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