Journal: Frontiers in endocrinology

Article Title: Melatonin prevents cyclophosphamide-induced primordial follicle loss by inhibiting ovarian granulosa cell apoptosis and maintaining AMH expression.

doi: 10.3389/fendo.2022.895095

Figure Lengend Snippet: FIGURE 4 Cyc induced apoptosis of granulosa cells in growing follicles and Mel prevented this apoptosis. (A) Three-week-old ICR mice were injected i.p. with Cyc 12 or 24 h after injection of PMSG, and apoptosis in ovarian tissues was detected by TUNEL kit at 8, 12, 24, and 36 h after Cyc injection. Regardless of whether Cyc was injected 12 or 24 h after PMSG injection, apoptotic peak of granulosa cells in ovary appeared 12 h after Cyc intervention, which was not related to the time of PMSG administration. Mice in control group were injected with Sal instead of Cyc, and the apoptotic peak of granulosa cells appeared 48 h (PMSG 12 h + Sal 36 h, or PMSG 24 h + Sal 24 h) after injection of PMSG. DNase I treatment was used for positive control of TUNEL assay. There were four three-week-old female mice in each group; Bar = 200 mm. (B) TUNEL fluorescence intensity in ovaries of 3-week-old ICR mice 8, 12, 24 and 36 hours after Cyc injection. The mice received Cyc chemotherapy 12 hours after injection of PMSG. (C) TUNEL fluorescence intensity in ovaries of 3-week-old ICR mice 8, 12 and 24 hours after Cyc injection. The mice received Cyc chemotherapy 24 hours after injection of PMSG. (D) Mel prevented apoptosis of granulosa cells 12 h after Cyc chemotherapy. There were four three-week-old female mice in each group; Bar = 200 mm. (E) TUNEL fluorescence intensity in ovaries of 3- week-old ICR mice in Sal, Cyc, Mel and Mel+Cyc groups 12 hours after Cyc injection.

Article Snippet: After deparaffinization of three-week-old female mouse ovary slides, the TUNEL BrightGreen Apoptosis Detection Kit Vazyme Code (Vazyme A112-02) was used to perform TUNEL analysis according to the manufacturer’s instructions.

Techniques: Injection, TUNEL Assay, Control, Positive Control

Journal: Frontiers in endocrinology

Article Title: Melatonin prevents cyclophosphamide-induced primordial follicle loss by inhibiting ovarian granulosa cell apoptosis and maintaining AMH expression.

doi: 10.3389/fendo.2022.895095

Figure Lengend Snippet: FIGURE 5 Mel inhibits Cyc-induced apoptosis of granulosa cells through mitochondrial apoptosis pathway. (A) Western blot analysis was performed on cleaved-caspase3, caspase3, Bax, and Bcl-2 in the ovaries of three-week-old ICR mice 12 h after Cyc (75 mg/kg) treatment with or without Mel (n = 6). a,b,c Bars with different letters are significantly different in each group (P < 0.05). (B) Western blot analysis was performed on mitochondrial Cyt-c (mito Cyt-c) and cytoplasm Cyt-c (cyto Cyt-c) in the ovaries of three-week-old ICR mice 12 h after Cyc (75 mg/kg) treatment with or without Mel (n = 6). *P < 0.05 compared with Sal group. #P < 0.05 compared with Cyc group.

Article Snippet: After deparaffinization of three-week-old female mouse ovary slides, the TUNEL BrightGreen Apoptosis Detection Kit Vazyme Code (Vazyme A112-02) was used to perform TUNEL analysis according to the manufacturer’s instructions.

Techniques: Western Blot

Journal: Frontiers in endocrinology

Article Title: Melatonin prevents cyclophosphamide-induced primordial follicle loss by inhibiting ovarian granulosa cell apoptosis and maintaining AMH expression.

doi: 10.3389/fendo.2022.895095

Figure Lengend Snippet: FIGURE 6 Mel prevents Cyc-induced over-activation of primordial follicles through inhibiting ovarian granulosa cell apoptosis and maintaining AMH expression. During normal follicle development, very few primordial follicles are selected and activated, whereas the vast majority of primordial follicles are maintained in a dormant state. AMH is mainly produced by granulosa cells in secondary follicles and early antral follicles. AMH can inhibit the activation of primordial follicles during recruitment and inhibit the sensitivity of antral follicles to FSH during recruitment cycle, thus maintaining AMH levels and selective activation of primordial follicle pool. Cyc exposure disturbs the balance of follicle activation by increasing apoptosis of AMH-secreting granulosa cells in growing follicles, resulting in further decrease of AMH levels and indirect over-activation of primordial follicle pool. Eventually, activated follicles undergo apoptosis and more primordial follicles are stimulated to become activated primary follicles, resulting in POF. With Mel co-treatment, activation of primordial follicles is reduced and apoptosis of AMH-secreting granulosa cells in growing follicles is decreased. Surviving granulosa cells maintained normal levels of AMH production, which regulates natural recruitment of primordial follicles and oogenesis. Hence, Mel restores the balance of follicle activation and returns ovaries to a healthy state.

Article Snippet: After deparaffinization of three-week-old female mouse ovary slides, the TUNEL BrightGreen Apoptosis Detection Kit Vazyme Code (Vazyme A112-02) was used to perform TUNEL analysis according to the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Produced

Journal: Molecular Oncology

Article Title: Characterization of PHGDH expression in bladder cancer: potential targeting therapy with gemcitabine/cisplatin and the contribution of promoter DNA hypomethylation

doi: 10.1002/1878-0261.12697

Figure Lengend Snippet: PHGDH inhibition by si‐RNA and inhibitor. (A) Immunoblotting analysis showed that PHGDH expression was elevated in all BC cells compared to breast cancer cells. PHGDH expression values normalized by β‐actin were indicated. (B) Cell proliferation after treatment with PHGDH si‐RNA (* P < 0.01, ** P < 0.001). The Bonferroni‐adjusted Mann–Whitney U ‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD. (C) Apoptosis levels after treatment with PHGDH si‐RNA (* P < 0.02, ** P < 0.001). The representative quadrant figures of apoptosis assay determined by flow cytometry are shown. Early apoptotic cells can be seen in the bottom right quadrant and late are in the upper right (lower). The Bonferroni‐adjusted Mann–Whitney U ‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD. (D) Decreased Ki‐67 and increased cleaved caspase3 levels in si‐ PHGDH ‐transfected BC cells. PHGDH expression values normalized by β‐actin are indicated. Each experiment was carried out in triplicate.

Article Snippet: Following xenografting, apoptotic cells in the tumor fraction were detected by the TUNEL method with a MEBSTAIN Apoptosis Kit Direct (code 8445 MBL, Woburn, MA, USA) per the directions of the manufacturer.

Techniques: Inhibition, Western Blot, Expressing, MANN-WHITNEY, Apoptosis Assay, Flow Cytometry, Transfection

Journal: Molecular Oncology

Article Title: Characterization of PHGDH expression in bladder cancer: potential targeting therapy with gemcitabine/cisplatin and the contribution of promoter DNA hypomethylation

doi: 10.1002/1878-0261.12697

Figure Lengend Snippet: PHGDH inhibition by a PHGDH inhibitor. (A) Cell proliferation after treatment with a PHGDH inhibitor (CBR‐5884) (* P < 0.01, ** P < 0.001). The Bonferroni‐adjusted Mann–Whitney U ‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD. (B) Apoptosis after treatment with CBR‐5884 (* P < 0.0001). The representative quadrant figures of apoptosis assay determined by flow cytometry are shown. Early apoptotic cells can be seen in the bottom right quadrant and late are in the upper right (right). Each experiment was carried out in triplicate. The Bonferroni‐adjusted Mann–Whitney U ‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD.

Article Snippet: Following xenografting, apoptotic cells in the tumor fraction were detected by the TUNEL method with a MEBSTAIN Apoptosis Kit Direct (code 8445 MBL, Woburn, MA, USA) per the directions of the manufacturer.

Techniques: Inhibition, MANN-WHITNEY, Apoptosis Assay, Flow Cytometry

Journal: Animals : an open access journal from MDPI

Article Title: Mechanism of circRNA_4083 Circularization and Its Role in Regulating Cell Viability.

doi: 10.3390/ani15111527

Figure Lengend Snippet: Figure 3. Overexpression of CircRNA_4083 down-regulates the level of cell apoptosis and promotes cell proliferation. The control was transfected with the pcDNA3.1 empty vector, #1 was transfected with the plasmid containing the genomic region of circRNA_4083 and its upstream and downstream intronic sequences cloned into the pcDNA3.1 vector, and #2 to #4 were transfected with deletion mu- tants constructed based on the MSH3 expression plasmid. (A) Flow cytometry analysis of apoptosis in DF-1 cells. (B) Flow cytometry-based cell cycle profiling. (C) Statistical analysis of cell viability. (D) qRT-PCR detection of apoptosis-related gene Bcl2. (E) qRT-PCR detection of cell cycle-related gene Cyclin D1. Data are expressed as mean ± SEM (n = 3). Group differences were analyzed using the one-way ANOVA (multi-group comparisons). (* p < 0.05, **** p < 0.0001).

Article Snippet: Cell cycle distribution was assessed using the Cell Cycle and Apoptosis Analysis Kit (Beyotime, code no. C1052, Shanghai, China).

Techniques: Over Expression, Control, Transfection, Plasmid Preparation, Clone Assay, Construct, Expressing, Flow Cytometry, Quantitative RT-PCR

Journal: Animals : an open access journal from MDPI

Article Title: Mechanism of circRNA_4083 Circularization and Its Role in Regulating Cell Viability.

doi: 10.3390/ani15111527

Figure Lengend Snippet: Figure 4. The silencing of CircRNA_4083 can upregulate the level of cell apoptosis and inhibit cell proliferation. (A) The interference efficiency after transfection of different siRNA-circRNA_4083 in DF- 1 cells. (B) qRT-PCR analysis of MSH3 expression after transfection of different siRNA-circRNA_4083 in DF-1 cells. (C) Apoptosis of DF-1 cells after transfection with siRNA-circRNA_4083. (D) The cell cycle of DF-1 cells after transfection with siRNA-circRNA_4083. Data are expressed as mean ± SEM (n = 3). Group differences were analyzed using the one-way ANOVA (multi-group comparisons). (ns, p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Cell cycle distribution was assessed using the Cell Cycle and Apoptosis Analysis Kit (Beyotime, code no. C1052, Shanghai, China).

Techniques: Transfection, Quantitative RT-PCR, Expressing

Journal: Molecules

Article Title: The n -Butanol Extract Obtained from the Inner Bark of Tabebuia rosea (Bertol.) DC, Specioside, and Catalposide Induce Leukemia Cell Apoptosis in the Presence of Apicidin

doi: 10.3390/molecules29173986

Figure Lengend Snippet: Gene expression: BCL2 and BAX genes in THP-1 cells treated with the n -butanol extract at different concentrations and pretreated with APC for 24 h. ( a ) BCL2 expression. ( b ) BAX expression. ( c ) Relative expression of the BAX/BCL2 genes. * p ≤ 0.05. Kruskal–Wallis, Dunn’s post hoc test.

Article Snippet: The expression of the MAPK14 (gene encoding p38), BCL2 , and BAX genes was evaluated by qRT–PCR and quantified with the 2 −ΔΔCt method in the Applied Biosystems StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), which uses predesigned TaqMan Gene Expression Assays (codes Hs00608023_m1, Hs00608023_m1, and Hs00180269_m1, respectively) (Applied Biosystems, Foster City, CA, USA) and the TaqMan ® RNA-to-CT TM 1-Step Kit (Applied Biosystems, Foster City, CA, USA, Cat No. 4392653).

Techniques: Gene Expression, Expressing

Journal: Molecules

Article Title: The n -Butanol Extract Obtained from the Inner Bark of Tabebuia rosea (Bertol.) DC, Specioside, and Catalposide Induce Leukemia Cell Apoptosis in the Presence of Apicidin

doi: 10.3390/molecules29173986

Figure Lengend Snippet: Gene expression: BCL2 and BAX in THP-1 cells treated with catalposide (Cat) and pretreated with APC for 24 h. ( a ) BCL2 expression; ( b ) BAX expression; ( c ) relative expression of the BAX/BCL2 genes. * p ≤ 0.05, ** p ≤ 0.01. Kruskal–Wallis, Dunn’s post hoc test.

Article Snippet: The expression of the MAPK14 (gene encoding p38), BCL2 , and BAX genes was evaluated by qRT–PCR and quantified with the 2 −ΔΔCt method in the Applied Biosystems StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), which uses predesigned TaqMan Gene Expression Assays (codes Hs00608023_m1, Hs00608023_m1, and Hs00180269_m1, respectively) (Applied Biosystems, Foster City, CA, USA) and the TaqMan ® RNA-to-CT TM 1-Step Kit (Applied Biosystems, Foster City, CA, USA, Cat No. 4392653).

Techniques: Gene Expression, Expressing

Journal: Molecules

Article Title: The n -Butanol Extract Obtained from the Inner Bark of Tabebuia rosea (Bertol.) DC, Specioside, and Catalposide Induce Leukemia Cell Apoptosis in the Presence of Apicidin

doi: 10.3390/molecules29173986

Figure Lengend Snippet: Gene expression of BCL2 and BAX in THP-1 cells treated with specioside (Sp) and pretreated with APC for 24 h. ( a ) BCL2 expression; ( b ) BAX expression; ( c ) relative expression of the BAX / BCL2 genes. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 Kruskal–Wallis, Dunn’s post hoc test.

Article Snippet: The expression of the MAPK14 (gene encoding p38), BCL2 , and BAX genes was evaluated by qRT–PCR and quantified with the 2 −ΔΔCt method in the Applied Biosystems StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), which uses predesigned TaqMan Gene Expression Assays (codes Hs00608023_m1, Hs00608023_m1, and Hs00180269_m1, respectively) (Applied Biosystems, Foster City, CA, USA) and the TaqMan ® RNA-to-CT TM 1-Step Kit (Applied Biosystems, Foster City, CA, USA, Cat No. 4392653).

Techniques: Gene Expression, Expressing

Journal: Italian Journal of Food Science

Article Title: Vanillic acid promotes keratinocyte migration, proliferation, and angiogenesis

doi: 10.15586/ijfs.v36i4.2753

Figure Lengend Snippet: Figure 2. VA inhibits the apoptosis of keratinocytes. (A) Flow cytometry analysis of HaCaT cell apoptosis was performed using Annexin V-FITC/ PI staining at 24 h. Annexin V-/PI- represents healthy cells, Annexin V+/PI- represents early apoptotic cells, Annexin V+/PI+ represents late apoptotic cells, and Annexin V-/PI+ represents necrotic cells. (B). The expression of Bax, Bcl2, and cleaved Caspase-3 was detected by western blotting. β-actin was used as the reference protein. A semi-quantitative analysis of Bax, Bcl2, and cleaved Caspase-3 expression was performed using ImageJ software. **p < 0.01 vs. 0 μg/mL VA group.

Article Snippet: Apoptosis assay To assess apoptosis in HaCaT cells, we used the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Code No. C1062L).

Techniques: Flow Cytometry, Staining, Expressing, Western Blot, Software

Journal: Italian Journal of Food Science

Article Title: Vanillic acid promotes keratinocyte migration, proliferation, and angiogenesis

doi: 10.15586/ijfs.v36i4.2753

Figure Lengend Snippet: Figure 6. Inhibition of the AMPK pathway reverses the effects of VA on keratinocytes. (A) HaCaT cell proliferation was assessed using the MTT assay. Optical density (OD570) values were measured at 0, 24, 48, and 72 h. (B) PCNA expression was determined by western blotting at 24 h. (C) Flow cytometry analysis of HaCaT cell apoptosis performed using Annexin V-FITC/ PI staining at 24 h. (D) HaCaT cell migration was evaluated using an in vitro scratch assay, and width measurements were per formed using ImageJ. (E) Secretion levels of FGF, PDGF, and VEGF were assessed using ELISA kits. **p < 0.01 vs. 0 μg/mL VA group. ^^vs. 100 μg/mL VA group.

Article Snippet: Apoptosis assay To assess apoptosis in HaCaT cells, we used the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Code No. C1062L).

Techniques: Inhibition, MTT Assay, Expressing, Western Blot, Flow Cytometry, Staining, Migration, In Vitro, Wound Healing Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Hematology & Oncology

Article Title: The first-in-class alkylating deacetylase inhibitor molecule tinostamustine shows antitumor effects and is synergistic with radiotherapy in preclinical models of glioblastoma

doi: 10.1186/s13045-018-0576-6

Figure Lengend Snippet: Tinostamustine increases the effects of radiotherapy in glioma models in vitro (part II). a Table summarizing subG1 cell percentage measured with propidium iodide (PI) by using Tali™ instrument (Thermo Fisher Scientifics, Carlsbad, CA, USA) in U87MG, U251, A172, and T98G treated with RT with or without TINO. b Western blot performed on Bcl2 and Bax expression performed in A172 cells treated with RT (0–6 Gy) with or without TINO 3.5 μM. c Caspase 3 activity measured in U87MG, U251, A172, and T98G treated with RT (0–6 Gy) with or without TINO. d Annexin V/PI positive apoptotic cells performed by using Tali™ Apoptosis Kit - Annexin V Alexa Fluor™ 488 & Propidium Iodide. e Clonogenic assay performed with AT101 (Gossypol, 10.0 μM), Bcl2 inhibitor in U87MG, U251, A172, and T98G. f Table summarizing DRE values calculated in U87MG, U251, A172, and T98G treated with RT (0–6 Gy) with or without 10.0 μM AT101. g Table summarizing DRE values calculated in U87MG, U251, A172, and T98G treated with RT (0–6 Gy) with or without 10.0 μM AT101 and TINO 3.5 μM (triple combination). Single results are representative of three different experiments performed in triplicate

Article Snippet: APOSTRAND™ ELISA apoptosis detection kit (code BML-AK120-0001) and p62 ELISA kit (code ADI-900-212-0001) were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA).

Techniques: In Vitro, Western Blot, Expressing, Activity Assay, Clonogenic Assay

Journal: Journal of Hematology & Oncology

Article Title: The first-in-class alkylating deacetylase inhibitor molecule tinostamustine shows antitumor effects and is synergistic with radiotherapy in preclinical models of glioblastoma

doi: 10.1186/s13045-018-0576-6

Figure Lengend Snippet: Effects of TINO with or without RT in patient derived glioma stem-like cells. a Effects of RT (0, 2, 4, and 6 Gy) with or without TINO in BT12M cell model. b DRE values calculated for BT12M, BT48EF, BT50EF, and CSCs-5 patient-derived glioma stem-like cells. c , d Western blot and ELISA determinations indicating the shift from authophagy to apoptosis. e DNA ladder in representative BT12M cells. f Table summarizing subG1 cell percentage measured with propidium iodide (PI) by using Tali™ instrument (Thermo Fisher Scientifics, Carlsbad, CA, USA) in BT12M, BT48EF, BT50EF, and CSCs-5 patient-derived glioma stem-like cells. g Caspase 3 activity performed in BT12M, BT48EF, BT50EF, and CSCs-5 patient-derived glioma stem-like cells. h Annexin V/PI-positive apoptotic cells performed by using Tali™ Apoptosis Kit - Annexin V Alexa Fluor™ 488 & Propidium Iodide. Single results are representative of three different experiments performed in triplicate

Article Snippet: APOSTRAND™ ELISA apoptosis detection kit (code BML-AK120-0001) and p62 ELISA kit (code ADI-900-212-0001) were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA).

Techniques: Derivative Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay