yb 1 sirna  (Cell Signaling Technology Inc)

Journal: Cancers

Article Title: Blocking Y-Box Binding Protein-1 through Simultaneous Targeting of PI3K and MAPK in Triple Negative Breast Cancers

doi: 10.3390/cancers12102795

Figure Lengend Snippet: S102 phosphorylation of YB-1 is differentially affected by the MAPK pathway and PI3K in KRAS -mutated MDA-MB-231 and PIK3CA / PTEN -mutated MDA-MB-453cells. ( A ) Protein samples from 2 parallel cultures were isolated, and the basal phosphorylation levels of AKT, ERK1/2 and YB-1 were detected by Western blotting. Blots were stripped and incubated with antibodies against total proteins. ( B , C ) Cells were transfected with control-siRNA (ctrl-siRNA), KRAS -siRNA or PTEN -siRNA. Protein samples were isolated at seventy-two hours after transfection ( B ) and 48 h after transfection ( C ). The indicated phospho- as well as total proteins were detected by Western blotting. The densitometry analysis shows the mean ratio of P-YB-1/YB-1 ± SD ( n = 4 data from 2 independent experiments) ( B ), normalized to 1 in ctrl-siRNA conditions ( B , C ). ( D ) Indicated cells were treated with the PI3K inhibitor LY294002 (LY, 10 μM), MEK inhibitor PD98059 (PD, 20 μM), or a combination of LY and PD for 2 h and protein samples were isolated. Protein samples exposed to the same treatment conditions were isolated from 2 parallel cultures. The indicated phospho- as well as total proteins were detected by Western blotting. ( E ) Densitometry values represent the mean P-YB-1/YB-1 ratio normalized to 1 in the DMSO-treated condition (MDA-MB-231: n = 6 data from 3 independent experiments; MDA-MB-453: n = 4 data from 3 independent experiments). (* p < 0.05, *** p < 0.001, Student’s t -test), n.s.: not significant.

Article Snippet: Antibodies against phospho-YB-1 (S102) (Cat.#2900), YB-1 (Cat.#4202), phospho-Akt (S473) (Cat.#9271), phospho-ERK1/2 (T202/Y204), (Cat.#4377), phospho-p90RSK (T359/S363) (Cat.#9344S), and ERK1/2 (Cat.#4695) as well as YB-1-siRNA (Cat #6206) were purchased from Cell Signaling (Frankfurt, Germany).

Techniques: Phospho-proteomics, Isolation, Western Blot, Incubation, Transfection, Control

Journal: Cancers

Article Title: Blocking Y-Box Binding Protein-1 through Simultaneous Targeting of PI3K and MAPK in Triple Negative Breast Cancers

doi: 10.3390/cancers12102795

Figure Lengend Snippet: Akt differentially affects S102 phosphorylation of YB-1 in PTEN wild-type vs. PTEN mutated cells. ( A ) The indicated cells were treated with the Akt inhibitor MK2206 (MK, 10 μM), PD98059 (PD, 20 μM), or a combination of MK and PD for 2 h. Control cells received DMSO. Protein samples were isolated, and the indicated proteins were detected by Western blotting. Protein samples exposed to the same treatment conditions were isolated from 2 parallel cultures, and the results are shown in the panel. ( B ) Densitometry values represent the mean P-YB-1/YB-1 ratio normalized to 1 in DMSO treated condition (MDA-MB-231: n = 7 data from 3 independent experiments; MDA-MB-453: n = 4 data from 3 independent experiments). (*** p < 0.001, Student’s t -test), n.s.: not significant. ( C ) Protein samples were isolated from MDA-MB-231 cells stably transfected with scrambled-shRNA (scr-sh), Akt1-shRNA, Akt2-shRNA, or Akt3-shRNA. Protein samples were isolated from three parallel cultures. ( D ) MDA-MB-231 cells stably expressing shRNA against endogenous Akt1, Akt2, or Akt3 were transiently transfected with plasmid expressing eGFP-tagged-Akt1, -Akt2, or -Akt3. Forty-eight hours after transfection, protein samples were isolated from two parallel cultures. ( E ) Cells were transfected with 50 nM of indicated siRNA, and protein samples were isolated after 48 h. Levels of phosphorylation and expression of indicated proteins were analyzed by Western blotting. Following detection of phospho-proteins, blots were stripped and re-incubated to detect total proteins. GAPDH was used as loading control.

Article Snippet: Antibodies against phospho-YB-1 (S102) (Cat.#2900), YB-1 (Cat.#4202), phospho-Akt (S473) (Cat.#9271), phospho-ERK1/2 (T202/Y204), (Cat.#4377), phospho-p90RSK (T359/S363) (Cat.#9344S), and ERK1/2 (Cat.#4695) as well as YB-1-siRNA (Cat #6206) were purchased from Cell Signaling (Frankfurt, Germany).

Techniques: Phospho-proteomics, Control, Isolation, Western Blot, Stable Transfection, Transfection, shRNA, Expressing, Plasmid Preparation, Incubation

Journal: Cancers

Article Title: Blocking Y-Box Binding Protein-1 through Simultaneous Targeting of PI3K and MAPK in Triple Negative Breast Cancers

doi: 10.3390/cancers12102795

Figure Lengend Snippet: Dual targeting of PI3K and MEK is an efficient approach to block proliferation of KRAS -mutated TNBC cells (MDA-MB-231), PIK3CA / PTEN -mutated TNBC cells (MDA-MB-453), and PIK3R1/HRAS -mutated TNBC (Hs578t) cells in a YB-1-dependent manner. ( A ) A proliferation assay was performed after cell treatment with the PI3K inhibitor LY (10 μM), MEK inhibitor PD (20 μM), or a combination of both inhibitors in 6-cm plates (30 × 10 3 cells for MDA-MB-231 and 300 × 10 3 cells for MDA-MB-453). The concentration of vehicle (DMSO) was similar under all conditions, including the control (0.2%). Asterisks indicate significant differences in PDT between the indicated treatment conditions (* p < 0.05, ** p < 0.01, and *** p < 0.001, Student’s t -test; n = 6 data points, 2 experiments). ( B ) Cells were counted in the proliferation assay on day 5 after indicated treatments, mean percentage of cells was calculated for each inhibitor-treated condition and normalized to the DMSO-treated condition. Bar graphs present the mean of 9 data points from 3 independent experiments in MDA-MB-231 and MDA-MB-453 cells and 7 data points from 3 independent experiments in Hs578t cells. (* p < 0.05, ** p < 0.01, and *** p < 0.001, Student’s t -test) ( C ) Cells were treated as indicated, protein samples were isolated on day 5 after treatment, and the levels of indicated proteins were detected by Western blotting. Densitometry values represent the P-YB-1/GAPDH, YB-1/GAPDH ratios normalized to 1 in the DMSO-treated control. GAPDH was detected as the loading control. Experiments were repeated 3 times in MDA-MB-231 and MDA-MB-453 cells and 2 times in Hs578t cells. ( D ) Representative light microscopy images of cells transfected with control (ctrl)-siRNA and YB-1-siRNA on day 6 after transfection. ( E ) MDA-MB-231(G13D) cells were transfected with 50 nM control (ctrl) siRNA or YB-1-siRNA, and 24 h after transfection, the cells were treated with the indicated inhibitors. Thereafter, the cells were counted on day 6, and the results were graphed. Bar graphs present the mean cell count of 6 parallel cultures obtained from 2 independent experiments. Asterisks indicate significant differences between the indicated treatment conditions (* p < 0.05, ** p < 0.01, and *** p < 0.001, Student’s t -test), n.s.: not significant. ( F ) Protein samples were isolated from the DMSO-treated cells, and the level of YB-1 knockdown by siRNA was confirmed by Western blotting.

Article Snippet: Antibodies against phospho-YB-1 (S102) (Cat.#2900), YB-1 (Cat.#4202), phospho-Akt (S473) (Cat.#9271), phospho-ERK1/2 (T202/Y204), (Cat.#4377), phospho-p90RSK (T359/S363) (Cat.#9344S), and ERK1/2 (Cat.#4695) as well as YB-1-siRNA (Cat #6206) were purchased from Cell Signaling (Frankfurt, Germany).

Techniques: Blocking Assay, Proliferation Assay, Concentration Assay, Control, Isolation, Western Blot, Light Microscopy, Transfection, Cell Counting, Knockdown