Journal: Cell Communication and Signaling : CCS

Article Title: The mTOR pathway controls phosphorylation of BRAF at T401

doi: 10.1186/s12964-024-01808-2

Figure Lengend Snippet: Dactolisib and rapamycin impair BRAF phosphorylation at T401. Individual Western blot analyses of WM3928 cells treated for 6 h with the indicated inhibitors variations are shown. Dactolisib was used in three experiments ( A , B , and D ) at a concentration of 2.5 µM. Naporafenib was used in ( A) and ( B ) at 1 µM. XL888 was used at either 1 µM ( A ) or 100 nM ( B and D ). Trametinib was applied at a concentration of 50 nM. Rapamycin was used at 100 nM and 1 µM in ( B ) and at 100 nM in ( D ). C shows a titration of dactolisib. Detection of tubulin, HSP90 and vinculin, as well as total proteins, serve as loading controls

Article Snippet: Inhibitors (dactolisib, torin1, alpelisib, rapamycin, trametinib, naporafenib, temsirolimus, everolimus, tacrolimus, afatinib, XL888 and ulixertinib) were purchased from SelleckChem.

Techniques: Phospho-proteomics, Western Blot, Concentration Assay, Titration

Journal: Cell Communication and Signaling : CCS

Article Title: The mTOR pathway controls phosphorylation of BRAF at T401

doi: 10.1186/s12964-024-01808-2

Figure Lengend Snippet: Dactolisib and torin1 impair BRAF phosphorylation at T401 across various cell types. Western Blot analysis of MCF10AecoR cells (A), HEK293T cells ( B ), and MEFs ( C ). Cells were treated with indicated inhibitors (all 1 µM, except trametinib, 50 nM) or control (DMSO) for 4 h and then subjected to Western blot analysis using the indicated antibodies. Phospho-BRAF T401 and total BRAF were quantified using FusionCapt Advance Software, normalized to α-Tubulin, and the ratio for pBRAF/BRAF was calculated for MCF10A ( D ), HEK393T ( E ), and MEF ( F ) cell lines. Ratios were normalized to DMSO. Statistical analysis: mean ± SD, n = 4, one-way ANOVA with Dunnett ‘s test for multiple comparisons, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Detection of tubulin as well as of total ERK serves as loading control

Article Snippet: Inhibitors (dactolisib, torin1, alpelisib, rapamycin, trametinib, naporafenib, temsirolimus, everolimus, tacrolimus, afatinib, XL888 and ulixertinib) were purchased from SelleckChem.

Techniques: Phospho-proteomics, Western Blot, Control, Software

Journal: Cell Communication and Signaling : CCS

Article Title: The mTOR pathway controls phosphorylation of BRAF at T401

doi: 10.1186/s12964-024-01808-2

Figure Lengend Snippet: Overexpression of oncogenic RHEB and mTOR mutants increases BRAF phosphorylation at T401, a process reversed by mTOR inhibitors. A Representative Western Blot analysis of HEK293T cells ectopically expressing the indicated wildtype (WT) or mutant myc-tagged RHEB or untagged mTOR proteins. Cells transfected with the empty vector (EV) pCDNA3.1 serve as negative control. B Quantification of phospho-BRAF T401 per total BRAF ( n = 4). C HEK293T cells were transfected with expression vectors for the indicated RHEB or mTOR proteins, or EV. Forty-eight hours post transfection, cells were treated with the indicated inhibitors for 4 h, lysed and subjected to Western blot analysis. Images represent three independent experiments. D Following quantification of Western blot signals for pT401 and total BRAF using FusionCapt Advance Software and normalization to α-Tubulin, the ratio for pBRAF/BRAF was calculated. Ratios were normalized to EV and DMSO. Statistical analysis: mean ± SD, n = 3 ( D ) or 4 ( B ), one-way ANOVA with Dunnett ‘s test for multiple comparisons, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Detection of tubulin and/or total ERK ( C ) serves as loading control

Article Snippet: Inhibitors (dactolisib, torin1, alpelisib, rapamycin, trametinib, naporafenib, temsirolimus, everolimus, tacrolimus, afatinib, XL888 and ulixertinib) were purchased from SelleckChem.

Techniques: Over Expression, Phospho-proteomics, Western Blot, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Negative Control, Software, Control

Journal: Cell Communication and Signaling : CCS

Article Title: The mTOR pathway controls phosphorylation of BRAF at T401

doi: 10.1186/s12964-024-01808-2

Figure Lengend Snippet: Raptor knock-down sensitizes HEK293T cells to torin1 treatment. Western blot analysis of HEK293T cells stably transduced with indicated dox-inducible pTRIPZ shRNAmir constructs. Following dox treatment for 72 h, cells were either treated with 10 nM torin1 for 4 h or DMSO control. A Representative Western blot using the indicated antibodies. B Quantification of Western blots normalized either to non-silencing control incubated with DMSO or non-silencing control treated with 10 nM torin1. Following quantification of phospho-BRAF T401 and total BRAF signals using FusionCapt Advance Software and normalization to α-Tubulin, the ratio for pBRAF/BRAF was calculated. Statistical analysis: mean ± SD, n = 4 or 5, one-way ANOVA with Dunnett ‘s test for multiple comparisons, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Detection of tubulin serves as a loading control

Article Snippet: Inhibitors (dactolisib, torin1, alpelisib, rapamycin, trametinib, naporafenib, temsirolimus, everolimus, tacrolimus, afatinib, XL888 and ulixertinib) were purchased from SelleckChem.

Techniques: Knockdown, Western Blot, Stable Transfection, Transduction, Construct, Control, Incubation, Software

Journal: Genome Medicine

Article Title: A case study of an integrative genomic and experimental therapeutic approach for rare tumors: identification of vulnerabilities in a pediatric poorly differentiated carcinoma

doi: 10.1186/s13073-016-0366-0

Figure Lengend Snippet: a Sensitivity of PDX tumors to the mTOR inhibitor, temsirolimus. Chemoresistance to carboplatin and JQ1 were observed following a transient period of response. Mean and standard error of the mean (SEM) are shown. b Phosphorylation level of RPS6 upon temsirolimus treatment. c C-MYC and N-MYC protein levels upon JQ1 treatment. d Temsirolimus treatment results in decreased Ki-67 staining with concomitant increase in cleaved caspase 3 (Cl. CASP 3) following short-term (3 days) and long-term (50 days) treatments. * p < 0.05, ** p < 0.01. e Tumor growth after temsirolimus treatment withdrawal. Mean and SEM are shown. f Temsirolimus treatment can successfully rescue and induce tumor regression in carboplatin-resistant tumors. Mean and SEM are shown. g Combination therapy (temsirolimus and irinotecan) does not result in increased anti-tumor activity. Tumor regrowth is observed with withdrawal of treatment. Mean and SEM are shown

Article Snippet: Selumetinib, carboplatin, irinotecan, and temsirolimus were purchased from MedChem Express.

Techniques: Phospho-proteomics, Staining, Activity Assay

Journal: bioRxiv

Article Title: 3D vascularized microtumors unveil aberrant ccRCC vasculature and differential sensitivity to targeted treatments

doi: 10.1101/2025.03.27.645644

Figure Lengend Snippet: HUVEC (A and B), RCC4 spheroids (C and D) or co-culture of HUVEC and RCC4 spheroids (E-G) were embedded in collagen I hydrogel and treated by temsirolimus (5, 10 or 25 µM), crizotinib (0.05 or 0.5 µM), sunitinib (0.05, 0.5 or 5 µM) or vehicle (0) in NHDF-conditioned medium for 2 days. A: Capillaries immunostained for CD31 (red) and stained bt DAPI for nuclei (blue). Scale bar: 100 µm. B: 3D quantification of total capillary length, branch point number and connectivity index of capillary network. Results were normalized to control condition (vehicle). C: RCC4 spheroids stained for F-actin by phalloidin (green) and for nuclei by DAPI (red). Scale bar: 100 µm. D: Quantification of area and perimeter of spheroids. Results were normalized to control condition (vehicle). E: Vascularized fluorescent RCC4 spheroids (green, mid- and bottom panels) immunostained for CD31 (red, top and bottom panels) and stained by DAPI for nuclei (blue, bottom panels). Scale bar: 100 µm. F: 3D quantification of total capillary length, branch point number and connectivity index of the capillary network in the vascularized microtumors. Results were normalized to control condition (vehicle). G: Quantification of area and perimeter of spheroids. Results were normalized to control condition (vehicle). Graphs represent the mean of 3 independent experiments +/- SEM. Statistical analyses were performed over control condition using Kruskal-Wallis test (B, D, F and G). ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, $ p<0.00001.

Article Snippet: To assess the effects of drugs on tumor invasion, capillary or pond formation, RCC4 spheroids and HUVECs, either alone or in co-culture were treated with vehicle (dimethyl sulfoxide, DMSO), temsirolimus (ThermoFisher Scientific, #J63654.MB; 5, 10 or 25 µM, crizotinib (Merck Millipore, #PZ0191; 0.05 or 0.5 µM) or sunitinib (Merck Millipore, #1642358; 0.05, 0.5 or 5 µM).

Techniques: Co-Culture Assay, Staining, Control