Journal: Neoplasia (New York, N.Y.)

Article Title: Amelioration of muscle wasting by gintonin in cancer cachexia

doi: 10.1016/j.neo.2021.11.008

Figure Lengend Snippet: GT protects C2C12 myotubes from TNFα/IFNγ-induced muscle cell atrophy. (A-C) C2C12 myotubes were treated with GT for 48 h at the indicated concentration and stained with anti-MHC Ab. (A) Representative images were shown. (B) Average myotube diameter was measured by ImageJ software. (C) The number of nuclei per myotube was quantified. The data were shown as mean ± SEM of more than 100 myotubes from 10 randomly chosen fields (* P ≤ 0.05; *** P ≤ 0.0001). (D-F) C2C12 myotubes were treated with GT at the indicated concentrations together with TNFα (20 ng/mL) and IFNγ (100U/mL) for 24 h, and then stained with anti-MHC Ab. (D) Representative images were shown. (E) Average myotube diameter was measured by ImageJ software. (F) The number of nuclei per myotube was quantified. The data were shown as mean ± SEM of more than 100 myotubes from 10 randomly chosen fields (* P ≤ 0.05; *** P ≤ 0.0001). (G-I) C2C12 myotubes were pre-treated with TNFα (20 ng/mL) and IFNγ (100U/mL) for 24 h, and then treated with GT (100 ng/mL) for 24 h. Cells were stained with anti-MHC Ab. (G) Representative images were shown. (H) Average myotube diameter was measured by ImageJ software. (I) The number of nuclei per myotube was quantified. The data were shown as mean ± SEM of more than 100 myotubes from 10 randomly chosen fields (* P ≤ 0.05; *** P ≤ 0.0001). (J-K) C2C12 myotubes were treated with GT (100 ng/mL) together with TNFα (20 ng/mL) and IFNγ (100U/mL). Cells were isolated, and the protein levels of Atrogin-1 and MuRF-1 were evaluated by western blot. (J) Representative images were shown. (K) Images were measured by ImageJ software. The data were shown as mean ± SEM ( n =3; * P ≤ 0.05).

Article Snippet: Then, myotubes were washed, incubated with 20 μM of DCFDA solution (ab113851, Abcam) for 45 min at 37°C in the darkness, and washed with the 1X buffer according to the manufacturer's protocol.

Techniques: Concentration Assay, Staining, Software, Isolation, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Amelioration of muscle wasting by gintonin in cancer cachexia

doi: 10.1016/j.neo.2021.11.008

Figure Lengend Snippet: GT protects against cellular atrophy through the lysophosphatidic acid receptor (LPAR) and Gαi2 activation. (A) C2C12 myotubes were treated with GT (100 ng/mL) for 24 h and then the expression of LPARs were quantified by RT-PCR. The data were shown as mean ± SEM of three independent experiments (* P ≤ 0.05; ns, not significant). (B–D) C2C12 myotubes were treated with GT (100 ng/mL), TNFα (20 ng/mL) and IFNγ (100U/mL), or Ki16425 (10 μM) for 24 h and then stained with anti-MHC Ab. (B) Representative images were shown. (C) Average myotube diameters and (D) the number of nuclei per myotube of more than 100 myotubes from 10 randomly chosen fields for each condition were measured by ImageJ software. The data were shown as mean ± SEM (* P ≤ 0.05; *** P ≤ 0.0001). (E-F) C2C12 cells were transfected with control (non-targeting) (siCtrl) or Gαi2 siRNA. (E) mRNA level of Gαi2 was evaluated by RT-PCR and (F, G) protein level of Gαi2 was evaluated by western blot. The data were shown as mean ± SEM of 2 independent experiments ((* P ≤ 0.05; *** P ≤ 0.0001). (H, I) The transfected cells were differentiated to myotubes for 4 days and treated with GT (100 ng/mL) for 48 h. (H) Representative images were shown. (I) Average myotube diameters of more than 100 myotubes from 10 randomly chosen fields of each condition were measured by ImageJ software. The data were shown as mean ± SEM (* P ≤ 0.05;*** P ≤ 0.001; ns, not significant).

Article Snippet: Then, myotubes were washed, incubated with 20 μM of DCFDA solution (ab113851, Abcam) for 45 min at 37°C in the darkness, and washed with the 1X buffer according to the manufacturer's protocol.

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Software, Transfection, Control, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Amelioration of muscle wasting by gintonin in cancer cachexia

doi: 10.1016/j.neo.2021.11.008

Figure Lengend Snippet: GT protects C2C12 myoblast from oxidative stress through the reduction of ROS and inflammation genes. (A, B) C2C12 myoblast was incubated for 4 h with TNFα (20 ng/mL), GT (100 ng/mL), or N-acetyl cysteine (NAC) as indicated, and then ROS levels were measured by flow cytometry. NAC was used as a negative control. (A) Representative FACS profiles were shown and (B) the data were presented as mean ± SEM of four independent experiments (* P ≤ 0.05; ***P ≤ 0.0001). (C, D) C2C12 myotube was incubated with TNFα (20 ng/mL) and GT (100 ng/mL) for 4 h as indicated, and then ROS levels were measured by fluorescence microscope. (C) Representative fluorescence images were shown and (D) fluorescence intensity were quantified using the ImageJ software. The data were presented as mean ± SEM of at least 10 randomly chosen fields of each condition (*** P ≤ 0.0001). (E, F) C2C12 myoblast was incubated for 24 h with TNFα (20 ng/mL) or GT (100ng/mL) as indicated, and mitochondria ROS were measured by flow cytometry. (E) Representative FACS profiles were shown and (F) the data were presented as mean ± SEM of four independent experiments (* P ≤ 0.05; *** P ≤ 0.0001). (G, H) C2C12 myoblast was incubated for 24 h with TNFα (20 ng/mL) or GT (100ng/mL) as indicated, and mitochondrial membrane potential (ΔѰm) were measured by flow cytometry. (G) Representative FACS profiles were shown and (H) the data were presented as mean ± SEM of five independent experiments (* P ≤ 0.05). (I) The effect of GT on scavenging of hydroxyl radical was analyzed using iron (II)-dependent TBA reactive substance. Ascorbic acid (AA) was used as a positive control. Data were shown as mean ± SEM of three independent experiments (* P ≤ 0.05; *** P ≤ 0.0001). (J, K) C2C12 myotubes were treated for 24 h with TI (TNFα at 20 ng/mL and IFNγ at 100U/mL) or GT (100 ng/mL) as indicated, and then the levels of IL-6 (J) and Nox-2 (K) were quantified by RT-PCR. The data were shown as mean ± SEM of three to four independent experiments (*, P ≤ 0.05).

Article Snippet: Then, myotubes were washed, incubated with 20 μM of DCFDA solution (ab113851, Abcam) for 45 min at 37°C in the darkness, and washed with the 1X buffer according to the manufacturer's protocol.

Techniques: Incubation, Flow Cytometry, Negative Control, Fluorescence, Microscopy, Software, Membrane, Positive Control, Reverse Transcription Polymerase Chain Reaction

Journal: Neoplasia (New York, N.Y.)

Article Title: Amelioration of muscle wasting by gintonin in cancer cachexia

doi: 10.1016/j.neo.2021.11.008

Figure Lengend Snippet: GT protects against the atrophy of primary normal Human Skeletal Myoblasts (HSkM). (A–C) HSkM myoblast were differentiated to myotube for 7 days in differentiation media. Differentiated cells were treated with different concentrations of GT in the presence or absence of TNFα (10 ng/mL) for 3 days and then stained with anti-MHC Ab. (A) Representative images of myotube cultures were captured with a phase-contrast microscope (100x magnification). (B) Average myotube diameters and (C) the number of nuclei per myotube were quantified from more than 100 myotubes in 10 randomly chosen fields of each condition using ImageJ software. The data were shown as mean ± SEM (* P ≤ 0.05; *** P ≤ 0.0001). (D, E) HSkM myotubes were treated with GT (100 ng/mL) together with TNFα (10 ng/mL) for 8 h. Cells then were isolated, and the protein levels of Atrogin-1 and MuRF-1 were evaluated by western blot. (D) Representative images were shown. (E) Images were measured by ImageJ software. The data were shown as mean ± SEM ( n =3; * P ≤ 0.05).

Article Snippet: Then, myotubes were washed, incubated with 20 μM of DCFDA solution (ab113851, Abcam) for 45 min at 37°C in the darkness, and washed with the 1X buffer according to the manufacturer's protocol.

Techniques: Staining, Microscopy, Software, Isolation, Western Blot

Journal: bioRxiv

Article Title: LIMD1 Loss Confers a GPX4-Dependent Cell State in Lung Cancer

doi: 10.1101/2025.06.06.658274

Figure Lengend Snippet: (A) Representative images of isogenic H1299 cells stained for lipid droplets with Oil Red O upon 24 h treatment with vehicle and BSA-conjugated Oleate (100 µM) (n=3). (B) Quantification of basal lipid droplet levels in isogenic H1299 cells. (C) ROS levels evaluated by H2DCFDA staining in isogenic H1299 cells following seeding in 6-well plates at a confluency of 1.25×10 6 cells per well (n=3). (D, E) Lipid peroxidation levels evaluated by C11-BODIPY 581/591 staining in isogenic H1299 cells under basal conditions (n=4) (D) and upon treatment (E) with RSL3 (8 nM, 10 nM, 20 nM), fer-1 (2 μM) and RSL3 (20 nM) combined with fer-1 (2 μM) for 24 h (n=3). (F) ROS levels evaluated by H2DCFDA staining in isogenic SAEC cells following seeding in 6-well plates at a confluency of 1.25×10 6 cells per well (n=3). (G, H) Lipid peroxidation levels evaluated by C11-BODIPY 581/591 staining in isogenic SAEC cells under basal conditions (n=4) (G) and upon treatment (H) with ML210 (0.1 µM, 0.2 µM), fer-1 (2 μM) for 24 h; H 2 O 2 (300 μM) and H 2 O 2 (300 μM) with fer-1 (2 μM) treatment was performed for the last 30 minutes (n=3). Data is shown as biological replicates ± S.D. in C-H and as biological replicates ± S.E.M. in B . One-way ANOVA with Dunnett’s multiple comparisons test was performed in B-D, F , whereas Two-way ANOVA with Bonferroni’s multiple comparisons test was performed in E and H . Two-tailed unpaired t-test was performed in G . * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Cells were trypsinised, neutralised with 2% FBS in PBS (FACS buffer), pelleted, and resuspended in 500 μL FACS buffer containing either 2 μM BODIPY 581/591 C11 (ThermoFisher #D3861) or 100 μM H2DCFDA (Tocris #5935).

Techniques: Staining, Two Tailed Test

Journal: International Wound Journal

Article Title: Topical effect of benzalkonium bromide on wound healing and potential cellular and molecular mechanisms

doi: 10.1111/iwj.13555

Figure Lengend Snippet: Viability and reactive oxygen species (ROS) production analysis of BB, PVP‐I, CHG treated cells. A, Growth inhibition curves of cells based on CCK‐8 analysis. B, ROS production of cells detected by flow cytometry. C, Mean fluorescence intensity of H2DCFDA probe in cells. D, Fluorescence images of cells after H2DCFDA probe staining. ** P < .01; *** P < .001

Article Snippet: ROS Fluorescent indicator (H2DCFDA) was purchased from Tocris Bioscience (Bristol, United Kingdom).

Techniques: Inhibition, CCK-8 Assay, Flow Cytometry, Fluorescence, Staining

Journal: Theranostics

Article Title: Targeting KDM4B that coactivates c-Myc-regulated metabolism to suppress tumor growth in castration-resistant prostate cancer

doi: 10.7150/thno.58729

Figure Lengend Snippet: KDM4B modulates energetic metabolism in C4-2B cells. (A) qRT-PCR analysis of gene expression in KDM4B-knockdown (sh4B#1 and sh4B#2) C4-2B cells as a log2 fold change of that in control (LKO) cells. The upregulated genes were marked in red and the downregulated genes were in blue. The values are means of three independent experiments. (B-F) Analysis of cellular metabolites in the energy pathways for control and KDM4B-knockdown cells. Cells (LKO, sh4B#1, and sh4B#2) were cultured for 24 h. Glutamine (B) , glutamate (C) , citrate (D) , glutathione (E) , and oxidized glutathione (F) in lysates were analyzed by LC-MS analysis. Data are presented as the average of n = 9 replicates ± SD. (G) PDH activity of KDM4B-knockdown (sh4B#1 and sh4B#2) and control (LKO). Cells were cultured for 24 h and were harvested, followed by the measurement of PDH activity according to the manufacture's instruction (PDH Activity Colorimetric Assay Kit). (H) Analysis of cellular ROS using the H2DCFDA (DCF) assay. Cells were stained with DCF to label ROS (green) and DAPI to label nuclei (blue), followed by capturing images with ZEISS LSM800 confocal microscopy. Scale bar, 50 μm. The relative fluorescence intensity of cells was determined by Image J. Statistical analysis was assessed by Student's t -test. (I) A schematic diagram depicts the KDM4B-regulated metabolism to maintain redox and support tumor growth. Upregulation (red) or downregulation (blue) of genes from (A) , metabolites (B-F) , PDH activity (G) , and ROS (H) in KDM4B-knockdown cells as compared with the LKO cells is indicated. The colored key (blue-to-red) represent Log2 fold changes normalized by the average of KDM4B-knockdown cells. GLUT1, glucose transporter 1; HK2, hexokinase 2; PFK, phosphofructokinase; ALDOA, aldolase A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGM1, phosphoglucomutase 1; ENO1, enolase 1; PKM2, pyruvate kinase M2; LDHA, lactate dehydrogenase; PDK1/2/3, pyruvate dehydrogenase kinase 1/2/3; IDH2, isocitrate dehydrogenase 2; KGDH, α-ketoglutarate dehydrogenase; SUCLG1, succinyl-CoA synthetase; SDHA, succinate dehydrogenase A; MDH1, malate dehydrogenase 1; GLS1, glutaminase; PCK1/2, phosphoenolpyruvate carboxykinase 1/2; Glucose 6-P, glucose-6-phosphate; PEP, phosphoenolpyruvate; α-KG, α-ketoglutarate; Asp, aspartate; OAA, oxaloacetate.

Article Snippet: The fluorescence signals of H2DCFDA were detected by ZEISS LSM800 confocal microscopy (ZEISS, Oberkochen, Germany) and the images were analyzed with Zen2.3 software and ImageJ.

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Colorimetric Assay, DCF Assay, Staining, Confocal Microscopy, Fluorescence