Journal: Cancers

Article Title: Ribociclib Induces Broad Chemotherapy Resistance and EGFR Dependency in ESR1 Wildtype and Mutant Breast Cancer.

doi: 10.3390/cancers13246314

Figure Lengend Snippet: Figure 1. ERα mutant cells are sensitive to various inhibitors of the cell cycle. (A) ERα-MutA and ERα-WT cells were cultured with ER degrader fulvestrant. After six days, cell viability was measured using CellTiter-Glo. Full circles depicted independent replicate (n = 3) values, while the lines represent the curve fitting for sigmoidal (4PL) model (black, ERα-WT; red, ERα-MutA). (B) ERα-MutA and ERα-WT cells were cultured with ERα degrader AZD-9496. After six days, cell viability was measured using CellTiter-Glo. Full circles depicted independent replicate (n = 3) values, while the lines represent the curve fitting for sigmoidal (4PL) model (black, ERα-WT; red, ERα-MutA). (C) Representative western blot showing expression of ERα in ERα-MutA and ERα-WT cell lines treated with 20 nM and 100 nM fulvestrant or AZD-9496 for 24 and 48 h, respectively. Hsp90 was used as loading control (n = 3). (D) Schematic representation of the drug screen: ERα-MutA cells were seeded and cultured in full medium. On the following day, the drugs from the library were added at the concentration of 1 µM. After six-day treatment, CellTiter Blue assay was performed and viability measured using fluorescence reader. (E) Ranked plot showing viability of ERα-MutA cells following a six-day with 1 µM of the library compounds. Drugs that reduce the viability below 0.7 are considered effective. Black dots and red shade around these represent the mean viability and SD per compound, respectively (n = 3). (F) Normalized viability per compound category of the drug screen in ERα-MutA cells. Red line represents the mean viability per category, and black dots represent viability value for each of the compounds (n = 3). Figures S5 and S6 contain the western blot original material including uncropped blot images and densitometry readings/intensity ratio of western blot bands, respectively.

Article Snippet: Antibodies for HSP90 (sc-7947) and ERK1/2 (sc514302) were purchased from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Cell Culture, Western Blot, Expressing, Control, Concentration Assay, CtB Assay

Journal: Signal transduction and targeted therapy

Article Title: IGF-1R inhibition induces MEK phosphorylation to promote survival in colon carcinomas.

doi: 10.1038/s41392-020-0204-0

Figure Lengend Snippet: Fig. 5 Inactivation of MEK1 or MEK2 stimulates AKT phosphorylation. a, b Knockdown of MEK1 or MEK2 boosts AKT phosphorylation. Cells were transfected with scrambled siRNA, mek1 siRNA, or mek2 siRNA. After 48 h, cell lysates were subjected to western blot analysis. a Representative images are shown. b Densitometric quantification of phospho-AKT levels in (a). The ratio of phospho-AKT/GAPDH of transfection with scrambled siRNA is designated as 1. Data from three independent experiments were analyzed by one-sample t-test (mean ± SD; *P < 0.05). c, d Inhibition of MEK1/2 activation induces AKT phosphorylation. HCT116 cells were treated with 0, 1, 5, and 10 μM of U0126 for 72 h. The cell lysates were used to examine the levels of phospho-AKT. c Representative western blot images are shown. b Densitometric quantification of phospho-AKT and phospho-p70S6K1 levels in (c). The ratio of phospho-AKT/GAPDH or phospho-p70S6K1 in cells with 0 μM of U0126 is designated as 1. Data from three independent experiments were analyzed by one-sample t-test (mean ± SD; *P < 0.05; **P < 0.01)

Article Snippet: The MEK2 (67410–1-Ig) antibody was purchased from Proteintech (1:4,000 dilution).

Techniques: Phospho-proteomics, Knockdown, Transfection, Western Blot, Inhibition, Activation Assay