Journal: Life

Article Title: Quercetin–Arctigenin Co-Treatment Induces Mitochondrial Dysfunction and Apoptotic Cell Death Through Metabolic Stress in Malignant Mesothelioma Cells

doi: 10.3390/life16050774

Figure Lengend Snippet: Effects of quercetin and arctigenin on relative metabolic activity in mesothelioma and mesothelial cells. ( A – C ) MSTO-211H and MeT-5A cells were treated with increasing concentrations of QUE or ATG for ( A ) 24 h, ( B ) 48 h, and ( C ) 72 h. Relative metabolic activity was measured using the MTT assay. Data are presented as the mean ± SD from at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05 vs. control).

Article Snippet: Human malignant mesothelioma MSTO-211H cells and human mesothelial MeT-5A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activity Assay, MTT Assay, Control

Journal: Life

Article Title: Quercetin–Arctigenin Co-Treatment Induces Mitochondrial Dysfunction and Apoptotic Cell Death Through Metabolic Stress in Malignant Mesothelioma Cells

doi: 10.3390/life16050774

Figure Lengend Snippet: Combined treatment with quercetin and arctigenin reduces metabolic activity and intracellular ATP production in mesothelioma cells. ( A – C ) MSTO-211H and MeT-5A cells were treated with QUE (40 μM), ATG (50 μM), or their combination for ( A ) 24 h, ( B ) 48 h, and ( C ) 72 h. Relative metabolic activity was measured using the MTT assay. ( D – F ) Intracellular ATP levels were measured after ( D ) 24 h, ( E ) 48 h, and ( F ) 72 h treatment. Data are presented as the mean ± SD from at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05, *** p < 0.001, **** p < 0.0001 vs. control).

Article Snippet: Human malignant mesothelioma MSTO-211H cells and human mesothelial MeT-5A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activity Assay, MTT Assay, Control

Journal: Life

Article Title: Quercetin–Arctigenin Co-Treatment Induces Mitochondrial Dysfunction and Apoptotic Cell Death Through Metabolic Stress in Malignant Mesothelioma Cells

doi: 10.3390/life16050774

Figure Lengend Snippet: Combination treatment suggests a potential synergistic interaction, reduces cell growth capacity, and increases caspase activity in mesothelioma cells. ( A ) Combination index (CI) values were calculated for QUE (40 μM) and ATG (50 μM) co-treatment in MeT-5A and MSTO-211H cells at 24, 48, and 72 h. CI values < 1 may be indicative of a potential synergistic interaction under the experimental conditions tested. ( B ) Cell growth capacity was assessed by crystal violet staining following 48 h treatment with QUE, ATG, or their combination. ( C ) Caspase 3/7 activity was measured after 48 h of treatment to evaluate apoptosis-associated protease activation. Data are presented as the mean ± SD from at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control).

Article Snippet: Human malignant mesothelioma MSTO-211H cells and human mesothelial MeT-5A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activity Assay, Staining, Activation Assay, Control

Journal: Life

Article Title: Quercetin–Arctigenin Co-Treatment Induces Mitochondrial Dysfunction and Apoptotic Cell Death Through Metabolic Stress in Malignant Mesothelioma Cells

doi: 10.3390/life16050774

Figure Lengend Snippet: Combined treatment with quercetin and arctigenin suggests increased apoptotic cell populations in mesothelioma cells. Representative Annexin V dot plots showing apoptotic cell populations in MeT-5A and MSTO-211H cells following treatment with QUE (40 μM), ATG (50 μM), or their combination for 48 h. Dot plots represent cells stained with Annexin V and PI, where color intensity indicates cell density and quadrants correspond to live, early apoptotic, and late apoptotic cell populations. Percentages of live, early apoptotic, and late apoptotic cells are indicated. Lower panels show quantitative analysis of live cells (%) and total apoptotic cells (%). Data are presented as the mean ± SD from at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (** p < 0.01, **** p < 0.0001 vs. control).

Article Snippet: Human malignant mesothelioma MSTO-211H cells and human mesothelial MeT-5A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Staining, Control

Journal: Life

Article Title: Quercetin–Arctigenin Co-Treatment Induces Mitochondrial Dysfunction and Apoptotic Cell Death Through Metabolic Stress in Malignant Mesothelioma Cells

doi: 10.3390/life16050774

Figure Lengend Snippet: Co-treatment with quercetin and arctigenin is associated with modulation of mitochondrial apoptosis-associated signaling and energy stress-related protein expression in mesothelioma cells. ( A ) Representative Western blot analysis of apoptosis-associated proteins in MeT-5A and MSTO-211H cells treated with quercetin (QUE, 40 μM), arctigenin (ATG, 50 μM), or their combination for 48 h. Expression levels of anti-apoptotic proteins Mcl-1, Bcl-xL, and Bcl-2, as well as apoptosis execution markers cleaved caspase-3 and cleaved PARP, were examined. β-actin was used as the loading control. ( B ) Representative Western blot analysis of energy stress- and mitochondrial protein expression-related markers. Expression levels of phosphorylated AMPK (p-AMPK), total AMPK, and selected oxidative phosphorylation (OXPHOS) complex subunits (ATP5A, UQCRC2, SDHB, COX II, and NDUFB8) were evaluated in MeT-5A and MSTO-211H cells following treatment with QUE, ATG, or their combination for 48 h. Relative protein expression levels were normalized to β-actin, and the p-AMPK/AMPK ratio was calculated to evaluate AMPK activation. Densitometric values are presented as mean ± SD from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control). Faint background signals may be present due to exposure sonditions and do not affect the interpretation of the results.

Article Snippet: Human malignant mesothelioma MSTO-211H cells and human mesothelial MeT-5A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Western Blot, Control, Phospho-proteomics, Activation Assay

Journal: BMC cancer

Article Title: Induction of tumor initiation is dependent on CD44s in c-Met⁺ hepatocellular carcinoma.

doi: 10.1186/s12885-015-1166-4

Figure Lengend Snippet: Figure 3 c-Met regulates CD44s expression through AKT signaling. (A) MHCC97-H cells were treated with 1 μM of PHA665752 for 24 h. Immunoblot analysis of CD44v6 (~160 kDa), CD44s (~85 kDa), and B-actin. (B) MHCC97-H cells were treated with 25 μM DMSO, LY294002 (PI3K inhibitor) or PD98059 (MEK inhibitor) for 24 h, and immunoblot analysis was performed. (C) MHCC97-H cells were stably transfected with c-Met shRNA. Lysates were collected after 2 passages and CD44s, fibronectin, and E-cadherin expression was via immunoblotting. (D) Tumorsphere formation (40X magnification) assay of stably transfected MHCC97-H cells with c-Met shRNA compared to scrambled control. The data represent the mean ± SEM of triplicates, *p < 0.01. Phase-contrast images are representative of triplicates (40X magnification).

Article Snippet: Stable shRNA: TG320418 HuSH 29mer shRNA constructs against c-Met in the pGFP-V-RS vector was purchase from OriGene (Rockville, MD).

Techniques: Expressing, Western Blot, Stable Transfection, Transfection, shRNA, Control

Journal: BMC cancer

Article Title: Induction of tumor initiation is dependent on CD44s in c-Met⁺ hepatocellular carcinoma.

doi: 10.1186/s12885-015-1166-4

Figure Lengend Snippet: Figure 4 CD44s regulates mesenchymal and tumor-initiating stem-like characteristics. (A) Tumorsphere assay of MHCC97-H cells treated with DMSO, LY294002 or PD98059 (25 μM) for 24 h followed by trypan blue exclusion. 1x10^5 cells were plated in triplicates in 6-well low adherent plates for an additional 2 weeks. The data represent the mean ± SEM of triplicates, *p < 0.05. Phase contrast images are at 40X magnification. (B) Endogenous protein levels of CD44s and the mesenchymal markers E-cadherin and Fibronectin in two pooled stable CD44s shRNA cell lines. (C-D) Heatmap of MHCC97-H CD44s shRNA compared to scrambled control cells. Relative mRNA expression of stem cell genes in CD44s shRNA #1 compared with the scrambled control normalized to GAPDH. The data represent the mean ± SEM of triplicates, *p < 0.001.

Article Snippet: Stable shRNA: TG320418 HuSH 29mer shRNA constructs against c-Met in the pGFP-V-RS vector was purchase from OriGene (Rockville, MD).

Techniques: shRNA, Control, Expressing

Journal: BMC cancer

Article Title: Induction of tumor initiation is dependent on CD44s in c-Met⁺ hepatocellular carcinoma.

doi: 10.1186/s12885-015-1166-4

Figure Lengend Snippet: Figure 5 c-Met induces tumor-initiating characteristics through CD44s. (A) 5x104 MHCC97-H cells were plated onto 6-well low adherent culture plates in triplicates. Tumorspheres were collected by centrifugation after 2 weeks . For monolayer cultured cells, 5x104 MHCC97-H were plated on 10 cm plate and cultured for 2 weeks. Media was changed every 2–3 days. MHCC97-H cells were plated in low-adherent cell or monolayer cell culture dishes for two weeks and immunoblotting analysis was confirmed on tumorsphere lysates. The data are representative of three independent experiments. (B) 5x103 MHCC97-H scrambled, c-Met shRNA and CD44s shRNA cells were grown in 6-well low-adherent culture plates for two weeks and the numbers of tumorspheres were counted (40X magnification). The data are representative of two independent experiments and are shown as the mean ± SEM of triplicate plates. (C) CD44s recovers tumorsphere formation. MHCC97-H cells were transfected with c-Met siRNA (25pM) for 24 hrs followed by overexpression of CD44s or pBabe empty vector retrovirus for an additional 48 h (immunoblot) or two weeks (tumorsphere assay). (D) Immunoblot data are representative of two independent experiments. The data for the tumorsphere assay data are representative of two independent experiments and are shown as the mean ± SEM of triplicate wells (40X magnification).

Article Snippet: Stable shRNA: TG320418 HuSH 29mer shRNA constructs against c-Met in the pGFP-V-RS vector was purchase from OriGene (Rockville, MD).

Techniques: Centrifugation, Cell Culture, Western Blot, shRNA, Transfection, Over Expression, Plasmid Preparation

Journal: BMC cancer

Article Title: Induction of tumor initiation is dependent on CD44s in c-Met⁺ hepatocellular carcinoma.

doi: 10.1186/s12885-015-1166-4

Figure Lengend Snippet: Figure 6 CD44s regulates tumor initiation in vivo. (A) Immunoblot of MHCC97-H scrambled and CD44s shRNA #1 before cells were injected into athymic nude mice. (B-D) Tumor initiation graph of MHCC97-H CD44s shRNA compared with the scrambled shRNA control. Bilateral subcutaneous injections of 1x102, 1x103, or 1x104 cells were inoculated into athymic nude mice, and the number of tumors formed and the percent tumor initiation were calculated (1x102, N = 10; 1x103, N = 8; or 1x104, N = 6). Tumor volume was calculated at the end of the experiment and. Data represent the mean ± SD. Confirmation of down-regulation of CD44s and c-Met signaling was performed by immunoblotting.

Article Snippet: Stable shRNA: TG320418 HuSH 29mer shRNA constructs against c-Met in the pGFP-V-RS vector was purchase from OriGene (Rockville, MD).

Techniques: In Vivo, Western Blot, shRNA, Injection, Control

Journal: Oncogene

Article Title: An ErbB-3 antibody, MP-RM-1, inhibits tumor growth by blocking ligand-dependent and independent activation of ErbB-3/Akt signaling.

doi: 10.1038/onc.2011.322

Figure Lengend Snippet: Figure 1 NRG-1b-dependent ErbB-3/Akt activation is inhibited by MP-RM-1. (a) pSuper 4Mut and shErbB-3 infected MDA-MB- 435 and IR-8 cells were grown in 0.2% FBS RPMI 1640 for 24 h and then stimulated with 10 ng/ml of NRG-1b for 5 min before lysis. Cell lysates where then probed with the indicated antibodies. (b) MDA-MB-435 cells were grown in 0.2% FBS RPMI 1640 for 24 h and then incubated with 10 mg/ml of MP-RM-1 for the indicated time before NRG-1b stimulation. Cell lysates were immunoblotted for phosphorylated ErbB-3 and Akt expression levels. The same filter was reprobed with anti-ErbB-3 and anti-Akt antibodies for a loading control. (c) MDA-MB-435 cells were grown in 0.2% FBS RPMI 1640 for 24 h and then incubated with the indicated amount of MP- RM-1 for 2 h before NRG-1b stimulation. Cell lysates were immunoblotted for phosphorylated ErbB-3 and Akt expression levels. The same filter was reprobed with anti-ErbB-3 and anti-Akt antibodies for a loading control. Anti-p-ErbB-3 and anti-p-Akt immunoreactivity was quantified and plotted after normalization for total ErbB-3 and Akt immunoreactiviy (right panels). (d) IR-8 human melanoma cancer cells were grown in 0.2% FBS RPMI 1640 for 24 h and then incubated with the 10 mg/ml of MP-RM-1 for 2 h before NRG-1b stimulation. Cell lysates were immunoblotted for phosphorylated ErbB-3 and Akt expression levels. The same filter was reprobed with anti-ErbB-3 and anti-Akt antibodies for a loading control.

Article Snippet: Antibodies were as follows: phosphorylated ErbB-3 (Tyr1289), phosphorylated Akt (Ser473), Akt, PLCg1, phosphorylated MET (Tyr1234 1235) from Cell Signaling (Boston, MA, USA); C-17 ErbB-3 and MET from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Actin from Sigma-Aldrich (St Louis, MO, USA); anti-ErbB-2 antibody 300G9 was obtained as described in Digiesi et al. (1992) rhodamine[125I]-labeled phalloidin from Sigma; Draq5 from Alexis; Yo-Pro dye from Invitrogen (Carlsbad, CA, USA).

Techniques: Activation Assay, Infection, Lysis, Incubation, Expressing, Control

Journal: Oncogene

Article Title: An ErbB-3 antibody, MP-RM-1, inhibits tumor growth by blocking ligand-dependent and independent activation of ErbB-3/Akt signaling.

doi: 10.1038/onc.2011.322

Figure Lengend Snippet: Figure 5 MP-RM-1inhibits ligand-indipendent activation of ErbB-3. (a) pSuper 4Mut and shErbB-3 infected MKN-45 cancer cells were grown in serum-free DMEM for 24 h and then lysed for immunoblot analysis. (b) MKN-45 cells were grown in serum-free DMEM for 24 h and then exposed to MP-RM-1 for the indicated time. Lysates were analyzed by immunoblot for phosphorylated ErbB-3 and phosphorylated Akt. The same filter was reprobed with the indicated antibodies for a loading control. (c) MKN-45 cells were grown in serum free DMEM for 24 h and then incubated for 2 h with trastuzumab (10 mg/ml), MP-RM-1 (10 mg/ml) or increasing amount of MET inhibitor SU11274. Lysates were analyzed by immunoblot with the indicated antibodies. (d) MKN-45 cells were grown in serum-free DMEM for 24 h and then incubated with 10 mg/ml of MP-RM-1 for 15 and 120 min. Lysates were analysed by immunoblot with the indicated antibodies (lower panel) or immunoprecipitated with an anti-phosphorylated MET antibody and then probed with an anti-ErbB-3 antibody (upper panel). (e) MKN-45 cells were grown in serum free DMEM for 24 h and then incubated with 1 mg/ml of SU1174 alone or together with 10 mg/ml of MP-RM-1. After 2 h, NRG-1b (10 ng/ml) was added, as indicated.

Article Snippet: Antibodies were as follows: phosphorylated ErbB-3 (Tyr1289), phosphorylated Akt (Ser473), Akt, PLCg1, phosphorylated MET (Tyr1234 1235) from Cell Signaling (Boston, MA, USA); C-17 ErbB-3 and MET from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Actin from Sigma-Aldrich (St Louis, MO, USA); anti-ErbB-2 antibody 300G9 was obtained as described in Digiesi et al. (1992) rhodamine[125I]-labeled phalloidin from Sigma; Draq5 from Alexis; Yo-Pro dye from Invitrogen (Carlsbad, CA, USA).

Techniques: Activation Assay, Infection, Western Blot, Control, Incubation, Immunoprecipitation

Journal: Cell reports

Article Title: Mislocalization of protein kinase A drives pathology in Cushing’s syndrome

doi: 10.1016/j.celrep.2022.111073

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: phospho-CREB/phospho-ATF1 , CST , 9198; RRID: AB_2561044.

Techniques: Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Stable Transfection, Software

Journal: Biochemical Journal

Article Title: Autoregulation of the MET receptor tyrosine kinase by its intracellular juxtamembrane domain

doi: 10.1042/BCJ20253378

Figure Lengend Snippet: ( A ) Linear domain schematic of the MET intracellular fragment with key regions and amino acid numbers annotated. Key regulatory phospho-sites are indicated for reference: S985, which is phosphorylated by PKC, and Y1003, which recruits c-Cbl when phosphorylated. TMD stands for the transmembrane domain. ( B ) Conservation of the MET JM sequence was assessed using the Aminode algorithm. Values on Y-axis represent the relative substitution score for each amino acid number, with higher numbers indicating less sequence conservation. Yellow bars mark evolutionary conserved regions (ECRs) within the JM. ( C ) Disorder prediction for the MET JM using the IUPred2A algorithm. Dotted line indicates the order-disorder threshold; higher values reflect greater disorder. N-terminal region of the kinase domain fold is included for comparison. ( D ) AlphaFold3 model of the MET JM domain, colored by pLDDT (predicted local distance difference test) confidence scores. The location of the predicted N-terminal JM helix, the helix αJM, and the boundary residues of the N- and C-terminal JM residues are annotated. ( E ) JM domain sequence alignment of selected MET orthologs is shown with subdomain regions annotated as in ( A ). Known phosphorylation sites are marked in red or orange. Dotted box marks the predicted N-terminal JM helix. Relative secondary structure predictions (marked red for α helix and green for β sheet) and the corresponding confidence scores are shown below the sequence alignment.

Article Snippet: Phosphorylation at MET Y1003 (#3135, CST) was also measured to confirm complete dephosphorylation of the recombinant MET protein (Supp ).

Techniques: Sequencing, Comparison, Phospho-proteomics