Journal: PLoS ONE

Article Title: The Activation of ERK1/2 and JNK MAPK Signaling by Insulin/IGF-1 Is Responsible for the Development of Colon Cancer with Type 2 Diabetes Mellitus

doi: 10.1371/journal.pone.0149822

Figure Lengend Snippet: MC38 cells were cultured with insulin and IGF-1 alone or both together for 72 hours. ERK1/2 inhibitor PD98059 (B), JNK inhibitor SP600125 (C) or their vehicle DMSO added to the cultures when MC38 cells were treated with both insulin and IGF-1. (A) Cells were collected for proliferation analysis with CCK-8 assay at 24, 48 and 72 hour. **P<0.01; ***P<0.001 between the indicated two groups, n = 3 per group. (B and C) Western blotting analysis for p-ERK1/2, ERK1/2, p-JNK, JNK, Cyclin D1, Bcl2, Bax and Caspase3 protein expression in the treated MC38 cells. GAPDH served as a loading control. The blots shown are representative of three separate experiments.

Article Snippet: The primary antibodies of GAPDH (Cat#4695s), ERK1/2 (Cat#5174), p-ERK1/2 Thr202/Tyr204 (Cat#4370), JNK (Cat#9252), p-JNK Thr183/Tyr185 (Cat#9251), P38 (Cat#8690), p-P38 Thr180/Tyr182 (Cat#4511) and Caspase3 (Cat#9664) were from Cell Signaling Technology (Beverly, MA), Bcl-2 (Cat# sc-509), Bax (Cat# sc-20067) and Cyclin D1 (Cat# sc-753) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Cell Culture, CCK-8 Assay, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: The Activation of ERK1/2 and JNK MAPK Signaling by Insulin/IGF-1 Is Responsible for the Development of Colon Cancer with Type 2 Diabetes Mellitus

doi: 10.1371/journal.pone.0149822

Figure Lengend Snippet: 2 × 10 6 MC38 cells suspended in 0.1 ml of PBS were subcutaneously injected into the db/db and db/+ mice to initiate tumor growth in vivo . (A) Tumor size was measured every 3 days. *P<0.05; ***P<0.001. (B) The tumors were excised and weighted 3 weeks after cell injection. ***P< 0.001. A representative tumor mass from each group was shown in the inset (scale bar = 1cm). (C and E) Western blotting analysis of p-ERK1/2, ERK1/2, p-JNK, JNK, Cyclin D1, Bcl-2, Bax and Caspase3 protein expression in tumors. GAPDH served as a loading control. The blots shown are representative of three separate experiments. (D and F) Semi-quantitation for the expressions of ERK1/2 and pERK1/2, JNK and p-JNK, Cyclin D1, Bcl-2, Bax and Caspase3 protein. Fold changes were normalized by control groups. *P<0.05; **P<0.01 versus control, n = 3 per group.

Article Snippet: The primary antibodies of GAPDH (Cat#4695s), ERK1/2 (Cat#5174), p-ERK1/2 Thr202/Tyr204 (Cat#4370), JNK (Cat#9252), p-JNK Thr183/Tyr185 (Cat#9251), P38 (Cat#8690), p-P38 Thr180/Tyr182 (Cat#4511) and Caspase3 (Cat#9664) were from Cell Signaling Technology (Beverly, MA), Bcl-2 (Cat# sc-509), Bax (Cat# sc-20067) and Cyclin D1 (Cat# sc-753) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Injection, In Vivo, Western Blot, Expressing, Control, Quantitation Assay

Journal: PLoS ONE

Article Title: The Activation of ERK1/2 and JNK MAPK Signaling by Insulin/IGF-1 Is Responsible for the Development of Colon Cancer with Type 2 Diabetes Mellitus

doi: 10.1371/journal.pone.0149822

Figure Lengend Snippet: 2 × 10 6 MC38 cells suspended in 0.1 ml of PBS were subcutaneously injected into the db/db mice to initiate tumor growth in vivo . 10 mg/kg PD98059 or 30 mg/kg SP600125 was administered intraperitoneally every 3 days when tumor volume reached 100mm 3 . 1% DMSO was used as control treatment. (A) Tumor size was measured every 3 days. *P<0.05; ***P<0.001. (B) The tumors were excised and weighted 3 weeks after cell injection. ***P<0.001. A representative tumor mass from each group was shown in the inset (scale bar = 1cm). (C and E) Western blotting analysis of p-ERK1/2, ERK1/2, p-JNK, JNK, Cyclin D1, Bcl-2, Bax and Caspase3 protein expression in tumors. GAPDH served as a loading control. The blots shown are representative of three separate experiments. (D and F) Semi-quantitation for the expressions of ERK1/2 and pERK1/2, JNK and p-JNK, Cyclin D1, Bcl-2, Bax and Caspase3 protein. Fold changes were normalized by control groups. **P<0.01; ***P<0.001 versus control, n = 3 per group.

Article Snippet: The primary antibodies of GAPDH (Cat#4695s), ERK1/2 (Cat#5174), p-ERK1/2 Thr202/Tyr204 (Cat#4370), JNK (Cat#9252), p-JNK Thr183/Tyr185 (Cat#9251), P38 (Cat#8690), p-P38 Thr180/Tyr182 (Cat#4511) and Caspase3 (Cat#9664) were from Cell Signaling Technology (Beverly, MA), Bcl-2 (Cat# sc-509), Bax (Cat# sc-20067) and Cyclin D1 (Cat# sc-753) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Injection, In Vivo, Control, Western Blot, Expressing, Quantitation Assay

Journal: Cell Research

Article Title: The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression

doi: 10.1038/s41422-019-0263-3

Figure Lengend Snippet: a qRT-PCR analysis of GPX4, SLC7A11, and ACSL4 expression in A549 and H460 cell lines at 24 h after exposure to 6 Gy of IR. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. b Western blotting analysis of ACSL4, SLC7A11, and GPX4 expression in A549 and H460 cell lines at 2 h, 24 h, and 48 h after exposure to 6 Gy of IR or no radiation. c Western blotting analysis of ACSL4 expression in sg Control (sg C), sg ACSL4-1, sg ACSL4-2, and sg ACSL4-3 A549 and H460 cell lines. d Cell viability measurements in sg C, sg ACSL4-2, and sg ACSL4-3 A549 and H460 cell lines treated with or without 10 μM erastin for 24 h. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. e, f Lipid peroxidation assessment in sg C, sg ACSL4-2, and sg ACSL4-3 A549 (e) and H460 (f) cell lines at 24 h after exposure to 6 Gy of IR. Bar graph showing IR-induced relative fold changes of lipid peroxidation by C11-BODIPY staining in the indicated cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. g qRT-PCR analysis of PTGS2 expression in sg C, sg ACSL4-2, and sg ACSL4-3 A549 and H460 cell lines at 24 h after exposure to 6 Gy of IR. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. h Clonogenic survival assay in sg C, sg ACSL4-2, and sg ACSL4-3 A549 and H460 cell lines that were pretreated with 5 μM ferrostatin-1 or DMSO for 24 h followed by exposure to 6 Gy of IR. The survival data were normalized to those of unirradiated control cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test.

Article Snippet: Details for the reagents and resources in this experiment and others below are listed in Table . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Vinculin Sigma-Aldrich Cat#V4505 SLC7A11/xCT (D2M7A) Cell Signaling Technology Cat#12619 phospho-p53 (Ser15) Cell Signaling Technology Cat#9284 ACSL4 Santa Cruz Cat#sc-271800 KEAP1 Santa Cruz Cat#sc-365626 GPX4 R&D Systems Cat#MAB5457 Tubulin Cell Signaling Technology Cat#2144 phospho-Chk2 (Thr68) Cell Signaling Technology Cat#2197 phospho-histone H2A.X (Ser139) EMD Millipore Cat#05-636 Anti-4 Hydroxynonenal (4-HNE) Abcam Cat#ab46545 Ki-67 (D2H10) Cell Signaling Technology 9027S Cleaved Caspase-3 (Asp175) Cell Signaling Technology 9661s Chemicals Erastin Sigma Cat#E7781 Ferrostatin-1 Sigma Cat#SML0583 Liproxstatin-1 Sigma Cat#SML1414 N-acetyl-L-cysteine Sigma Cat#A9165 Sulfasalazine Sigma Cat#S0883 Z-VAD-fmk R&D Systems Cat#FMK001 Necrostatin-1s BioVision Cat#2263 RSL3 Selleckchem Cat#S8155 FIN56 Cayman Chemical Cat#25180 ML162 Cayman Chemical Cat#20455 CM-H2DCFDA ThermoFisher Cat#C6827 BODIPY 581/591 C11 Invitrogen Cat#D3861 Critical Commercial Assays Cell Counting Kit-8 (CCK8) reagent Dojindo Molecular Technologies Cat#CK04 Experimental Models: Cell Lines A549 ATCC Cat#CCL-185 H460 ATCC Cat#HTB-177 H1299 ATCC Cat#CRL-5803 H23 ATCC Cat#CRL-5800 MCF7 ATCC Cat#HTB-22 HT-1080 ATCC Cat#CCL-121 FLO-1 Dr. Steven H. Lin at MD Anderson Cancer Center UMRC6 Dr. William G. Kaelin at Dana-Farber Cancer Institute HEK293T ATCC Cat#CRL-11268 Experimental Models: Organisms/Strains Mouse: athymic nude: Foxn1nu/nu The facility in the Department of Experimental Radiation Oncology at MD Anderson Cancer Center NOD scid gamma (NSG) mice The facility in the Department of Experimental Radiation Oncology at MD Anderson Cancer Center Sequence-Based Reagents For primer and guide RNA sequences, please see Supplementary information, Table S 2 .

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Western Blot, Staining, Clonogenic Cell Survival Assay

Journal: Cell Research

Article Title: The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression

doi: 10.1038/s41422-019-0263-3

Figure Lengend Snippet: a Western blotting analysis showing SLC7A11 levels in H1299 and H23 cell lines with stable expression of empty vector (EV) and SLC7A11. b Lipid peroxidation assessment in EV- and SLC7A11-expressing H1299 or H23 cells at 24 h after exposure to 6 Gy or 2 Gy of IR, respectively. Bar graph showing the relative levels of lipid peroxidation by C11-BODIPY staining in the indicated cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. c qRT-PCR analysis of PTGS2 expression in EV- and SLC7A11-expressing H1299 or H23 cells at 24 h after exposure to 6 Gy or 2 Gy of IR, respectively. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. d Representative images of clonogenic survival assays in EV- and SLC7A11-expressing H1299 or H23 cells that were pretreated with 5 μM ferrostatin-1 or DMSO for 24 h followed by exposure to 6 Gy or 2 Gy of IR, respectively. e The quantified clonogenic survival assay in EV- and SLC7A11-expressing H1299 or H23 cells that were pretreated with 5 μM ferrostatin-1 or DMSO for 24 h followed by exposure to IR at a dose of 6 Gy or 2 Gy, respectively. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. f The quantified clonogenic survival curve in EV- and SLC7A11-expressing H1299 cells that were pretreated with 5 μM ferrostatin-1 or DMSO for 24 h followed by exposure to IR at the indicated doses. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using multiple t-test. g Western blotting analysis of KEAP1, SLC7A11 and ACSL4 expression in sg Control (sg C), sg KEAP1-2, and sg KEAP1-2-sg SLC7A11 (sg SLC) H1299 cell lines. h, i Lipid peroxidation assessment in sg C, sg KEAP1-2, and sg KEAP1-2-sg SLC H1299 cells at 24 h after exposure to 6 Gy of IR h. Bar graph showing IR-induced relative fold changes of lipid peroxidation by C11-BODIPY staining in the indicated cells (i). Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. j qRT-PCR analysis of PTGS2 expression in sg C, sg KEAP1-2, and sg KEAP1-2-sg SLC H1299 cells at 24 h after exposure to 6 Gy of IR. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. k Clonogenic survival assays in sg C, sg KEAP1-2, and sg KEAP1-2-sg SLC H1299 cells that were pretreated with 5 μM ferrostatin-1 or DMSO for 24 h followed by exposure to 6 Gy of IR. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test.

Article Snippet: Details for the reagents and resources in this experiment and others below are listed in Table . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Vinculin Sigma-Aldrich Cat#V4505 SLC7A11/xCT (D2M7A) Cell Signaling Technology Cat#12619 phospho-p53 (Ser15) Cell Signaling Technology Cat#9284 ACSL4 Santa Cruz Cat#sc-271800 KEAP1 Santa Cruz Cat#sc-365626 GPX4 R&D Systems Cat#MAB5457 Tubulin Cell Signaling Technology Cat#2144 phospho-Chk2 (Thr68) Cell Signaling Technology Cat#2197 phospho-histone H2A.X (Ser139) EMD Millipore Cat#05-636 Anti-4 Hydroxynonenal (4-HNE) Abcam Cat#ab46545 Ki-67 (D2H10) Cell Signaling Technology 9027S Cleaved Caspase-3 (Asp175) Cell Signaling Technology 9661s Chemicals Erastin Sigma Cat#E7781 Ferrostatin-1 Sigma Cat#SML0583 Liproxstatin-1 Sigma Cat#SML1414 N-acetyl-L-cysteine Sigma Cat#A9165 Sulfasalazine Sigma Cat#S0883 Z-VAD-fmk R&D Systems Cat#FMK001 Necrostatin-1s BioVision Cat#2263 RSL3 Selleckchem Cat#S8155 FIN56 Cayman Chemical Cat#25180 ML162 Cayman Chemical Cat#20455 CM-H2DCFDA ThermoFisher Cat#C6827 BODIPY 581/591 C11 Invitrogen Cat#D3861 Critical Commercial Assays Cell Counting Kit-8 (CCK8) reagent Dojindo Molecular Technologies Cat#CK04 Experimental Models: Cell Lines A549 ATCC Cat#CCL-185 H460 ATCC Cat#HTB-177 H1299 ATCC Cat#CRL-5803 H23 ATCC Cat#CRL-5800 MCF7 ATCC Cat#HTB-22 HT-1080 ATCC Cat#CCL-121 FLO-1 Dr. Steven H. Lin at MD Anderson Cancer Center UMRC6 Dr. William G. Kaelin at Dana-Farber Cancer Institute HEK293T ATCC Cat#CRL-11268 Experimental Models: Organisms/Strains Mouse: athymic nude: Foxn1nu/nu The facility in the Department of Experimental Radiation Oncology at MD Anderson Cancer Center NOD scid gamma (NSG) mice The facility in the Department of Experimental Radiation Oncology at MD Anderson Cancer Center Sequence-Based Reagents For primer and guide RNA sequences, please see Supplementary information, Table S 2 .

Techniques: Western Blot, Expressing, Plasmid Preparation, Staining, Two Tailed Test, Quantitative RT-PCR, Clonogenic Cell Survival Assay

Journal: Cell Research

Article Title: The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression

doi: 10.1038/s41422-019-0263-3

Figure Lengend Snippet: Reagents and resources.

Article Snippet: Details for the reagents and resources in this experiment and others below are listed in Table . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Vinculin Sigma-Aldrich Cat#V4505 SLC7A11/xCT (D2M7A) Cell Signaling Technology Cat#12619 phospho-p53 (Ser15) Cell Signaling Technology Cat#9284 ACSL4 Santa Cruz Cat#sc-271800 KEAP1 Santa Cruz Cat#sc-365626 GPX4 R&D Systems Cat#MAB5457 Tubulin Cell Signaling Technology Cat#2144 phospho-Chk2 (Thr68) Cell Signaling Technology Cat#2197 phospho-histone H2A.X (Ser139) EMD Millipore Cat#05-636 Anti-4 Hydroxynonenal (4-HNE) Abcam Cat#ab46545 Ki-67 (D2H10) Cell Signaling Technology 9027S Cleaved Caspase-3 (Asp175) Cell Signaling Technology 9661s Chemicals Erastin Sigma Cat#E7781 Ferrostatin-1 Sigma Cat#SML0583 Liproxstatin-1 Sigma Cat#SML1414 N-acetyl-L-cysteine Sigma Cat#A9165 Sulfasalazine Sigma Cat#S0883 Z-VAD-fmk R&D Systems Cat#FMK001 Necrostatin-1s BioVision Cat#2263 RSL3 Selleckchem Cat#S8155 FIN56 Cayman Chemical Cat#25180 ML162 Cayman Chemical Cat#20455 CM-H2DCFDA ThermoFisher Cat#C6827 BODIPY 581/591 C11 Invitrogen Cat#D3861 Critical Commercial Assays Cell Counting Kit-8 (CCK8) reagent Dojindo Molecular Technologies Cat#CK04 Experimental Models: Cell Lines A549 ATCC Cat#CCL-185 H460 ATCC Cat#HTB-177 H1299 ATCC Cat#CRL-5803 H23 ATCC Cat#CRL-5800 MCF7 ATCC Cat#HTB-22 HT-1080 ATCC Cat#CCL-121 FLO-1 Dr. Steven H. Lin at MD Anderson Cancer Center UMRC6 Dr. William G. Kaelin at Dana-Farber Cancer Institute HEK293T ATCC Cat#CRL-11268 Experimental Models: Organisms/Strains Mouse: athymic nude: Foxn1nu/nu The facility in the Department of Experimental Radiation Oncology at MD Anderson Cancer Center NOD scid gamma (NSG) mice The facility in the Department of Experimental Radiation Oncology at MD Anderson Cancer Center Sequence-Based Reagents For primer and guide RNA sequences, please see Supplementary information, Table S 2 .

Techniques: CCK-8 Assay, Sequencing, Software

Journal: Experimental and therapeutic medicine

Article Title: PHLDA3 inhibition attenuates endoplasmic reticulum stress-induced apoptosis in myocardial hypoxia/reoxygenation injury by activating the PI3K/AKT signaling pathway.

doi: 10.3892/etm.2021.10045

Figure Lengend Snippet: Figure 4. Blockage of the p‑PI3K/AKT pathway inhibits the protective effects of PHLDA3 inhibition following H/R injury. (A) Cell Counting Kit‑8 assays were used to evaluate cell viability. (B) LDH release was detected by ELISA. (C) Apoptotic rate in each group. mRNA levels of ERS markers, including (D) CHOP, (E) GRP78 and (F) caspase‑12, were measured by reverse transcription‑quantitative PCR. Data are presented as the mean ± standard deviation. n=4/group. *P<0.05 vs. H/R + AdshPHLDA3 + PBS. #P<0.05 vs. H/R + AdshRNA + PBS. PHLDA3, pleckstrin homology‑like domain family A member 3; H/R, hypoxia/ reoxygenation; LDF, lactate dehydrogenase; GRP78, 78 kDa glucose‑regulated protein; AdshRNA, adenovirus encoding scrambled short hairpin RNA; AdshPHLDA3, adenoviral vectors encoding PHLDA3 shRNA; NS, not significant.

Article Snippet: Antibodies against p‐AKT (cat. no. 4060; 1:600), AKT (cat. no. 4691; 1:400) and CHOP (cat. no. 5554; 1:600) were purchased from Cell Signaling Technology, Inc. Horseradish peroxidase‐conjugated goat‐anti rabbit secondary antibodies were obtained from BIOSS.

Techniques: Inhibition, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, shRNA

Journal: Aging (Albany NY)

Article Title: Betulinic acid induces autophagy-dependent apoptosis via Bmi-1/ROS/AMPK-mTOR-ULK1 axis in human bladder cancer cells

doi: 10.18632/aging.203441

Figure Lengend Snippet: BA induces autophagy in human bladder cancer cells. ( A ) Confocal fluorescence microscopy of EJ and T24 cells transfected with mCherry-GFP-LC3B lentivirus and exposed to 25 μM BA for 24 h. ( B ) Transmission electron microscopy (magnification: 5000× and 20,000×) images of EJ and T24 cells exposed to 25 μM BA for 24 h. Arrows indicate autophagic vesicles. ( C ) Luminescent determination of ADP/ATP ratio in EJ and T24 cells exposed to DMSO (control) or BA for 24 h. ( D , E ) Western blot analysis of changes in AMPK-mTOR-ULK1 phosphorylation status and p62 and LC3B-II expression upon treatment with the specified BA doses. * p <0.05, ** p <0.01.

Article Snippet: Antibodies against p-ULK1 (Ser555; Cat. 5869), cleaved poly (ADP-ribose) polymerase (PARP) (Cat. 9541), p-AMPKα (Cat. 2535), SQSTM1/p62 (Cat. 8025), and cleaved caspase 3 (Cat. 9664) were procured from Cell Signaling Technology, USA.

Techniques: Fluorescence, Microscopy, Transfection, Transmission Assay, Electron Microscopy, Control, Western Blot, Phospho-proteomics, Expressing

Journal: Aging (Albany NY)

Article Title: Betulinic acid induces autophagy-dependent apoptosis via Bmi-1/ROS/AMPK-mTOR-ULK1 axis in human bladder cancer cells

doi: 10.18632/aging.203441

Figure Lengend Snippet: BA-mediated ROS generation induces caspase-dependent apoptosis and autophagy in bladder cancer cells. ( A ) Flow cytometry analysis of ROS generation in DCFH-DA-loaded EJ and T24 cells exposed to BA at 0, 10, and 25 μM for 24 h. ( B ) Flow cytometry analysis of ROS generation in EJ and T24 cells pre-exposed to NAC. ( C ) Flow cytometry analysis of apoptosis. ( D ) The CCK-8 assay was employed to evaluate NAC effect on BA-triggered cytotoxicity. ( E ) Western blot analysis of p-AMPKα (Thr172), cleaved PARP, p-mTOR (Ser2448), p-ULK1 (Ser555), Bcl-2, p62, and LC3B II levels. * p <0.05, ** p <0.01.

Article Snippet: Antibodies against p-ULK1 (Ser555; Cat. 5869), cleaved poly (ADP-ribose) polymerase (PARP) (Cat. 9541), p-AMPKα (Cat. 2535), SQSTM1/p62 (Cat. 8025), and cleaved caspase 3 (Cat. 9664) were procured from Cell Signaling Technology, USA.

Techniques: Flow Cytometry, CCK-8 Assay, Western Blot

Journal: Aging (Albany NY)

Article Title: Betulinic acid induces autophagy-dependent apoptosis via Bmi-1/ROS/AMPK-mTOR-ULK1 axis in human bladder cancer cells

doi: 10.18632/aging.203441

Figure Lengend Snippet: BA-triggered autophagy-dependent apoptosis requires activation of the AMPK-mTOR-ULK1 axis. Cell viability and western blot assay results for EJ and T24 cells pre-exposed to 10 μM dorsomorphin (ComC) ( A ) or 500 μM AICAR ( B ) for 4 h and incubated with DMSO (control) or 25 μM BA for another 24 h. The CCK-8 assay was used for analysis of cell viability. Western blot was employed to determine the expression of cleaved PARP, p-AMPKα (Thr172), p-mTOR (Ser2448), p-ULK1 (Ser555), Bcl-2, cleaved caspase-3, and LC3B-II. * p <0.05, ** p <0.01.

Article Snippet: Antibodies against p-ULK1 (Ser555; Cat. 5869), cleaved poly (ADP-ribose) polymerase (PARP) (Cat. 9541), p-AMPKα (Cat. 2535), SQSTM1/p62 (Cat. 8025), and cleaved caspase 3 (Cat. 9664) were procured from Cell Signaling Technology, USA.

Techniques: Activation Assay, Western Blot, Incubation, Control, CCK-8 Assay, Expressing

Journal: Aging (Albany NY)

Article Title: Betulinic acid induces autophagy-dependent apoptosis via Bmi-1/ROS/AMPK-mTOR-ULK1 axis in human bladder cancer cells

doi: 10.18632/aging.203441

Figure Lengend Snippet: Bmi-1 overexpression partially reverses BA-induced ROS overproduction, migration inhibition, and autophagy-dependent apoptosis. Stable overexpression of Bmi-1 was established in EJ and T24 cells (EJ-con/EJ-Bmi-1 and T24-con/T24-Bmi-1) via lentiviral transduction before exposure to BA. ( A ) RT-qPCR analysis of relative Bmi-1 expression in control and Bmi-1-transduced cells. ( B ) Assessment of intracellular ROS contents. ( C ) Wound healing assay results. ( D ) Transwell migration analysis. ( E ) Western blot analysis of p-AMPKα, p-mTOR, p-ULK1, Bcl-2, cleaved caspase-3, Bmi-1, LC3B-II, p62, and cleaved PARP expression. ( F ) Apoptosis determination by flow cytometry. * p <0.05, ** p <0.01.

Article Snippet: Antibodies against p-ULK1 (Ser555; Cat. 5869), cleaved poly (ADP-ribose) polymerase (PARP) (Cat. 9541), p-AMPKα (Cat. 2535), SQSTM1/p62 (Cat. 8025), and cleaved caspase 3 (Cat. 9664) were procured from Cell Signaling Technology, USA.

Techniques: Over Expression, Migration, Inhibition, Transduction, Quantitative RT-PCR, Expressing, Control, Wound Healing Assay, Western Blot, Flow Cytometry

Journal: International Journal of Molecular Medicine

Article Title: Irisin attenuates acute lung injury by suppressing the pyroptosis of alveolar macrophages

doi: 10.3892/ijmm.2023.5235

Figure Lengend Snippet: Primers used for the reverse transcription-quantitative polymerase chain reaction in the present study.

Article Snippet: The primary antibodies used for western blotting were as follows: NLRP3 (cat. no. D4D8T), pro-caspase-1 (cat. no. E2Z1C), cleaved caspase-1 (cat. no. Asp296), IL-1β (cat. no. 3A6) were purchased from Cell Signaling Technology, Inc.; GSDMD (cat. no. AF4012) was purchased from Affinity Biosciences; HSP90 (cat. no. A5027) was purchased from ABclonal Biotech Co., Ltd.; β-actin (cat. no. 20536-1-AP); was purchased from Proteintech Group, Inc.

Techniques: Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Irisin attenuates acute lung injury by suppressing the pyroptosis of alveolar macrophages

doi: 10.3892/ijmm.2023.5235

Figure Lengend Snippet: LPS-induced acute lung infection in mice (magnification, ×200). (A) FNDC5/irisin was expressed in damaged lung tissue following LPS (2 mg/kg) stimulation. The brown areas shown by the red arrows represent the cytoplasmic expression of FNDC5/irisin. (B) The level of irisin in the serum of mice. (C) The bands of NLRP3 and β-actin obtained using western blotting. (D) Protein expression of NLRP3 at different time periods of LPS (2 mg/kg) stimulation. Data are expressed as the mean ± standard deviation, n=6. * P<0.05 vs. Con group; # P<0.05 vs. 12 h group; & P<0.05 vs. 24 h group. LPS, lipopolysaccharide; Con, control; NLRP3, nucleotide-binding and oligomerization domain-like receptor protein 3; FNDC5, fibronectin type III repeat-containing protein.

Article Snippet: The primary antibodies used for western blotting were as follows: NLRP3 (cat. no. D4D8T), pro-caspase-1 (cat. no. E2Z1C), cleaved caspase-1 (cat. no. Asp296), IL-1β (cat. no. 3A6) were purchased from Cell Signaling Technology, Inc.; GSDMD (cat. no. AF4012) was purchased from Affinity Biosciences; HSP90 (cat. no. A5027) was purchased from ABclonal Biotech Co., Ltd.; β-actin (cat. no. 20536-1-AP); was purchased from Proteintech Group, Inc.

Techniques: Infection, Expressing, Western Blot, Standard Deviation, Control, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Irisin attenuates acute lung injury by suppressing the pyroptosis of alveolar macrophages

doi: 10.3892/ijmm.2023.5235

Figure Lengend Snippet: Irisin protects against LPS-induced cell damage. The data are expressed as mean ± SD, n=3. (A) MH-S cells were treated with various concentrations (0, 0.01, 0.1, 1, 10 and 100 µ g/ml) of LPS for 24 h and analyzed using CCK-8 assay. * P<0.05 vs. 0 µ g/ml group. (B) CCK-8 assay results of LPS (10 µ g/ml)-treated MH-S cells measured at different time points. * P<0.05 vs. 2 h group. (C and D) CCK-8 assay to detect the cytotoxicity of irisin (0, 100, 200 and 400 ng/ml) and 17-AAG (0, 50, 100 and 200 ng/ml) in MH-S cells. (E and F) Quantitative analysis of NLRP3 protein expression at different time points of LPS stimulation (10 µ g/ml). * P<0.05 vs. Con group; # P<0.05 vs. 2 h group; & P<0.05 vs. 4 h group; ns, not significant vs. 4 h group. LPS, lipopolysaccharide; Con, control; 17-AAG, 17-N-allylamino-17-demethoxygeldanamycin.

Article Snippet: The primary antibodies used for western blotting were as follows: NLRP3 (cat. no. D4D8T), pro-caspase-1 (cat. no. E2Z1C), cleaved caspase-1 (cat. no. Asp296), IL-1β (cat. no. 3A6) were purchased from Cell Signaling Technology, Inc.; GSDMD (cat. no. AF4012) was purchased from Affinity Biosciences; HSP90 (cat. no. A5027) was purchased from ABclonal Biotech Co., Ltd.; β-actin (cat. no. 20536-1-AP); was purchased from Proteintech Group, Inc.

Techniques: CCK-8 Assay, Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: Irisin attenuates acute lung injury by suppressing the pyroptosis of alveolar macrophages

doi: 10.3892/ijmm.2023.5235

Figure Lengend Snippet: Irisin inhibits the release of pro-inflammatory cytokines. Mice were randomly assigned to the control, LPS (2 mg/kg), LPS + IR (LPS 2 mg/kg, irisin 0.5 mg/kg), and LPS + MCC950 (LPS 2 mg/kg, MCC950 50 mg/kg) groups (n=6). Following treatment, serum and BALF samples were collected. MH-S cells were co-cultured with LPS (10 µ g/ml) and IR (200 ng/ml) or 17-AAG (100 ng/ml) for 4 h (n=3). ELISA was used to detect IL-1β, IL-18, and TNF-α secretion in (A) BALF, (B) serum, and (C) cell supernatant samples. The data are expressed as the mean ± SD. * P<0.05 vs. Con group; # P<0.05 vs. LPS group; & P<0.05 vs. LPS + IR group; ns, not significant vs. LPS + IR group. LPS, lipopolysaccharide; Con, control; 17-AAG, 17-N-allylamino-17-demethoxygeldanamycin; IL, interleukin; TNF-α, tumor necrosis factor α.

Article Snippet: The primary antibodies used for western blotting were as follows: NLRP3 (cat. no. D4D8T), pro-caspase-1 (cat. no. E2Z1C), cleaved caspase-1 (cat. no. Asp296), IL-1β (cat. no. 3A6) were purchased from Cell Signaling Technology, Inc.; GSDMD (cat. no. AF4012) was purchased from Affinity Biosciences; HSP90 (cat. no. A5027) was purchased from ABclonal Biotech Co., Ltd.; β-actin (cat. no. 20536-1-AP); was purchased from Proteintech Group, Inc.

Techniques: Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Medicine

Article Title: Irisin attenuates acute lung injury by suppressing the pyroptosis of alveolar macrophages

doi: 10.3892/ijmm.2023.5235

Figure Lengend Snippet: Irisin inhibits the mRNA expression of pro-inflammatory cytokines. The grouping of mice and cells is the same as that in . Reverse transcription-quantitative PCR was used to detect the mRNA expression of IL-1β, IL-18 and TNF-α in (A-C) lung tissues and (D-F) MH-S cells. The data are expressed as the mean ± SD. * P<0.05 vs. Con group; # P<0.05 vs. LPS group; & P<0.05 vs. LPS + IR group; ns, not significant vs. LPS + IR group. LPS, lipopolysaccharide; Con, control; M, MCC950; IR, irisin; 17, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin).

Article Snippet: The primary antibodies used for western blotting were as follows: NLRP3 (cat. no. D4D8T), pro-caspase-1 (cat. no. E2Z1C), cleaved caspase-1 (cat. no. Asp296), IL-1β (cat. no. 3A6) were purchased from Cell Signaling Technology, Inc.; GSDMD (cat. no. AF4012) was purchased from Affinity Biosciences; HSP90 (cat. no. A5027) was purchased from ABclonal Biotech Co., Ltd.; β-actin (cat. no. 20536-1-AP); was purchased from Proteintech Group, Inc.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: International Journal of Molecular Medicine

Article Title: Irisin attenuates acute lung injury by suppressing the pyroptosis of alveolar macrophages

doi: 10.3892/ijmm.2023.5235

Figure Lengend Snippet: Irisin inhibits caspase-1 activity in vivo (n=6) and in vitro (n=3). Mice were randomly assigned to the control, LPS (2 mg/kg), and LPS + IR (LPS 2 mg/kg, irisin 0.5 mg/kg) groups. (A) FLIVO detected caspase-1 enzyme activity in mice. (B) Quantitative analysis of in vivo staining in mice. (C) The immunofluorescence staining of frozen sections of mouse lung tissue. The merged image represents the overlap of FLIVO- and DAPI-stained sections. Green color indicates FLIVO staining, blue color indicates DAPI staining. (D) Statistical analysis of caspase-1 fluorescence value in mice lung tissue. (E) FLICA detected caspase-1 enzyme activity in cells. (F) Statistical analysis of caspase-1 fluorescence value in vivo . The data are expressed as the mean ± SD. * P<0.05 vs. Con group; # P<0.05 vs. LPS group; ns, not significant vs. LPS + IR group. LPS, lipopolysaccharide; Con, control.

Article Snippet: The primary antibodies used for western blotting were as follows: NLRP3 (cat. no. D4D8T), pro-caspase-1 (cat. no. E2Z1C), cleaved caspase-1 (cat. no. Asp296), IL-1β (cat. no. 3A6) were purchased from Cell Signaling Technology, Inc.; GSDMD (cat. no. AF4012) was purchased from Affinity Biosciences; HSP90 (cat. no. A5027) was purchased from ABclonal Biotech Co., Ltd.; β-actin (cat. no. 20536-1-AP); was purchased from Proteintech Group, Inc.

Techniques: Activity Assay, In Vivo, In Vitro, Control, Staining, Immunofluorescence, Fluorescence

Journal: International Journal of Molecular Medicine

Article Title: Irisin attenuates acute lung injury by suppressing the pyroptosis of alveolar macrophages

doi: 10.3892/ijmm.2023.5235

Figure Lengend Snippet: Irisin inhibits caspase-1-mediated pyroptosis by regulating the HSP90/NLRP3 signaling pathway. The grouping of mice and cells is the same as in . (A and B) After drug treatment, proteins were extracted from (A) lung tissue and (B) MH-S cells and subjected to western blotting to determine the expression levels of NLRP3, HSP90, GSDMD, caspase-1, cleaved caspase-1 and IL-1β proteins. (C-N) Relative quantitative analysis of protein expression. The data are expressed as the mean ± SD. * P<0.05 vs. Con group; # P<0.05 vs. LPS group; & P<0.05 vs. LPS + IR group; ns, not significant vs. LPS + IR group. LPS, lipopolysaccharide; Con, control; NLRP3, nucleotide-binding and oligomerization domain-like receptor protein 3; HSP90, heat shock protein 90; GSDMD, gasdermin D; IL, interleukin; M, MCC950; IR, irisin; 17, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin).

Article Snippet: The primary antibodies used for western blotting were as follows: NLRP3 (cat. no. D4D8T), pro-caspase-1 (cat. no. E2Z1C), cleaved caspase-1 (cat. no. Asp296), IL-1β (cat. no. 3A6) were purchased from Cell Signaling Technology, Inc.; GSDMD (cat. no. AF4012) was purchased from Affinity Biosciences; HSP90 (cat. no. A5027) was purchased from ABclonal Biotech Co., Ltd.; β-actin (cat. no. 20536-1-AP); was purchased from Proteintech Group, Inc.

Techniques: Western Blot, Expressing, Control, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Irisin attenuates acute lung injury by suppressing the pyroptosis of alveolar macrophages

doi: 10.3892/ijmm.2023.5235

Figure Lengend Snippet: RT-qPCR analysis of (A and E) NLRP3, (B and F) HSP90, (C and G) GSDMD, and (D and H) Caspase-1 mRNA expression in vivo (n=6, A-D) and in vivo (n=3, E-H). The data are expressed as the mean ± SD. * P<0.05 vs. Con group; # P<0.05 vs. LPS group; & P<0.05 vs. LPS + IR group; ns, not significant vs. LPS + IR group. LPS, lipopolysaccharide; Con, control; M, MCC950; IR, irisin; 17, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin); GSDMD, gasdermin D; NLRP3, nucleotide-binding and oligomerization domain-like receptor protein 3; HSP90, heat shock protein 90.

Article Snippet: The primary antibodies used for western blotting were as follows: NLRP3 (cat. no. D4D8T), pro-caspase-1 (cat. no. E2Z1C), cleaved caspase-1 (cat. no. Asp296), IL-1β (cat. no. 3A6) were purchased from Cell Signaling Technology, Inc.; GSDMD (cat. no. AF4012) was purchased from Affinity Biosciences; HSP90 (cat. no. A5027) was purchased from ABclonal Biotech Co., Ltd.; β-actin (cat. no. 20536-1-AP); was purchased from Proteintech Group, Inc.

Techniques: Quantitative RT-PCR, Expressing, In Vivo, Control, Binding Assay

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Re regulates PFKFB3-mediated glycolysis to inhibit endothelial cell migration to ameliorate atherosclerosis

doi: 10.1016/j.jgr.2025.11.012

Figure Lengend Snippet: Re suppresses ox-LDL-induced HUVEC proliferation and migration. (A, B) Results of the CCK-8 assay, n = 6. (C) Representative images of HUVECs at 0 h and 12 h after ox-LDL induction in wound healing experiments, bar = 50 μm. (D) Quantification of EC migration in the wound healing assay, n = 3. (E) Western blot assay and quantitative data of VE-cadherin and VEGFR2 in HUVECs, n = 3. ### p < 0.001, ## p < 0.01, # p < 0.05, vs. control group, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, vs. ox-LDL group.

Article Snippet: Ginsenoside Re, (Cat. B21055 , Shanghai Yuanye Biotechnology Co., Ltd, purity≥ 98 %); Simvastatin tablets (Cat. 20210925, Shandong Xinqi Pharmaceutical Co., Ltd); PFKFB3 antibody (Cat. D7H4Q, Cell Signaling Technology, Inc.); HIF-1α Rabbit pAb (Cat. A11945, ABclonal Biotechnology Co., Ltd); HK2 polyclonal antibody (Cat. 22029-1-AP, Wuhan Sanying Biotechnology Co., Ltd); vascular endothelial cadherin (VE-cadherin) antibody (Cat. 2158S, Cell Signaling Technology, Inc.); VEGFA Rabbit mAb (Cat. ab214424, abcam); VEGFR2 Rabbit mAb (Cat. 2479, Cell Signaling Technology, Inc.).

Techniques: Migration, CCK-8 Assay, Wound Healing Assay, Western Blot, Control

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Re regulates PFKFB3-mediated glycolysis to inhibit endothelial cell migration to ameliorate atherosclerosis

doi: 10.1016/j.jgr.2025.11.012

Figure Lengend Snippet: Re inhibits endothelial cell migration via the PFKFB3-HIF-1α/VEGFA-VEGFR2 signaling pathways. (A) Western blot assay and quantitative analysis of PFKFB3 in HUVECs, n = 3. (B) Cell viability of PFKFB3 overexpressing ECs measured by CCK-8 assay, n = 6. (C) Western blot assay and quantitative analysis of PFKFB3 in HUVECs, n = 3. (D–E) Representative images of HUVEC induced by ox-LDL for 0 h and 12 h in wound healing experiments (bar = 50 μm) and quantification of EC migration, n = 3. (F, G) Western blot assay and quantitative data of VE-cadherin, HIF-1α, VEGFA, and VEGFR2 in HUVECs, n = 3. ### p < 0.001, ## p < 0.01, # p < 0.05, vs. control group; ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, vs. ox-LDL group.

Article Snippet: Ginsenoside Re, (Cat. B21055 , Shanghai Yuanye Biotechnology Co., Ltd, purity≥ 98 %); Simvastatin tablets (Cat. 20210925, Shandong Xinqi Pharmaceutical Co., Ltd); PFKFB3 antibody (Cat. D7H4Q, Cell Signaling Technology, Inc.); HIF-1α Rabbit pAb (Cat. A11945, ABclonal Biotechnology Co., Ltd); HK2 polyclonal antibody (Cat. 22029-1-AP, Wuhan Sanying Biotechnology Co., Ltd); vascular endothelial cadherin (VE-cadherin) antibody (Cat. 2158S, Cell Signaling Technology, Inc.); VEGFA Rabbit mAb (Cat. ab214424, abcam); VEGFR2 Rabbit mAb (Cat. 2479, Cell Signaling Technology, Inc.).

Techniques: Migration, Protein-Protein interactions, Western Blot, CCK-8 Assay, Control

Journal: Molecular Medicine Reports

Article Title: CDCA2 promotes tumorigenesis and induces radioresistance in oesophageal squamous cell carcinoma cells

doi: 10.3892/mmr.2021.12169

Figure Lengend Snippet: The expression of CDCA2 is upregulated in ESCC tissues. (A) Heatmap of the gene expression profiles of ESCC tissues and normal tissues basing on the data downloaded from TCGA. (B) Volcano map of the ESCC-related data extracted from TCGA. (C) The expression of CDCA2 in ESCC samples was significantly upregulated. ****P<0.0001. CDCA2, cell division cycle-associated 2; ESCC, oesophageal squamous cell carcinoma; TCGA, The Cancer Genome Atlas; FC, fold change; FDR, false discovery rate.

Article Snippet: The next day, HRP-linked anti-rabbit secondary antibodies (1:3,000; cat. no. 7074; Cell Signaling Technology, Inc.) were incubated at room temperature for 2 h. Western blotting analysis was conducted using a rabbit anti-CDCA2 monoclonal antibody (1:1,000; cat. no. 14976; Cell Signaling Technology, Inc.).

Techniques: Expressing, Gene Expression

Journal: Molecular Medicine Reports

Article Title: CDCA2 promotes tumorigenesis and induces radioresistance in oesophageal squamous cell carcinoma cells

doi: 10.3892/mmr.2021.12169

Figure Lengend Snippet: The expression of CDCA2 is upregulated in ESCC cell lines. (A) CDCA2 expression levels in the ESCC cell lines were detected by RT-qPCR. Mean ± standard deviation, n=3, **P<0.01. (B and C) Knockdown efficiency was verified by RT-qPCR in cells transfected with shCDCA2 lentivirus. Mean ± standard deviation, n=3, ***P<0.001. (D) CDCA2 expression levels in the ESCC cell lines as detected by western blot analysis. (E and F) Western blot analysis validated the transfection efficiency in cells transfected with shCDCA2 lentivirus. CDCA2, cell division cycle-associated 2; ESCC, oesophageal squamous cell carcinoma; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin.

Article Snippet: The next day, HRP-linked anti-rabbit secondary antibodies (1:3,000; cat. no. 7074; Cell Signaling Technology, Inc.) were incubated at room temperature for 2 h. Western blotting analysis was conducted using a rabbit anti-CDCA2 monoclonal antibody (1:1,000; cat. no. 14976; Cell Signaling Technology, Inc.).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Knockdown, Transfection, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: CDCA2 promotes tumorigenesis and induces radioresistance in oesophageal squamous cell carcinoma cells

doi: 10.3892/mmr.2021.12169

Figure Lengend Snippet: The knockdown of CDCA2 suppresses cell proliferation. CCK-8 assays were conducted to measure the rate of ESCC cell proliferation in (A) KYSE450/shCDCA2 and (B) TE13/shCDCA2 cells compared to that of the control groups. Mean ± standard deviation, n=3, **P<0.01, ***P<0.001. Typical images of the EdU incorporation assays and mean percentage of EdU positive cells in (C and D) KYSE450 cells and (E and F) TE13 cells with CDCA2 knockdown compared with the controls (blue fluorescence, cell nuclei; red fluorescence, EdU-positive cells) (magnification, ×40). Mean ± standard deviation, n=3, **P<0.01, ***P<0.001. CDCA2, cell division cycle-associated 2; ESCC, oesophageal squamous cell carcinoma; sh, short hairpin; OD, optical density; sh, short hairpin; NC, negative control; EdU, 5-Ethynyl-2′-deoxyuridine.

Article Snippet: The next day, HRP-linked anti-rabbit secondary antibodies (1:3,000; cat. no. 7074; Cell Signaling Technology, Inc.) were incubated at room temperature for 2 h. Western blotting analysis was conducted using a rabbit anti-CDCA2 monoclonal antibody (1:1,000; cat. no. 14976; Cell Signaling Technology, Inc.).

Techniques: Knockdown, CCK-8 Assay, Control, Standard Deviation, Fluorescence, Negative Control

Journal: Molecular Medicine Reports

Article Title: CDCA2 promotes tumorigenesis and induces radioresistance in oesophageal squamous cell carcinoma cells

doi: 10.3892/mmr.2021.12169

Figure Lengend Snippet: CDCA2 could regulate radiosensitivity in ESCC cells. The clonogenic survival assays was performed to compare the (A) KYSE450/shCDCA2 and (B) TE13/shCDCA2 cells to the control groups. The survival curves were calculated and fitted to a multi-target single-hit model. Mean ± standard deviation, n=3, **P<0.01. (C and E) Immunofluorescence staining of γH2AX. (D and F) The mean numbers of γH2AX foci were employed to evaluate radiation-induced double-strand break levels in ESCC cells (results from C and E). Mean ± standard deviation, n=3, *P<0.05, **P<0.01. CDCA2, cell division cycle-associated 2; ESCC, oesophageal squamous cell carcinoma; sh, short hairpin.

Article Snippet: The next day, HRP-linked anti-rabbit secondary antibodies (1:3,000; cat. no. 7074; Cell Signaling Technology, Inc.) were incubated at room temperature for 2 h. Western blotting analysis was conducted using a rabbit anti-CDCA2 monoclonal antibody (1:1,000; cat. no. 14976; Cell Signaling Technology, Inc.).

Techniques: Control, Standard Deviation, Immunofluorescence, Staining

Journal: Molecular Medicine Reports

Article Title: CDCA2 promotes tumorigenesis and induces radioresistance in oesophageal squamous cell carcinoma cells

doi: 10.3892/mmr.2021.12169

Figure Lengend Snippet: Inhibition of CDCA2 changes the cell cycle distribution in X ray-exposed ESCC cells. Cell cycle distribution of (A) KYSE450/shCDCA2 and (B) TE13/shCDCA2 cells. (C and D) Phase percentage with CDCA2 knockdown following radiation. (E and F) Downregulation of CDCA2 had a higher G 2 /M phase percentage. Mean ± standard deviation, n=3, **P<0.01, ***P<0.001, ****P<0.0001. CDCA2, cell division cycle-associated 2; ESCC, oesophageal squamous cell carcinoma; sh, short hairpin; NC, negative control; IR, ionising radiation; ns, not significant.

Article Snippet: The next day, HRP-linked anti-rabbit secondary antibodies (1:3,000; cat. no. 7074; Cell Signaling Technology, Inc.) were incubated at room temperature for 2 h. Western blotting analysis was conducted using a rabbit anti-CDCA2 monoclonal antibody (1:1,000; cat. no. 14976; Cell Signaling Technology, Inc.).

Techniques: Inhibition, Knockdown, Standard Deviation, Negative Control

Journal: Molecular Medicine Reports

Article Title: CDCA2 promotes tumorigenesis and induces radioresistance in oesophageal squamous cell carcinoma cells

doi: 10.3892/mmr.2021.12169

Figure Lengend Snippet: GSEA analysis of CDCA2. The knockdown of CDCA2 suppresses cell proliferation in vivo . (A and B) GSEA analysis showed that CDCA2 was associated with cell cycle phase transition pathways. (C) Representative xenografts each group were shown (n=3). (D) Tumour weight was recorded and presented as mean ± standard deviation. ***P<0.001, ****P<0.0001. GSEA, gene set enrichment analysis; CDCA2, cell division cycle-associated 2; sh, short hairpin; NC, negative control; IR, ionising radiation.

Article Snippet: The next day, HRP-linked anti-rabbit secondary antibodies (1:3,000; cat. no. 7074; Cell Signaling Technology, Inc.) were incubated at room temperature for 2 h. Western blotting analysis was conducted using a rabbit anti-CDCA2 monoclonal antibody (1:1,000; cat. no. 14976; Cell Signaling Technology, Inc.).

Techniques: Knockdown, In Vivo, Sublimation, Standard Deviation, Negative Control

Journal: Cell Death & Disease

Article Title: Upregulated PPARG2 facilitates interaction with demethylated AKAP12 gene promoter and suppresses proliferation in prostate cancer

doi: 10.1038/s41419-021-03820-7

Figure Lengend Snippet: A CCK-8 assay of the proliferation rates of recombinant PCA cell lines transfected with EV- and PPARG2-overexpression lentivirus vectors, respectively. * P < 0.01. B , C EDU assays were performed in EV and PPARG2-transfected PCA cells. * P < 0.05 vs. EV group. D , E Cell cycle profiles were examined by flow cytometry with propidium iodide (PI) staining; cell numbers were counted according to DNA content of G1, S, and G2 phases. * P < 0.05 vs. EV group. F – H Western blot analysis of cyclinD1, cyclinB1, p21 Cip1 and p27 Kip1 , Bcl-2, p-AKT, and AKT protein expressions in indicated cells. * P < 0.05 vs. EV group.

Article Snippet: The primary antibodies used were as follows: PPARG2 (1 : 800; catalog number sc-166731, Santa Cruz, CA), cyclinD1 (1 : 1000; catalog number sc-166731, Santa Cruz, CA), cyclinB1 (1 : 1000; catalog number 12231, Cell Signaling Technology, Inc.), p21 Cip1 (1 : 1000; catalog number 2947, Cell Signaling Technology, Inc.), p27 Kip1 (1 : 1000; catalog number 3686, Cell Signaling Technology, Inc.), Bcl-2 (1 : 1000; catalog number 4223, Cell Signaling Technology, Inc.), AKT (1 : 1000; catalog number SAB4500797, Sigma-Aldrich, Saint Louis, MO, USA), p-AKT (1 : 1000; catalog number 05–802 R, Sigma-Aldrich, Saint Louis, MO, USA), DNA methyltransferase 3A (DNMT3A (1 : 1000; catalog number 32578, Cell Signaling Technology, Inc.), and DNMT3B (1 : 1000; catalog number 72335, Cell Signaling Technology, Inc.). β-Actin (1 : 1000; catalog number 3700, Cell Signaling Technology, Inc.) was used as an internal reference.

Techniques: CCK-8 Assay, Recombinant, Transfection, Over Expression, Flow Cytometry, Staining, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Exosomes Derived From Hypoxia-Conditioned Stem Cells of Human Deciduous Exfoliated Teeth Enhance Angiogenesis via the Transfer of let-7f-5p and miR-210-3p

doi: 10.3389/fcell.2022.879877

Figure Lengend Snippet: Hypo-exos significantly enhance the growth and migration of endothelial cells in vitro compared with Norm-exos. (A) Effects of Hypo-exos and Norm-exos on the viability of endothelial cells analyzed using the CCK-8 assay. (B) Effects of Hypo-exos and Norm-exos on the proliferation of endothelial cells assessed by immunofluorescence staining of Ki67; scale bars = 100 μm. (C) Percentage of Ki67-positive cells (%) quantified in (B) . (D) Significantly increased closure of the scratched area in Hypo-exos treated group was observed in wound healing assays; scale bars = 200 μm. (E) Quantification of the rate of scratch closure (%) in (D) . (F) Significantly higher number of endothelial cells in Hypo-exos group migrated to the bottom than Norm-exos group verified by Transwell migration assays; scale bars = 200 μm. (G) Quantification of the number of migrated HUVECs in (F) . ** p < 0.01, *** p < 0.001.

Article Snippet: Next, cells were incubated with the Ki67 primary antibody (Cat. Ab15580, Abcam) overnight at 4°C.

Techniques: Migration, In Vitro, CCK-8 Assay, Immunofluorescence, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fucoidan-Supplemented Diet Potentiates Immune Checkpoint Blockage by Enhancing Antitumor Immunity

doi: 10.3389/fcell.2021.733246

Figure Lengend Snippet: Fucoidan diet enhances the antitumor efficacy of PD-1 antibodies. (A) Experimental scheme for fucoidan-supplemented diet combined with PD-1 immunotherapy in the B16 melanoma model. C57BL/6 mice were subcutaneously inoculated with B16 cells, fed with fucoidan A or F from the inoculation day (day 0) to the end point (day 20). Each mouse was administered with 200 μg PD-1 antibody at day 7, 10, and 13. i.p., intraperitoneally. (B) Tumor volumes recorded at indicated times are shown. (C) Tumor images and (D) weights of harvested tumors at day 20 are shown. (E) Flow cytometry analysis of spleen resident and tumor infiltrating lymphocytes (TILs). Spleens, tumor-draining lymph nodes, and tumor tissues were collected respectively for flow cytometry analysis. Top: Representative FCM plots (left) and analysis (right) of CD8 + and CD4 + T cells in spleen. Middle: Representative FCM plots (left) and analysis (right) of NK cells in spleen. Bottom: FCM plots (left) and analysis (right) of CD45 + CD3 + TILs. A, B, C, D ( n = 8 mice per group) and E ( n = 6 mice per group) are representative of two independent experiments. Fuco, fucoidan. Error bars, mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., non-significant.

Article Snippet: CD3E was detected with CD3E (CD3-12) rat mAb (CST Signaling, cat. #4443) at 1:1000 dilution and goat anti-rat IgG (H + L) HRP conjugates (Proteintech, cat. #SA00001) at 1:2000 dilution.

Techniques: Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fucoidan-Supplemented Diet Potentiates Immune Checkpoint Blockage by Enhancing Antitumor Immunity

doi: 10.3389/fcell.2021.733246

Figure Lengend Snippet: Fucoidan alone is insufficient to inhibit melanoma cell growth. (A) B16 cells were cultured with fucoidan A or F with indicated concentration for 48 h in 96-well plates. CCK8 assay was used to monitor cell proliferation. (B) B16 cells were cultured with 100 μg/ml fucoidan A or F for different durations. Cell apoptosis (C) was assessed by Annexin V/PI staining and cell cycle (D) was measured using PI staining. B16 cells were incubated with 100 μg/ml fucoidan A or F for 48 hours. Growth (E) and weights (F) of B16 tumors harvested from control mice, or mice with dietary fucoidan A or F treatments. The flow cytometry analysis of splenic CD8 + /CD4 + T cells (G) , CD3 + TILs (H) , or splenic NK cells (I) , in fucoidan A treated or untreated B16-bearing mice. In vitro experiments were repeated at least three times. For in vivo experiments, data in E-F ( n = 7 mice per group) and G-I ( n = 6 mice per group) are representative of two independent replicates. Error bars, mean ± SEM. * P < 0.05, non-significant.

Article Snippet: CD3E was detected with CD3E (CD3-12) rat mAb (CST Signaling, cat. #4443) at 1:1000 dilution and goat anti-rat IgG (H + L) HRP conjugates (Proteintech, cat. #SA00001) at 1:2000 dilution.

Techniques: Cell Culture, Concentration Assay, CCK-8 Assay, Staining, Incubation, Control, Flow Cytometry, In Vitro, In Vivo

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fucoidan-Supplemented Diet Potentiates Immune Checkpoint Blockage by Enhancing Antitumor Immunity

doi: 10.3389/fcell.2021.733246

Figure Lengend Snippet: Fucoidan activates the JAK-STAT pathway and promotes T cell proliferation and activation. (A) IFNγ and TNFα expression in primary CD8 + T cells cultured with anti-CD3/CD28 beads, with or without fucoidan (25 μg/ml) treatments. Numbers adjacent to the outlined areas indicate the frequency of cells expressing IFNγ and TNFα. All data are quantified on the right. (B) IFNγ expression levels in CD8 + T cells treated by indicated concentrations of fucoidan. (C) CFSE assays quantifying the proliferation of CD8 + T cells stimulated by indicated concentrations of fucoidan. (D) Gene set enrichment analysis (GSEA) of the RNAseq data of fucoidan-treated (10 μg/ml) and control CD8 + T cells. (E) Heatmap of fucoidan-regulated genes involved in the JAK-STAT pathway. Ctrl, control. A1, A2, two biological replicates of fucoidan A-treated samples. F1, F2, two biological replicates of fucoidan F-treated samples. (F) Volcano plots of differentially expressed genes from RNAseq data of fucoidan-treated and control CD8 + T cells. The x axis represents log2 of fold changes (FC) of gene expression levels in fucoidan-treated cells relative to control cells, and the y axis represents log10 of corresponding P values. Genes within the JAK-STAT pathway were highlighted. (G) qRT-PCR analysis of the mRNA expression of indicated genes in fucoidan-treated and control CD8 + T cells. (H,I) IFNγ expression in CD8 + T cells activated by anti-CD3/CD28 beads, treated with fucoidan (10 μg/ml) and/or AZD1480 (50 nM). Data are representative of three independent experiments. Error bars, mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: CD3E was detected with CD3E (CD3-12) rat mAb (CST Signaling, cat. #4443) at 1:1000 dilution and goat anti-rat IgG (H + L) HRP conjugates (Proteintech, cat. #SA00001) at 1:2000 dilution.

Techniques: Activation Assay, Expressing, Cell Culture, Control, Gene Expression, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fucoidan-Supplemented Diet Potentiates Immune Checkpoint Blockage by Enhancing Antitumor Immunity

doi: 10.3389/fcell.2021.733246

Figure Lengend Snippet: Fucoidan interacts with the T cell receptor complex to enhance T cell activity. (A) Genes Ontology (GO) analysis showing the enrichment of specific pathways regulated by indicated transcription factors, based on upregulated hallmark genes in fucoidan-treated CD8 + T cells. (B) Flow cytometry analysis of CD8 + T cells unstained or stained with FITC-labeled Ulex Europaeus Agglutinin I (FITC-UEA-I), supplemented with or without 100 μg/ml fucoidan. (C) Representative fluorescent images of Jurkat cells overexpressing RFP-CD3E (red) stained with FITC-UEA-I (green), supplemented with or without 100 μg/ml fucoidan. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. (D) Lysates from primary CD8 + T and Jurkat cells treated with or without fucoidan were subjected to pull-down assays with Ulex Europaeus Agglutinin I (UEA I) conjugated agarose, followed by immunoblotting analysis with CD3E antibodies. (E,F) Granzyme B expression levels in Jurkat cells activated by anti-human CD3/CD28 supplemented with fucoidan (10 μg/ml) and/or AZD1480 (40 μM). (G) Knockdown efficiencies of CD3E mRNA and protein using indicated shRNAs in Jurkat cells. (H) qRT-PCR analysis of GZMB and IL2 mRNA expression in Jurkat cells transfected with CD3E shRNA#2 or shNC. GZMB, Granzyme B. NC, non-targeted control. Data are representative of two independent experiments. Error bars, mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and n.s., non-significant.

Article Snippet: CD3E was detected with CD3E (CD3-12) rat mAb (CST Signaling, cat. #4443) at 1:1000 dilution and goat anti-rat IgG (H + L) HRP conjugates (Proteintech, cat. #SA00001) at 1:2000 dilution.

Techniques: Activity Assay, Flow Cytometry, Staining, Labeling, Western Blot, Expressing, Knockdown, Quantitative RT-PCR, Transfection, shRNA, Control