Journal: International journal of molecular sciences

Article Title: V600E BRAF Inhibition Induces Cytoprotective Autophagy through AMPK in Thyroid Cancer Cells.

doi: 10.3390/ijms22116033

Figure Lengend Snippet: Figure 2. Inhibition of V600EBRAF activates the AMPK pathway and inhibits mTOR in thyroid cancer cells. Phosphorylation levels of AMPK (p-AMPK), ACC (p-ACC), LKB1 (p-LKB1) and S6 (p-S6), together with total protein levels of AMPK and LKB1 in 8505C and BHT101 cells treated with DMSO (-) or PLX4720 (PLX) (A) or U0126 (U0) (C) for 24 h. (B) p-AMPK, p-ACC, p-S6 and total AMPK levels in cells transfected with a scrambled oligo control (sc) or a specific siRNA for BRAF (siBRAF) for 72 h. (D) p-AMPK, p-ACC and p-S6 expression levels in cells incubated with DMSO or PLX4720, alone or with Dorsomorphin, for 24 h. (E) Expression of LKB1 and p-ACC in cells transfected with an oligo control or specific siRNA for LKB1 (siLKB1) for 48 h, and then incubated in the absence or presence of PLX4720 for 24 h. (F) p-S6 levels in cells incubated with DMSO or PLX4720, in the absence or presence of Rapamycin, for 24 h. (G) Levels of p-AMPK, AMPK, p-S6, HA-BRAF and phosphorylated ERK (p-ERK) in both WRO-mock (-) and WRO-VE (VE) cells treated with DMSO or PLX4720 for 24 h. For each experiment, membranes were reprobed with anti-β-Tubulin as a loading control. Blots are representative of experiments performed three times with similar results.

Article Snippet: Human ATC-derived cell lines 8505C and BHT101, both harbouring V600EBRAF mutation, were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) WRO-mock (control) and WRO-VE (stably overexpressing V600EBRAF) cells were generated by lentiviral infection of the follicular thyroid cancer (FTC) derived cell line WRO, that harbour wtBRAF, as described in Baquero et al. [12].

Techniques: Inhibition, Phospho-proteomics, Transfection, Control, Expressing, Incubation

Journal: International journal of molecular sciences

Article Title: V600E BRAF Inhibition Induces Cytoprotective Autophagy through AMPK in Thyroid Cancer Cells.

doi: 10.3390/ijms22116033

Figure Lengend Snippet: Figure 3. V600EBRAF inhibition increases autophagy through AMPK-ULK1 signalling. Expression levels of phosphorylated ULK1 at Ser555 (p-ULK), p62 and LC3 (left blots), and quantitative analysis of LC3-II/LC3-I ratios (right graphs) in 8505C and BHT101 cells treated with DMSO (-) or PLX4720 (PLX), in the absence or presence of Dorsomorphin (Dorso) (A) or Rapamycin (Rapa) (B) for 24 h. For each experiment, membranes were reprobed with anti-β-Tubulin as a loading control. Graphic bars represent the LC3-II/LC3-I ratio, calculated after quantitation of LC3-II and LC3-I bands of the blots, and are presented as fold induction relative to the untreated cells. Blots are from one representative experiment and data shown represent the mean ± SEM of the quantitation of at least three independent experiments performed with similar results. Significant differences compared to the corresponding controls: * 0.01 < p < 0.05, ** 0.001 < p < 0.01, *** p < 0.001, compared to untreated cells; # 0.01 < p < 0.05, ### p < 0.001, compared to Dorsomorphin or Rapamycin, respectively.

Article Snippet: Human ATC-derived cell lines 8505C and BHT101, both harbouring V600EBRAF mutation, were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) WRO-mock (control) and WRO-VE (stably overexpressing V600EBRAF) cells were generated by lentiviral infection of the follicular thyroid cancer (FTC) derived cell line WRO, that harbour wtBRAF, as described in Baquero et al. [12].

Techniques: Inhibition, Expressing, Control, Quantitation Assay

Journal: International journal of molecular sciences

Article Title: V600E BRAF Inhibition Induces Cytoprotective Autophagy through AMPK in Thyroid Cancer Cells.

doi: 10.3390/ijms22116033

Figure Lengend Snippet: Figure 4. Autophagy blockage induces cell death and sensitize thyroid cancer cells to V600EBRAF inhibition. Cell viability measured by MTT assay in 8505C (A) and BHT101 (B) cells following 24, 48, or 72 h treatment with PLX4720, Bafilomycin A1 or Chloroquine alone, or with PLX4720 in combination with each of the autophagy inhibitors. Results shown are the mean ± SEM of three independent experiments performed in triplicate. Apoptosis was measured in 8505C (C,D) BHT101 cells treated as in (A,B), respectively, by quantitation of the sub-G1 fractions of PI-stained cells by flow cytometry. Results shown are the mean ± SEM of three independent experiments performed in duplicate. Significant differences compared to the corresponding controls: * 0.01 < p < 0.05, ** 0.001 < p < 0.01, *** p < 0.001.

Article Snippet: Human ATC-derived cell lines 8505C and BHT101, both harbouring V600EBRAF mutation, were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) WRO-mock (control) and WRO-VE (stably overexpressing V600EBRAF) cells were generated by lentiviral infection of the follicular thyroid cancer (FTC) derived cell line WRO, that harbour wtBRAF, as described in Baquero et al. [12].

Techniques: Inhibition, MTT Assay, Quantitation Assay, Staining, Cytometry

Journal: International journal of molecular sciences

Article Title: V600E BRAF Inhibition Induces Cytoprotective Autophagy through AMPK in Thyroid Cancer Cells.

doi: 10.3390/ijms22116033

Figure Lengend Snippet: Figure 5. Autophagy inhibition enhances antitumor activity of PLX4720 BRAFi in vivo. Mice bearing subcutaneous BHT101 cells-derived xenografts on both flanks were randomly divided into four groups and treated intraperitoneal with vehicle (control), 25 mg/kg/day PLX4720 (PLX), 60 mg/kg Chloroquine (CQ) or combination of both (PLX + CQ) for 12 days. (A) Curves of tumour growth monitored by measuring the tumour volumes over time after the treatments. Data are shown as the mean ± SEM; n = 6. (B) Representative images showing mice from different treatment groups with tumours on day 12 of treatment. Significant differences compared to the corresponding untreated control: * 0.01 < p < 0.05, ** 0.001 < p < 0.01, *** p < 0.001.

Article Snippet: Human ATC-derived cell lines 8505C and BHT101, both harbouring V600EBRAF mutation, were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) WRO-mock (control) and WRO-VE (stably overexpressing V600EBRAF) cells were generated by lentiviral infection of the follicular thyroid cancer (FTC) derived cell line WRO, that harbour wtBRAF, as described in Baquero et al. [12].

Techniques: Inhibition, Activity Assay, In Vivo, Derivative Assay, Control