Journal: Cell Death & Disease

Article Title: Artesunate improves venetoclax plus cytarabine AML cell targeting by regulating the Noxa/Bim/Mcl-1/p-Chk1 axis

doi: 10.1038/s41419-022-04810-z

Figure Lengend Snippet: A Five AML cell lines were treated with 0.05–0.8 μM cytarabine for 72 h. Cell growth inhibition was measured by counting cell number and compared to the untreated group. B THP-1 and MOLM-13 cells were treated with cytarabine at the indicated concentrations and times. Cell cycle distribution assessed by FACS after PI staining. C Apoptotic cells determined by FACS with staining of Annexin V/PI. D Basal levels of Mcl-1, Bcl-2, Bim, and Noxa in five AML cell lines. E Relative levels of the indicated proteins of THP-1 and MOLM-13 cells after treatment with cytarabine determined by Western blotting. F THP-1 cells were transfected with two pairs of MCL1 siRNAs for 24 h, then treated with 8 μM cytarabine for 24 h, and the levels of γ-H2A.X and p-Chk1 were determined by Western blotting. The column graphs are the mean ± SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001 comparing to the control group by t -test.

Article Snippet: NOXA (sc-37305), BIM (sc-29802), MCL1 (sc-35877)siRNA, and a control siRNA were purchased from Santa Cruz Biotechnology, Inc. NOXA (s10708), BIM (s195011), MCL1 (s8583)siRNA was purchased from Thermo Fisher Scientific.

Techniques: Inhibition, Staining, Western Blot, Transfection, Control

Journal: Cell Death & Disease

Article Title: Artesunate improves venetoclax plus cytarabine AML cell targeting by regulating the Noxa/Bim/Mcl-1/p-Chk1 axis

doi: 10.1038/s41419-022-04810-z

Figure Lengend Snippet: A THP-1 and MOLM-13 cells were treated with artesunate and venetoclax at the indicated concentrations and times. Relative levels of the indicated proteins were determined by Western blotting. B THP-1 cells were pretreated with 25 μM Q-VD-OPh for 4 h, following by treatment with 1.6 μM artesunate and 0.1 μM venetoclax for 24 h. C THP-1 cells were treated with 1.6 μM artesunate and 0.1 μM venetoclax for 18 h, following by treatment with 10 μM MG132 for 6 h. D THP-1 cells were treated with 0.8 μM artesunate and 0.1 μM venetoclax for 24 h. MOLM-13 cells were treated with 0.2 μM artesunate and 0.01 μM venetoclax for 12 h. Cell lysates were prepared and immunoprecipitated with anti-Bim or anti-Noxa antibody, and then probed for Bcl-2 and Mcl-1. E THP-1 cells treated with 1.6 μM artesunate, 0.1 μM venetoclax and in combination for 24 h. The activated Bak and Bax determined with the anti-Bak(Ab-1) or anti-Bax (6A7) antibody(detecting the active forms). F THP-1 cells were transfected with two pairs of NOXA and BIM siRNAs for 24 h, then treated with 1.6 μM artesunate and 0.1 μM venetoclax for 24 h. Apoptotic cells were quantified using FACS after staining with Annexin V-FITC. G Levels of PARP, Bcl-2, Mcl-1, Bim, and Noxa were measured by Western blotting. The column graphs are the mean ± SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to the control group by t -test. ## P < 0.01; ### P < 0.001 by two-way ANOVA test.

Article Snippet: NOXA (sc-37305), BIM (sc-29802), MCL1 (sc-35877)siRNA, and a control siRNA were purchased from Santa Cruz Biotechnology, Inc. NOXA (s10708), BIM (s195011), MCL1 (s8583)siRNA was purchased from Thermo Fisher Scientific.

Techniques: Western Blot, Immunoprecipitation, Transfection, Staining, Control

Journal: Cell Death & Disease

Article Title: Artesunate improves venetoclax plus cytarabine AML cell targeting by regulating the Noxa/Bim/Mcl-1/p-Chk1 axis

doi: 10.1038/s41419-022-04810-z

Figure Lengend Snippet: THP-1 cells were transfected with two pairs of NOXA and BIM siRNAs for 24 h, then treated with the combination of 0.8 μM artesunate, 0.1 μM venetoclax, and 8 μM cytarabine for 24 h. A Relative levels of the indicated proteins were determined by Western blotting. B Apoptotic cells were quantified using FACS after staining with Annexin V-FITC. C Cell cycle distribution was assessed after PI staining by flow cytometry. Values are mean ± SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to the control group by t -test.

Article Snippet: NOXA (sc-37305), BIM (sc-29802), MCL1 (sc-35877)siRNA, and a control siRNA were purchased from Santa Cruz Biotechnology, Inc. NOXA (s10708), BIM (s195011), MCL1 (s8583)siRNA was purchased from Thermo Fisher Scientific.

Techniques: Transfection, Western Blot, Staining, Flow Cytometry, Control

Journal: Cell Death & Disease

Article Title: PTEN-regulated AKT/FoxO3a/Bim signaling contributes to reactive oxygen species-mediated apoptosis in selenite-treated colorectal cancer cells

doi: 10.1038/cddis.2013.3

Figure Lengend Snippet: Selenite-induced inhibition of AKT directly activated FoxO3a in CRC cells, an event closely correlated with apoptosis. ( a , b ) Inhibition of AKT with either LY294002 or AKT siRNA led to an inhibition of FoxO3a and increased apoptosis in selenite-treated HCT116 and SW480 CRC cells. Cells were treated with LY294002 for 1 h prior to selenite treatment or were transfected with AKT siRNA followed by treatment with either selenite or PBS for 24 h. Cells were then collected, and total cellular lysates were immunoblotted for p-AKT (Thr308) and p-Foxo3a (Ser253), Bim, cleaved PARP and cleaved caspase 9 and β -actin. ( c ) Activation of AKT protected cells from selenite-induced apoptosis and modulation of AKT/FoxO3a/Bim signaling. HCT116 and SW480 CRC cells were transfected with constitutively activated (c.a.) AKT constructs to restore AKT prior to selenite treatment for 24 h and were then subjected to western blot assays using antibodies against p-AKT, AKT, p-FoxO3a, Bim, cleaved PARP and cleaved caspase 9. β -Actin was used as a loading control. ( d , e ) The apoptotic rate of cells was determined by FACS in cells transfected with constitutively activated (c.a.) AKT constructs or AKT siRNA and treated with selenite. The results were calculated and are statistically represented as bar graphs. * P <0.05 indicates statistical significance between groups. ( f , g ) Foxo3a-mediated selenite-induced apoptosis downstream of AKT. Cells were transfected with FoxO3a siRNA prior to treatment with selenite for 24 h, after which time apoptosis was analyzed using western blotting or FACS

Article Snippet: AKT1 siRNA (5′-AAGGAGGGUUGGCUGCACAAA-3′); FOXO3a siRNA (5′-AAUGUGACA-UGGAGUCCAUUA-3′); Bim siRNA (5′-AAGGUAGACAAUUGCAGCCUG-3′); PTEN si RNA (5′-GACUUGAAGGCGUAUACAGtt-3′) and the control siRNA (5′-UUCUCCGAACGUGUCA-CGUTT3′) were chemically synthesized by GenePharm (Shanghai, China).

Techniques: Inhibition, Transfection, Activation Assay, Construct, Western Blot, Control

Journal: Cell Death & Disease

Article Title: PTEN-regulated AKT/FoxO3a/Bim signaling contributes to reactive oxygen species-mediated apoptosis in selenite-treated colorectal cancer cells

doi: 10.1038/cddis.2013.3

Figure Lengend Snippet: FoxO3a promoted the transcription and expression of bim, thereby enhancing apoptosis. ( a ) Selenite facilitated FoxO3a binding to the bim promoter. HCT116 and SW480 CRC cells were treated with or without selenite for 24 h followed by subjection to the ChIP assay. The relative band intensity of PCR products revealed the binding of FoxO3a to the bim promoter. ( b , c ) Selenite increased the transcription of bim in both HCT116 and SW480 CRC cells. Cells were treated with selenite for the indicated time periods followed by reverse transcription PCR and western blotting. ( d ) Selenite treatment caused the translocation of Bim from the cytoplasm to the mitochondria. HCT116 and SW480 CRC cells were treated with selenite for the indicated time periods, and the mitochondria were then isolated and immunoblotted for Bim. VDAC and β -actin were used as markers of the mitochondria and cytoplasm, respectively. ( e ) Colocalization results of Bim in selenite-treated HCT116 and SW480 CRC cells. Cells were treated with selenite for 24 h, and Mitotracker (red) solution was added to the medium to stain the mitochondria in cells. Cells were collected and stained with a Bim primary antibody and a FITC-conjugated secondary antibody (green). Nuclei are shown as blue signals. ( f , g ) Knockdown of Bim attenuated apoptosis in selenite-treated CRC cells. Bim siRNA was used to silence the expression of bim in HCT116 and SW480 CRC cells that subsequently were treated with selenite for 24 h. All groups of cells were analyzed using western blotting and FACS

Article Snippet: AKT1 siRNA (5′-AAGGAGGGUUGGCUGCACAAA-3′); FOXO3a siRNA (5′-AAUGUGACA-UGGAGUCCAUUA-3′); Bim siRNA (5′-AAGGUAGACAAUUGCAGCCUG-3′); PTEN si RNA (5′-GACUUGAAGGCGUAUACAGtt-3′) and the control siRNA (5′-UUCUCCGAACGUGUCA-CGUTT3′) were chemically synthesized by GenePharm (Shanghai, China).

Techniques: Expressing, Binding Assay, Reverse Transcription, Western Blot, Translocation Assay, Isolation, Staining, Knockdown

Journal: Cell Death & Disease

Article Title: PTEN-regulated AKT/FoxO3a/Bim signaling contributes to reactive oxygen species-mediated apoptosis in selenite-treated colorectal cancer cells

doi: 10.1038/cddis.2013.3

Figure Lengend Snippet: Selenite-induced PTEN modulated the AKT/FoxO3a/Bim signaling pathway. ( a ) Selenite treatment facilitated the binding of FoxO3a to the PTEN promoter. HCT116 and SW480 CRC cells were treated with selenite for 24 h and then were subjected to ChIP analysis. The binding ability of FoxO3a to the PTEN promoter was calculated and analyzed. ( b ) Selenite treatment enhanced the synthesis of PTEN mRNA through FoxO3a accumulation in the nucleus. HCT116 and SW480 CRC cells were treated for the indicated time periods with or without actinomycin D1 to inhibit new mRNA synthesis. Total cellular mRNA was then extracted and subjected to reverse transcription PCR. The PTEN mRNA level was determined and calculated from three independent experiments. ( c ) The expression of PTEN was enhanced in selenite-treated CRC cells. Western blotting was performed to determine the expression of PTEN in HCT116 and SW480 CRC cells after selenite treatment. ( d ) Selenite enhanced PTEN phosphatase activity in HCT116 and SW480 CRC cells. Twenty-four hours after selenite treatment, cells were collected, and PTEN phosphatase activity in each sample was determined as described in the Materials and Methods section. ( e , f ) PTEN perturbed the selenite-regulated AKT/FoxO3a signaling axis. Cells were transfected with phosphatase-dead PTEN (C124S) plasmids or PTEN siRNA to efficiently extinguish PTEN activity. Subsequently, cells were treated with or without selenite for 24 h, and then the expression levels of p-AKT (Thr308), p-FoxO3a (Ser253), FoxO3a, Bim, cleaved PARP and cleaved caspase 9 were detected using western blotting. β -Actin was used as a loading control. ( g ) Inhibition of PTEN abrogated the further inhibitory effect of PTEN on the AKT/FoxO3a/Bim signaling pathway. HCT116 and SW480 cells were treated with SF1670, a PTEN inhibitor, followed by selenite or PBS for 24 h. The altered expression patterns of p-AKT, AKT, p-FoxO3a, FoxO3a, cleaved PARP and cleaved caspase 9 were determined using western blotting. β -Actin was used as a control for equal loading

Article Snippet: AKT1 siRNA (5′-AAGGAGGGUUGGCUGCACAAA-3′); FOXO3a siRNA (5′-AAUGUGACA-UGGAGUCCAUUA-3′); Bim siRNA (5′-AAGGUAGACAAUUGCAGCCUG-3′); PTEN si RNA (5′-GACUUGAAGGCGUAUACAGtt-3′) and the control siRNA (5′-UUCUCCGAACGUGUCA-CGUTT3′) were chemically synthesized by GenePharm (Shanghai, China).

Techniques: Binding Assay, Reverse Transcription, Expressing, Western Blot, Activity Assay, Transfection, Control, Inhibition

Journal: Oncology Letters

Article Title: Regulation of the FOXO3a/Bim signaling pathway by 5,7-dihydroxy-8-nitrochrysin in MDA-MB-453 breast cancer cells

doi: 10.3892/ol.2012.1077

Figure Lengend Snippet: Effects of FOXO3a downregulation by siRNA transfection on FOXO3a expression and apoptosis in MDA-MB-453 cells. (A) MDA-MB-453 cells were transfected with 100 nM siRNA control or the siRNA duplexes against FOXO3a mRNA. Forty-eight hours after transfection, the cells were treated with 4.0 μ M NOC for 24 h. Western blotting of p-FOXO3a, FOXO3a, caspase-3 and caspase-9 was completed to confirm the downregulation of FOXO3a and the effects of caspase-3 and -9 cleavage by siRNA transfection. β-actin was used as the loading control. (B) MDA-MB-453 cells were transfected with 100 nM siRNA control or the siRNA duplexes against FOXO3a mRNA. Forty-eight hours after transfection, cells were treated with 8.0 μ M NOC for 24 h. The apoptotic rate was analyzed by FCM using PI staining. Data shown are means ± SD (n=3). * P<0.05; ** P<0.01 vs. 0.1% DMSO; # P<0.05 vs. the same concentration of NOC in combination with siRNA FOXO3a transfection. FOXO3a, forkhead box O3a; NOC, 7-dihydroxy-8-nitrochrysin; FCM, flow cytometry; PI, propidium iodide; SD, standard deviation; DMSO, dimethyl sulfoxide.

Article Snippet: FOXO3a siRNA (ACUCCGGGUCCAGCUCCAC) and Bim siRNA (5′-GATCCGT TCTGAGTGTGACCGAGA-3′) were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Transfection, Expressing, Control, Western Blot, Staining, Concentration Assay, Flow Cytometry, Standard Deviation

Journal: Oncology Letters

Article Title: Regulation of the FOXO3a/Bim signaling pathway by 5,7-dihydroxy-8-nitrochrysin in MDA-MB-453 breast cancer cells

doi: 10.3892/ol.2012.1077

Figure Lengend Snippet: Effects of NOC on the expression of Bcl-2 family proteins in MDA-MB-453 cells. (A) MDA-MB-453 cells were treated with the indicated concentrations of NOC or chrysin for 24 h. Expressions of Bax, PUMA, Noxa, Bim, Bcl-2 and Bcl-XL proteins were examined by western blotting of total cell lysates and β-actin was used as the loading control. (B) MDA-MB-453 cells were transfected with 100 nM siRNA control or the siRNA duplexes against FOXO3a mRNA. Forty-eight hours after transfection, cells were treated with 4.0 μ M NOC for 24 h. Expression of Bim protein was determined by western blotting of total cell lysates and β-actin was used as the loading control. (C) MDA-MB-453 cells were transfected with 100 nM siRNA control or the siRNA duplexes against Bim mRNA. Forty-eight hours after transfection, cells were treated with 4.0 μ M NOC for 24 h. Expression of Bim protein was determined by western blotting of total cell lysates and β-actin was used as the loading control. (D) MDA-MB-453 cells were transfected with 100 nM siRNA control or siRNA duplexes against Bim mRNA. Forty-eight hours after transfection, the cells were treated with 4.0 μ M NOC for 24 h. The apoptotic rate was analyzed by FCM using PI staining. Data shown are means ± SD (n=3). * P<0.05; ** P<0.01 vs. 0.1% DMSO; # P<0.05 vs. the same concentration of NOC in combination with siRNA Bim transfection. NOC, 7-dihydroxy-8-nitrochrysin; Bcl-2, B cell lymphoma 2; Bax, Bcl-2 associated X protein; PUMA, p53 upregulated modulator of apoptosis; FCM, flow cytometry; PI, propidium iodide; SD, standard deviation; DMSO, dimethyl sulfoxide.

Article Snippet: FOXO3a siRNA (ACUCCGGGUCCAGCUCCAC) and Bim siRNA (5′-GATCCGT TCTGAGTGTGACCGAGA-3′) were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Expressing, Western Blot, Control, Transfection, Staining, Concentration Assay, Flow Cytometry, Standard Deviation