Journal: Cell Death & Disease

Article Title: Dopamine D3 receptor signaling alleviates mouse rheumatoid arthritis by promoting Toll-like receptor 4 degradation in mast cells

doi: 10.1038/s41419-022-04695-y

Figure Lengend Snippet: A – C After D3R knockdown, phosphorylated mTOR, AKT, and AMPK content was checked by laser confocal microscopy in p815 mast cells. Mean fluorescence intensity was used to represent the expression. D Phosphorylated mTOR, AKT, and AMPK were checked by western blotting after 7-OH treatment. E – G TLR4 expression was checked by western blotting after the p815 mast cells were treated with the AKT antagonist GDC0068 (1 μM), mTOR inhibitor rapamycin (50 μM), and AMPK agonist A769662 (100 μM) after D3R knockdown. H The cytokines in activated p815 mast cells were measured after above the antagonists and agonist treatment. I Schematic diagram showing how TLR4 degradation is regulated by D3R and potent signaling pathways. Student’s t-test was used for ( F ) and one-way ANOVA (Tukey’s post hoc) was used for other panels; *, p < 0.05; **, p < 0.01; ***, p < 0.001, n = 3. Scale bar = 5 μm.

Article Snippet: Antibodies against D3R (#sc-136170), GAPDH (#sc-47724), p-p38 (#4511), p-ERK (#4370), p-JNK (#9255), p-c-jun (#3270), p-p65 (#3033), TLR4 (#14358), ubiquitin (#3936), LC3-I/II (#12741), mTOR (#2983), p-mTOR (#2971), Akt (#4691), p-Akt (#4060), p-AMPK (#4186) and AMPK (#sc-398861) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) or Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Confocal Microscopy, Fluorescence, Expressing, Western Blot, Protein-Protein interactions

Journal: Oncotarget

Article Title: Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings

doi: 10.18632/oncotarget.9969

Figure Lengend Snippet: HCT-116 cells or patient-1-derived primary CRC cells were treated with or without applied ODE, cells were further cultured, expressions of listed proteins were tested by Western blots A , B , E and F . Stable HCT-116 cells expressing scramble control shRNA (“scr-shRNA”), AMPKα1-shRNA or dominant negative (dn)-AMPKα1 (“dnAMPKα1”) were treated with or without applied ODE, cells were further cultured for 6 h C. or 24 h D ., expressions of listed proteins were tested by Western blots. Kinase phosphorylations and Bcl-2/HIF-1α expressions were quantified. Data in this figure were repeated three times, and similar results were obtained.

Article Snippet: As described in our previous studies [ ], lentiviral particles containing the p53-shRNA-1 (Santa Cruz, sc-29435-V), p53 shRNA-2 (Genechem, Shanghai, China) or AMPKα1 shRNA (Santa Cruz, sc-29673-V) (10 μL/well) were added to cultured CRC cells for 24 h. Afterwards, cell culture medium was replaced with puromycin (0.5 μg/mL)-containing complete medium.

Techniques: Derivative Assay, Cell Culture, Western Blot, Expressing, Control, shRNA, Dominant Negative Mutation

Journal: Oncotarget

Article Title: Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings

doi: 10.18632/oncotarget.9969

Figure Lengend Snippet: Stable HCT-116 cells expressing scramble control shRNA (“scr-shRNA”), AMPKα1-shRNA or dominant negative (dn)-AMPKα1 (“dnAMPKα1”) as well as their parental cells were treated with or without applied ODE, cells were further cultured, cell viability ( A. , MTT assay), cell death ( B. , trypan blue staining assay) and cell apoptosis ( C. , Histone DNA ELISA assay) were tested. Primary CRC cells (patient-1-dervied), transfected with scramble control siRNA (“scr-siRNA”) or AMPKα1-siRNA (−1/−2), were treated with ODE for indicated time, expressions of listed proteins were shown D ., cell viability E . and apoptosis F . were tested similarly. Kinase phosphorylations were quantified (D). Data in this figure were repeated three times, and similar results were obtained. * p < 0.05 vs. “C” of “scr-shRNA”/“scr-siRNA” group. # p < 0.05 vs. “ODE” of “scr-shRNA”/“scr-siRNA” group.

Article Snippet: As described in our previous studies [ ], lentiviral particles containing the p53-shRNA-1 (Santa Cruz, sc-29435-V), p53 shRNA-2 (Genechem, Shanghai, China) or AMPKα1 shRNA (Santa Cruz, sc-29673-V) (10 μL/well) were added to cultured CRC cells for 24 h. Afterwards, cell culture medium was replaced with puromycin (0.5 μg/mL)-containing complete medium.

Techniques: Expressing, Control, shRNA, Dominant Negative Mutation, Cell Culture, MTT Assay, Staining, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Oncotarget

Article Title: Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings

doi: 10.18632/oncotarget.9969

Figure Lengend Snippet: HCT-116 cells were treated with or without ODE at applied concentrations, cells were further cultured, expressions of listed proteins were tested by Western blots A and C ., the association between AMPKα1 (regular and p-) and p53 (regular and p-) was examined by co-immunoprecipitation (“Co-IP”) assay B ., IgG was also included as a Co-IP control (B). Stable HCT-116 cells expressing scramble-shRNA (“scr-shRNA”), AMPKα1-shRNA or dominant negative (dn)-AMPKα1 (“dnAMPKα1”) were treated with applied ODE, p53 (regular and p-) and Tubulin expressions were tested by Western blots D. Stable HCT-116 cells expressing scramble-shRNA (“scr-shRNA”) or p53-shRNA (“−1/−-2”) as well as their parental cells were treated with applied ODE, cell viability (MTT assay, E. ) and cell apoptosis (Histone DNA ELISA assay, F. ) were tested, expression of p53 in these cells was also shown (F, upper panel). p53 (regular and p-) and Tubulin expressions in ODE (50 μg/mL)-treated primary CRC cells (patient-1 derived) were shown G . p53 (regular and p-) and AMPKα1 expressions in ODE (50 μg/mL)-treated primary CRC cells with scramble control siRNA (“scr-siRNA”) or AMPKα1 siRNA (“−1/−2”) were shown H. Kinase phosphorylations and p53 expression were quantified. Data in this figure were repeated three times, and similar results were obtained. * p < 0.05 vs. “C” of “scr-shRNA” group. # p < 0.05 vs. “ODE” of “scr-shRNA” group.

Article Snippet: As described in our previous studies [ ], lentiviral particles containing the p53-shRNA-1 (Santa Cruz, sc-29435-V), p53 shRNA-2 (Genechem, Shanghai, China) or AMPKα1 shRNA (Santa Cruz, sc-29673-V) (10 μL/well) were added to cultured CRC cells for 24 h. Afterwards, cell culture medium was replaced with puromycin (0.5 μg/mL)-containing complete medium.

Techniques: Cell Culture, Western Blot, Co-Immunoprecipitation Assay, Control, Expressing, shRNA, Dominant Negative Mutation, MTT Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay