzymes in a system reflects the total antioxidant capacity of the bacterial cells. Rosup is a reactive oxygen positive-control reagent. Asterisks indicate a statistically significant difference in the value compared to that of untreated cells (one-way ANOVA, Dunnett’s multiple-comparison test; ***, P ≤ 0.001; *, P ≤ 0.05). Experiments were performed at least three times, and the data are presented as the means ± SEM.

Article Snippet: The primary polyclonal antibody against MsrA was produced by Genscript Biotechnology Company based on the MsrA protein sequence (Genscript, Nanjing, China).

Techniques: Serial Dilution, Mutagenesis, Transformation Assay, Plasmid Preparation, Positive Control, Comparison

Journal: Applied and Environmental Microbiology

Article Title: A Novel Small RNA, DsrO, in Deinococcus radiodurans Promotes Methionine Sulfoxide Reductase ( msrA ) Expression for Oxidative Stress Adaptation

doi: 10.1128/aem.00038-22

Figure Lengend Snippet: A noncoding RNA, DsrO, directly binds to the stem-loop structure of msrA mRNA. (A) Predicted binding pattern between msrA mRNA and DsrO based on the bioinformatics analysis. (B) Affinity coefficient between DsrO and msrA mRNA detected by the microscale thermophoresis (MST) method. (C to F) The transcriptional level of DsrO in response to the treatments of 80 mM H 2 O 2 (C), UV radiation (D), heat (48°C) (E), and cold (4°C) (F) in D. radiodurans . Asterisks indicate statistically significant differences compared to untreated cells (one-way ANOVA, Dunnett’s multiple-comparison test; ***, P ≤ 0.001; *, P ≤ 0.05). Experiments were performed at least three times, and data are presented as means ± SEM.

Article Snippet: The primary polyclonal antibody against MsrA was produced by Genscript Biotechnology Company based on the MsrA protein sequence (Genscript, Nanjing, China).

Techniques: Binding Assay, Microscale Thermophoresis, Comparison

Journal: Applied and Environmental Microbiology

Article Title: A Novel Small RNA, DsrO, in Deinococcus radiodurans Promotes Methionine Sulfoxide Reductase ( msrA ) Expression for Oxidative Stress Adaptation

doi: 10.1128/aem.00038-22

Figure Lengend Snippet: The key base mutation of the binding region between DsrO and msrA confers a phenotype sensitive to oxidative stress. (A and C) Survival of DsrO (A) or msrA (C) point-mutation strains upon exposure to 80 mM H 2 O 2 . (B and D) Total antioxidant capacity and ROS level of different DsrO (B) or msrA (D) point-mutant strains upon exposure to 80 mM H 2 O 2 . WT, wild-type strain; ΔDsrO/ msrA , DsrO/ msrA -deleted mutant; DsrO/ msrA-pRADZ3 , the DsrO/ msrA mutant with pRADZ3 empty plasmid; DsrO/ msrA -com, DsrO mutant supplemented with DsrO/ msrA gene; DsrO/ msrA -m3, DsrO/ msrA _com strain with the 1st mutated base, DsrO/ msrA _m4, DsrO_com strain with the 2nd mutated base; Rosup is a reagent that acts as a positive control. Asterisks indicate statistically significant differences compared to untreated cells. One-way ANOVA and Dunnett’s multiple-comparison test; ***, P ≤ 0.001; *, P ≤ 0.05. Experiments were performed at least three times, and the data are presented as the means ± SEM.

Article Snippet: The primary polyclonal antibody against MsrA was produced by Genscript Biotechnology Company based on the MsrA protein sequence (Genscript, Nanjing, China).

Techniques: Mutagenesis, Binding Assay, Plasmid Preparation, Positive Control, Comparison

Journal: Applied and Environmental Microbiology

Article Title: A Novel Small RNA, DsrO, in Deinococcus radiodurans Promotes Methionine Sulfoxide Reductase ( msrA ) Expression for Oxidative Stress Adaptation

doi: 10.1128/aem.00038-22

Figure Lengend Snippet: DsrO promotes the stability of msrA mRNA and enhances MsrA protein translation. (A) Time of msrA mRNA reaching its half-life in different strains. (B) Western blot of MsrA in different strains; bands of target protein (MsrA) and reference protein (GroEL) are indicated by arrows. WT, wild-type strain; ΔDsrO, DsrO deleted mutant; DsrO -pRADZ3 , the DsrO mutant transformed with pRADZ3 empty plasmid; DsrO-com, DsrO mutant supplemented with DsrO gene. (C) Intracellular carbonylation level and the percentage reduction of dabsyl-MetSO peak area in different samples. (D) The contents of intracellular Met-SO in DsrO mutants. One-way ANOVA and Dunnett’s multiple-comparison test; ***, P ≤ 0.001; *, P ≤ 0.05.

Article Snippet: The primary polyclonal antibody against MsrA was produced by Genscript Biotechnology Company based on the MsrA protein sequence (Genscript, Nanjing, China).

Techniques: Western Blot, Mutagenesis, Transformation Assay, Plasmid Preparation, Comparison

Journal: Applied and Environmental Microbiology

Article Title: A Novel Small RNA, DsrO, in Deinococcus radiodurans Promotes Methionine Sulfoxide Reductase ( msrA ) Expression for Oxidative Stress Adaptation

doi: 10.1128/aem.00038-22

Figure Lengend Snippet: DrRRA is the transcriptional regulator of the msrA gene in D. radiodurans . (A) Electrophoretic mobility shift assay of DrRRA binding to the Cy5-labeled msrA promoter specifically. (Lane 1 [left] and Lane 2 [right]) Detection result of dr_0997 promoter; lane 1 is the band of the single labeled dr_0997 probe; lane 2 is the result of the labeled dr_0997 probe with 6 μg DrRRA protein. Lanes 3 to 9) Detection result of msrA promoter; (lane 3) DrRRA protein; (lanes 4 to 8 reaction system contained 40 ng labeled msrA probe and 0 μg, 3 μg, 6 μg, 9 μg, and 12 μg DrRRA protein. (Lane 9) Reaction system containing 40 ng labeled msrA probe, 500 ng unlabeled msrA probe, and 6 μg DrRRA protein. (B) The deletion of the DrRRA gene in D. radiodurans resulted from the induction decrease of msrA expression activated by the oxidative stress. (C) H 2 O 2 sensitivity assays of different DrRRA strains. Spotted agar plates after H 2 O 2 treatment and serial dilution. Different strains were treated with 0 to 100 mM H 2 O 2 for 30 min and dotted in the plates. WT, wild-type strain, Δ drRRA , drRRA deleted mutant. One-way ANOVA, Dunnett’s multiple-comparison test; ***, P ≤ 0.001; *, P ≤ 0.05. Experiments were performed at least three times, and data are presented as means ± SEM.

Article Snippet: The primary polyclonal antibody against MsrA was produced by Genscript Biotechnology Company based on the MsrA protein sequence (Genscript, Nanjing, China).

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Labeling, Expressing, Serial Dilution, Mutagenesis, Comparison

Journal: Applied and Environmental Microbiology

Article Title: A Novel Small RNA, DsrO, in Deinococcus radiodurans Promotes Methionine Sulfoxide Reductase ( msrA ) Expression for Oxidative Stress Adaptation

doi: 10.1128/aem.00038-22

Figure Lengend Snippet: Diagram of DsrO posttranscriptionally regulating msrA in D. radiodurans . (i) D. radiodurans cells produce ROS under the treatment of exogenous H 2 O 2 or oxidative stresses. ROS oxidize the methionine residue of proteins into methionine sulfoxide, inactivating the protein or causing cell death. (ii) When exogenous H 2 O 2 enters the D. radiodurans cell, the sRNA DsrO and msrA are rapidly induced. At the same time, the transcription factor DrRRA upregulates the expression of msrA at the transcriptional level. (iii) DsrO binds to the stem-ring structure of msrA mRNA and thus enhances the stability of msrA mRNA and improves MsrA expression at the translational level. (iv) The increased MsrA reduces the methionine sulfoxide on the protein to methionine to restore protein function, thus promoting the oxidation resistance of D. radiodurans . The large gray-green circle represents the 50s large subunit of the ribosome, the small circle represents the 30 s small subunit of the ribosome, the blue stem-loop represents sRNA DsrO, the light purple stem-loop represents msrA mRNA, the gray fold represents DrRRA protein, and the bright purple fold represents MsrA protein. The dark blue folds represent proteins in the cell, and the rose dots above represent Met residues on the proteins.

Article Snippet: The primary polyclonal antibody against MsrA was produced by Genscript Biotechnology Company based on the MsrA protein sequence (Genscript, Nanjing, China).

Techniques: Residue, Expressing

Journal: Applied and Environmental Microbiology

Article Title: A Novel Small RNA, DsrO, in Deinococcus radiodurans Promotes Methionine Sulfoxide Reductase ( msrA ) Expression for Oxidative Stress Adaptation

doi: 10.1128/aem.00038-22

Figure Lengend Snippet: Primers used in this study

Article Snippet: The primary polyclonal antibody against MsrA was produced by Genscript Biotechnology Company based on the MsrA protein sequence (Genscript, Nanjing, China).

Techniques: Sequencing, Construct, Expressing