Journal: Oncotarget

Article Title: Crizotinib inhibits NF2-associated schwannoma through inhibition of focal adhesion kinase 1

doi: 10.18632/oncotarget.10248

Figure Lengend Snippet: ( A ) Western blot analysis of total FAK1 and p-FAKY 397 in protein extracts prepared from SC4 and HEI193 cells treated with crizotinib (10 μM for 10′ and 30′). Vinculin was used as a loading control. ( B ) Cell number counts of SC4 cells and western blot analysis of total FAK1 expression in cells treated with 2 independent shRNAs against FAK1 (shFAK-a, shFAK-b) or scrambled control (shCTRL). ( C ) Cell number counts of HEI193 cells and western blot analysis of total FAK1 expression in cells treated with 2 independent shRNAs against FAK1 (siFAK-a, siFAK-b) or scrambled control (siCTRL). ( D ) SC4 or HEI193 cells treated with defactinib at the indicated doses or with DMSO control, daily for 3 days. In all counting experiments cell numbers were scored daily and each time point was done in triplicate. The data shown represent the mean of 3 independent experiments. Error bars = SD.

Article Snippet: Specifically HEI193 cells were transfected with siRNA duplexes targeting FAK1 [ID# SR303877A- GCAAUGGAGCCGAGUAUUA AAGGUCT and SR303877B- AGAAGAUACUUACA CCAUGCCCUCA] as well as a non-targeting siRNA control [ID # SR30004- CGUUAAUCGCGUAUAAUACG CGUAT] (Origene, Rockville, USA) at a concentration of 30 nM.

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: Crizotinib inhibits NF2-associated schwannoma through inhibition of focal adhesion kinase 1

doi: 10.18632/oncotarget.10248

Figure Lengend Snippet: ( A ) Superimposition of the FAK1 (orange) and ALK (gray) kinase domains with crizotinib (ball and stick). Residues Glycine 509 (G509) and Serine 563 (S563) are highlighted (ball and stick). ( B ) Western blot analysis of the different FAK1 mutant expression in stably transfected SC4 cells. Vinculin was used as a loading control ( C ) 10-point dose response curves assessing EC50 of crizotinib in SC4 cells stably expressing crizotinib resistant FAK1 mutants. Calculated EC50 for each clone is indicated to the right. The data shown represents the mean of 3 independent experiments, each done in quadruplicate. Error bars = SD.

Article Snippet: Specifically HEI193 cells were transfected with siRNA duplexes targeting FAK1 [ID# SR303877A- GCAAUGGAGCCGAGUAUUA AAGGUCT and SR303877B- AGAAGAUACUUACA CCAUGCCCUCA] as well as a non-targeting siRNA control [ID # SR30004- CGUUAAUCGCGUAUAAUACG CGUAT] (Origene, Rockville, USA) at a concentration of 30 nM.

Techniques: Western Blot, Mutagenesis, Expressing, Stable Transfection, Transfection

Journal: PLoS Pathogens

Article Title: Periodontal pathogens promote cancer aggressivity via TLR/MyD88 triggered activation of Integrin/FAK signaling that is therapeutically reversible by a probiotic bacteriocin

doi: 10.1371/journal.ppat.1008881

Figure Lengend Snippet: (A) Representative immunoblots of three independent experiments showing integrin alpha V protein levels in UM-SCC-14A and HSC-3 cells challenged with control medium or media containing different MOIs of T . denticola , P . gingivalis or F . nucleatum for 2 h then cultured for 24 h. β-actin was used as a loading control for all immunoblots. (B) Representative immunoblots of three independent experiments showing integrin alpha V protein levels in cells transduced with control shRNA or alpha V shRNA. Lower panel represents the fold change values of alpha V expression using image J analysis. (C and D) Graphs show the total migratory distance of cells from the edges of the wounds. Measurements were made after 24 h. (E and F) Graphs show the fold changes in orasphere formation after 24 h. Data represent mean ± SD from three independent experiments. (G) Representative immunoblots of three independent experiments showing integrin alpha V protein levels in UM-SCC-14A cells challenged with control medium or media containing T . denticola ( 50 MOI) for 2 h then treated with nisin (50 μg/ml) for 24 h. (H) Representative immunoblots of three independent experiments showing alpha V protein levels in HSC-3 cells treated with nisin (50 μg/ml), Bepridil (1, 2, 5 μM), or nisin plus Bepridil (1, 2, 5 μM) for 24 h. (I) Representative immunoblots of three independent experiments showing phospho-FAK and FAK protein levels in HSC-3 cells challenged with control medium or media containing different MOIs of T . denticola for 2 h then cultured for 24 h. (J) Graphs show the total migratory distance of cells from the edges of the wounds. Measurements were made after 24 h. Data represent mean ± SD from three independent experiments. (Inset), Representative immunoblots of three independent experiments showing FAK protein levels in cells transduced with control shRNA or FAK shRNA. *Comparison between groups relative to their media controls * p ≤0.05; # Comparison between groups relative to their matching concentrations of pathogens alone # p ≤0.05. (K) Representative immunoblots of three independent experiments showing phospho-FAK and FAK protein levels in HSC-3 cells challenged with control medium or media containing T . denticola (50 MOI) for 2 h then treated with nisin (50 μg/ml) for 24 h.

Article Snippet: UM-SCC-14A and HSC-3 cells were transduced with alpha V-shRNA (SC-270259-V, Santa Cruz Biotechnology, Santa Cruz, CA) FAK shRNA (SC-29310-V, Santa Cruz Biotecnology, Santa Cruz, CA), MyD88 shRNA (SC-35986-V, Santa Cruz Biotechnology, Santa Cruz, CA) or scrambled-shRNA (SC-108080; Santa Cruz Biotechnology) lentiviral particles in 0.5 mL of serum-free media, then selected in 10 μg/mL puromycin (sc-108071; Santa Cruz Biotechnology, Santa Cruz, CA) for an additional 10 days.

Techniques: Western Blot, Cell Culture, Transduction, shRNA, Expressing

Journal: PLoS Pathogens

Article Title: Periodontal pathogens promote cancer aggressivity via TLR/MyD88 triggered activation of Integrin/FAK signaling that is therapeutically reversible by a probiotic bacteriocin

doi: 10.1371/journal.ppat.1008881

Figure Lengend Snippet: (A) Representative immunoblots of three independent experiments showing integrin alpha V, pFAK, FAK, TLR2, TLR4 and MyD88 protein levels in HSC-3 cells challenged with control medium or media containing T . denticola (50 MOI) for 2 h then cultured for 24 h. β-actin was used as a loading control. (B) Cells were challenged with control medium or media containing T . denticola (50 MOI) for 2 h then treated with nisin for 24 h and evaluated for changes in cell migration. Graphs show the total migratory distance of cells from the edges of the wounds. Measurements were made after 24 h. (Inset) Representative immunoblot of MyD88 protein levels in cells transduced with control shRNA or MyD88 shRNA. *Comparison between groups relative to their media controls * p ≤0.05; # Comparison between groups relative to their matching concentrations of pathogens alone # p ≤0.05. (C) Representative immunoblots of three independent experiments showing pFAK and FAK protein levels in cells challenged with control medium or media containing T . denticola (50 MOI) for 2 h then cultured for 24 h. (D) Model showing the crosstalk between TLR/MyD88 and integrin/FAK signaling pathways in pathogen-driven OSCC carcinogenesis. Periodontal pathogens ( T . denticola ) promote migration of OSCC cells via crosstalk between TLR/MyD88 and integrin/FAK signaling pathways, and thereby contribute to a more aggressive oral cancer phenotype.

Article Snippet: UM-SCC-14A and HSC-3 cells were transduced with alpha V-shRNA (SC-270259-V, Santa Cruz Biotechnology, Santa Cruz, CA) FAK shRNA (SC-29310-V, Santa Cruz Biotecnology, Santa Cruz, CA), MyD88 shRNA (SC-35986-V, Santa Cruz Biotechnology, Santa Cruz, CA) or scrambled-shRNA (SC-108080; Santa Cruz Biotechnology) lentiviral particles in 0.5 mL of serum-free media, then selected in 10 μg/mL puromycin (sc-108071; Santa Cruz Biotechnology, Santa Cruz, CA) for an additional 10 days.

Techniques: Western Blot, Cell Culture, Migration, Transduction, shRNA