Journal: The Journal of investigative dermatology

Article Title: Responses of Reconstructed Human Epidermis to Trichophyton rubrum Infection and Impairment of Infection by the Inhibitor PD169316.

doi: 10.1016/j.jid.2019.03.1147

Figure Lengend Snippet: Figure 3. p38 MAPK is constitutively phosphorylated in RHE and its activity can be inhibited by PD169316. (a) Levels of p38 MAPK and phosphorylated p38 MAPK investigated by western blotting, using antibodies specific for p38 MAPK or phosphorylated p38 MAPK, after protein extraction from RHE, during Trichophyton rubrum infection (0 to 4 days). (b) Levels of p38 MAPK and phosphorylated p38 MAPK, as well as levels of HSP27 and phosphorylated HSP27 assessed by western blotting, using specific antibodies, on protein extracts from RHE, either previously treated for 24 hours with PD169316 (15 mM), a p38 MAPK-specific inhibitor, or not treated, then exposed to H2O2 (1 mM) for 20 or 60 minutes. The detection of RPL13a protein was used as loading control. HSP27, heat shock protein 27; MAPK, mitogen- activated protein kinase; RHE, reconstructed human epidermis.

Article Snippet: Immunostaining, electron microscopy, RNA extraction, RT-qPCR ELISA, and western blot For detailed description of immunofluorescence staining of claudin- 1, scanning and transmission electron microscopy, total RNA extraction, reverse-transcription and quantitative PCR, ELISA, and protein extraction and western blot analysis, see the Supplementary Materials and Methods. p38 MAPK inhibitors BIRB796 was purchased from Tocris (Abingdon, UK), PD169316 from Santa Cruz biotechnology (Heidelberg, Germany), SB202190 and SB203580 from InvivoGen (Toulouse, France), and VX-702 from Selleckchem (Munich, Germany).

Techniques: Activity Assay, Western Blot, Protein Extraction, Infection, Control

Journal: The Journal of investigative dermatology

Article Title: Responses of Reconstructed Human Epidermis to Trichophyton rubrum Infection and Impairment of Infection by the Inhibitor PD169316.

doi: 10.1016/j.jid.2019.03.1147

Figure Lengend Snippet: Figure 6. Among five well-known p38 MAPK inhibitors, PD169316 is the only one that alters dermatophytes growth. Trichophyton rubrum arthroconidia were seeded on Sabouraud agar containing 15 mM of different p38 MAPK-specific inhibitors and incubated for 7 days at 27 C. (a) Growth percentage of T. rubrum evaluated by CFU counting (n ¼ 3; mean standard deviation) and (b) by microscopic observations of colonies in the presence of the inhibitors. Scale bar ¼ 1 mm. ***P < 0.001, Student t test. CFU, colony- forming unit; MAPK, mitogen-activated protein kinase.

Article Snippet: Immunostaining, electron microscopy, RNA extraction, RT-qPCR ELISA, and western blot For detailed description of immunofluorescence staining of claudin- 1, scanning and transmission electron microscopy, total RNA extraction, reverse-transcription and quantitative PCR, ELISA, and protein extraction and western blot analysis, see the Supplementary Materials and Methods. p38 MAPK inhibitors BIRB796 was purchased from Tocris (Abingdon, UK), PD169316 from Santa Cruz biotechnology (Heidelberg, Germany), SB202190 and SB203580 from InvivoGen (Toulouse, France), and VX-702 from Selleckchem (Munich, Germany).

Techniques: Incubation, Standard Deviation

Journal: International journal of antimicrobial agents

Article Title: Repurposing Niclosamide for Intestinal Decolonization of Vancomycin-resistant Enterococci

doi: 10.1016/j.ijantimicag.2018.02.003

Figure Lengend Snippet: Clinical isolates of vancomycin-resistant enterococci (VRE) utilized in this study.

Article Snippet: Ampicillin (IBI Scientific, Peosta, IA, USA), closantel (Ark Pharm, Libertyville, IL, USA), gentamicin sulfate (Fisher Bioreagents, Fairlawn, NJ, USA), linezolid (Chem-Impex International Inc., Wood Dale, IL, USA), niclosamide (Cayman Chemicals, Ann Arbor, MI, USA), oxyclozanide (Sigma-Aldrich, St. Louis, MO, USA), rafoxanide (TCI America, Portland, OR, USA), ramoplanin (Sigma-Aldrich, St. Louis, MO, USA), and vancomycin hydrochloride (Gold Biotechnology, St. Louis, MO, USA) were purchased from commercial vendors.

Techniques: Isolation, Infection

Journal: International journal of antimicrobial agents

Article Title: Repurposing Niclosamide for Intestinal Decolonization of Vancomycin-resistant Enterococci

doi: 10.1016/j.ijantimicag.2018.02.003

Figure Lengend Snippet: The minimum inhibitory concentration (MIC in μg/mL) of salicylanilide drugs (niclosamide, closantel, rafoxanide, and oxyclozanide) and control antibiotics against clinical isolates of vancomycin-resistant E. faecium and E. faecalis .

Article Snippet: Ampicillin (IBI Scientific, Peosta, IA, USA), closantel (Ark Pharm, Libertyville, IL, USA), gentamicin sulfate (Fisher Bioreagents, Fairlawn, NJ, USA), linezolid (Chem-Impex International Inc., Wood Dale, IL, USA), niclosamide (Cayman Chemicals, Ann Arbor, MI, USA), oxyclozanide (Sigma-Aldrich, St. Louis, MO, USA), rafoxanide (TCI America, Portland, OR, USA), ramoplanin (Sigma-Aldrich, St. Louis, MO, USA), and vancomycin hydrochloride (Gold Biotechnology, St. Louis, MO, USA) were purchased from commercial vendors.

Techniques: Concentration Assay

Journal: International journal of antimicrobial agents

Article Title: Repurposing Niclosamide for Intestinal Decolonization of Vancomycin-resistant Enterococci

doi: 10.1016/j.ijantimicag.2018.02.003

Figure Lengend Snippet: VRE strain 503 was incubated with test agents and samples were collected over a 24-hour incubation period. DMSO, solvent for niclosamide and linezolid, served as the negative control. The error bars represent standard deviation values obtained from triplicate samples used for each agent studied.

Article Snippet: Ampicillin (IBI Scientific, Peosta, IA, USA), closantel (Ark Pharm, Libertyville, IL, USA), gentamicin sulfate (Fisher Bioreagents, Fairlawn, NJ, USA), linezolid (Chem-Impex International Inc., Wood Dale, IL, USA), niclosamide (Cayman Chemicals, Ann Arbor, MI, USA), oxyclozanide (Sigma-Aldrich, St. Louis, MO, USA), rafoxanide (TCI America, Portland, OR, USA), ramoplanin (Sigma-Aldrich, St. Louis, MO, USA), and vancomycin hydrochloride (Gold Biotechnology, St. Louis, MO, USA) were purchased from commercial vendors.

Techniques: Incubation, Solvent, Negative Control, Standard Deviation

Journal: International journal of antimicrobial agents

Article Title: Repurposing Niclosamide for Intestinal Decolonization of Vancomycin-resistant Enterococci

doi: 10.1016/j.ijantimicag.2018.02.003

Figure Lengend Snippet: Fecal pellets were collected from mice treated with niclosamide (10 mg/kg), linezolid (10 mg/kg), or ramoplanin (10 mg/kg) once daily for eight days. Stool samples were collected before treatment (day 0), and on days 3, 5, and 7 after treatment and the viable CFU/g fecal content was determined. Data were analyzed via a two-way ANOVA with post-hoc Dunnet’s multiple comparisons test. One asterisk (*) indicates significant difference (P < 0.05) between mice treated with linezolid, niclosamide, or ramoplanin compared to untreated mice. Two asterisks (**) indicates significant difference (P < 0.05) between mice treated with either niclosamide or ramoplanin compared to mice treated with linezolid.

Article Snippet: Ampicillin (IBI Scientific, Peosta, IA, USA), closantel (Ark Pharm, Libertyville, IL, USA), gentamicin sulfate (Fisher Bioreagents, Fairlawn, NJ, USA), linezolid (Chem-Impex International Inc., Wood Dale, IL, USA), niclosamide (Cayman Chemicals, Ann Arbor, MI, USA), oxyclozanide (Sigma-Aldrich, St. Louis, MO, USA), rafoxanide (TCI America, Portland, OR, USA), ramoplanin (Sigma-Aldrich, St. Louis, MO, USA), and vancomycin hydrochloride (Gold Biotechnology, St. Louis, MO, USA) were purchased from commercial vendors.

Techniques:

Journal: International journal of antimicrobial agents

Article Title: Repurposing Niclosamide for Intestinal Decolonization of Vancomycin-resistant Enterococci

doi: 10.1016/j.ijantimicag.2018.02.003

Figure Lengend Snippet: a.) Log10 CFU/g of VRE in cecal content of mice treated with niclosamide (10 mg/kg), linezolid (10 mg/kg), or ramoplanin (10 mg/kg) once daily for eight days. Data were analyzed via a two-way ANOVA with post-hoc Dunnet’s multiple comparisons test. One asterisk (*) indicates significant difference (P < 0.05) between mice treated with either linezolid, niclosamide, or ramoplanin compared to untreated mice. Two asterisks (**) indicates significant difference (P < 0.05) between mice treated with either niclosamide or ramoplanin compared to mice treated with linezolid. b.) Log10 CFU/g E. faecium strain 503 in mice ileal contents after eighth day of treatment. Data were analyzed via a two-way ANOVA with post-hoc Dunnet’s multiple comparisons test. One asterisk (*) indicates significant difference (P < 0.05) between mice treated with either linezolid, niclosamide, or ramoplanin compared to untreated mice. Two asterisks (**) indicates significant difference (P < 0.05) between mice treated with either niclosamide or ramoplanin compared to mice treated with linezolid.

Article Snippet: Ampicillin (IBI Scientific, Peosta, IA, USA), closantel (Ark Pharm, Libertyville, IL, USA), gentamicin sulfate (Fisher Bioreagents, Fairlawn, NJ, USA), linezolid (Chem-Impex International Inc., Wood Dale, IL, USA), niclosamide (Cayman Chemicals, Ann Arbor, MI, USA), oxyclozanide (Sigma-Aldrich, St. Louis, MO, USA), rafoxanide (TCI America, Portland, OR, USA), ramoplanin (Sigma-Aldrich, St. Louis, MO, USA), and vancomycin hydrochloride (Gold Biotechnology, St. Louis, MO, USA) were purchased from commercial vendors.

Techniques:

Journal: Cancer Medicine

Article Title: Anti‐Metastatic Effects of Aprepitant on Glioblastoma Cells: Targeting the Substance P/Neurokinin‐1 Pathway

doi: 10.1002/cam4.71148

Figure Lengend Snippet: Evaluation of SP (500 nM) and aprepitant concentrations on U87‐MG cell viability. Cell viability was assessed by the WST‐1 method at 24 h and expressed as a percentage of the control. Statistical analysis was performed using one‐way ANOVA with Tukey's post hoc test. Results are presented as mean values ( n = 8) with significance levels indicated as p < 0.0001 (****). Data were analyzed relative to the control group (0.1% DMSO).

Article Snippet: Aprepitant was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Delaware Ave, California, USA).

Techniques: Control

Journal: Cancer Medicine

Article Title: Anti‐Metastatic Effects of Aprepitant on Glioblastoma Cells: Targeting the Substance P/Neurokinin‐1 Pathway

doi: 10.1002/cam4.71148

Figure Lengend Snippet: Invasion cell index (CI) curves from the RTCA‐DP system (A, C) and percentage slope graphs (B, D) were generated using the RTCA‐DP system to evaluate aprepitant concentrations (IC 25 : 38.5 μM, IC 50 : 77 μM, and IC 75 : 115.5 μM) alone and in combination with SP (500 nM) in U87‐MG cells. The percentage slope graphs (B and D) were plotted in GraphPad using the corresponding cell index curves (A, C). Data are presented as mean ± standard deviation ( n = 6), with significance levels indicated as p < 0.01 (**), and p < 0.001 (***).

Article Snippet: Aprepitant was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Delaware Ave, California, USA).

Techniques: Generated, Standard Deviation

Journal: Cancer Medicine

Article Title: Anti‐Metastatic Effects of Aprepitant on Glioblastoma Cells: Targeting the Substance P/Neurokinin‐1 Pathway

doi: 10.1002/cam4.71148

Figure Lengend Snippet: Migration cell index (CI) curves from the RTCA‐DP system (A, C) and the percentage slope graphs from GraphPad (B, D) were determined using the RTCA‐DP system for aprepitant concentrations (IC 25 : 38.5 μM, IC 50 : 77 μM, and IC 75 : 115.5 μM) alone and in combination with SP (500 nM) in U87‐MG cells. The percentage slope graphs (B, D) were plotted in GraphPad using the cell index curves (A, C). Data are presented as mean ± standard deviation ( n = 6), with significance levels indicated as p < 0.0001 (****).

Article Snippet: Aprepitant was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Delaware Ave, California, USA).

Techniques: Migration, Standard Deviation

Journal: Cancer Medicine

Article Title: Anti‐Metastatic Effects of Aprepitant on Glioblastoma Cells: Targeting the Substance P/Neurokinin‐1 Pathway

doi: 10.1002/cam4.71148

Figure Lengend Snippet: VEGF levels were determined by the ELISA method. The results are expressed as mean values ( n = 7) in pg/mL, with significance levels indicated as p < 0.001 (***). Statistical analysis was performed relative to the control group (control: 0.1% DMSO, SP control: 500 nM SP, SP‐aprepitant IC 25 : SP (500 nM) + aprepitant IC 25 (38.5 μM), SP‐aprepitant IC 50 : SP (500 nM) + aprepitant IC 50 (77 μM)).

Article Snippet: Aprepitant was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Delaware Ave, California, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer Medicine

Article Title: Anti‐Metastatic Effects of Aprepitant on Glioblastoma Cells: Targeting the Substance P/Neurokinin‐1 Pathway

doi: 10.1002/cam4.71148

Figure Lengend Snippet: IL‐6 (A) and IL‐8 (B) levels were determined by the ELISA method. The results are expressed as mean values ( n = 7), with significance levels indicated as p < 0.01 (**) and p < 0.001 (***). Statistical analysis was performed relative to the control group (control: 0.1% DMSO, SP control: 500 nM SP, SP‐aprepitant IC 25 : SP (500 nM) + aprepitant IC 25 (38.5 μM), SP‐aprepitant IC 50 : SP (500 nM) + aprepitant IC 50 (77 μM)).

Article Snippet: Aprepitant was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Delaware Ave, California, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer Medicine

Article Title: Anti‐Metastatic Effects of Aprepitant on Glioblastoma Cells: Targeting the Substance P/Neurokinin‐1 Pathway

doi: 10.1002/cam4.71148

Figure Lengend Snippet: MMP‐2 levels determined by the ELISA method. The results are expressed as mean values ( n = 7) in pg/mL, with significance levels indicated as p < 0.001 (***). Statistical analysis was performed relative to the control group (control: 0.1% DMSO, SP control: 500 nM SP, SP‐aprepitant IC 25 : SP (500 nM) + aprepitant IC 25 (38.5 μM), SP‐aprepitant IC 50 : SP (500 nM) + aprepitant IC 50 (77 μM)).

Article Snippet: Aprepitant was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Delaware Ave, California, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer Medicine

Article Title: Anti‐Metastatic Effects of Aprepitant on Glioblastoma Cells: Targeting the Substance P/Neurokinin‐1 Pathway

doi: 10.1002/cam4.71148

Figure Lengend Snippet: Morphological migration analysis results. The 24‐h impact of control, SP, SP‐aprepitant IC 25 , and SP‐aprepitant IC 50 on U87‐MG cell migration is visually presented (objective: 10X). The image of the well at 0 h was taken from a randomly selected well after the removal of the stopper apparatus following the application of SP and aprepitant concentrations. Before capturing images of the wells at 24 h, a mask was applied to delineate the well dimensions as observed at 0 h.

Article Snippet: Aprepitant was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Delaware Ave, California, USA).

Techniques: Migration, Control

Journal: PLOS ONE

Article Title: Identification of drug-like molecules targeting the ATPase activity of dynamin-like EHD4

doi: 10.1371/journal.pone.0302704

Figure Lengend Snippet: A One-hour time course of the MLG-based enzymatic ATPase assay in the presence of 50 μM inhibitor. EHD4 ΔN (blue curve) represents the positive control. B Primary hits were validated at 50 μM inhibitor concentration in an HPLC setup to exclude assay bias. ATPase assays done in A and B were in standard conditions. C Primary hits were tested against EHD2. Only MS8 had no effect on EHD2 ATPase activity. ATPase assay was done at an EHD2 concentration of 5 μM in the presence of 500 μg/ml Folch liposomes and 50 μM ATP in assay buffer at 30°C. D Changes in melting temperature of 5 μM EHD4 ΔN were studied in the presence of 50 μM inhibitor by thermal shift assay. MS1 and MS10 destabilized the protein, as evident by negative ΔT m , whereas MS8 had no effect on the stability of EHD4 ΔN . E MS8 did not inhibit the GTPase activity of DNM1L. Shown is an IC 50 curve plotted with an inhibitor concentration range from 0.125 μM to 50 μM. F MS8 substructure obtained by dissecting the diphenlyether bond. This substructure was used in our chemical search to have the highest diversity of the chemical space and the optimal coverage of chemical vectors to gain a structure-activity relationship.Data points represents the mean of two or more independent experiments and the error bar signifies the range or standard deviation. When the range/standard deviation is smaller than the size of the data point, it is not displayed.

Article Snippet: The identified EHD4 inhibitors are N-(2-hydroxyphenyl)-6-(3-oxo-2,3-dihydro-1,2-benzothiazol-2-yl)hexanamide (MS1, Molport, 008-333-699), N-(4-methyl-1,3-thiazol-2-yl)-3-phenoxybenzamide (MS8, Enamine, Z30820224) and 6-(Morpholino(4-(Trifluormethyl)phenyl)methyl)-1,3-Benzodioxol-5-ol (MS10, Enamine, Z99601030).

Techniques: ATPase Assay, Positive Control, Concentration Assay, Activity Assay, Liposomes, Thermal Shift Assay, Standard Deviation

Journal: Microbiology (Reading, England)

Article Title: Stratifying macrophages based on their infectious burden identifies novel host targets for intervention during Crohn's disease associated adherent-invasive Escherichia coli infection.

doi: 10.1099/mic.0.001470

Figure Lengend Snippet: Fig. 7. Evaluation of the effects of different chemical inhibitors on intracellular bacterial load in RAW 264.7 cells. RAW 264.7 cells were infected with LF82 for 1 h followed by treatment with different chemical inhibitors for a further 6 or 24 hpi; ST034307 (a), Clomipramine (b), Necrosulfonamide (c), Trametinib (d), GSK2636771 (e). Bacterial recovery is displayed as c.f.u. per gram of protein. Data points represent the mean of three technical repeats plus the standard deviation at a timepoint of 6 or 24 hpi. Each treatment was compared to the DMSO group. Statistical significance was determined by multiple comparisons with Brown-Forsythe and Welch ANOVA tests.

Article Snippet: After washing, cells were provided with fresh RPMI media (3 % FBS, 50 μg ml−1 gentamicin) containing indicated concentrations of different chemical inhibitors (ST034307 [SML2109, Sigma- Aldrich], GSK2636771 [HY- 15245, MedChemExpress], necrosulfonamide [HY- 100573, MedChemExpress], trametinib [HY- 10999, MedChemExpress], and clomipramine [17321- 11- 6, Sigma- Aldrich]).

Techniques: Infection, Standard Deviation

Journal: Microbiology (Reading, England)

Article Title: Stratifying macrophages based on their infectious burden identifies novel host targets for intervention during Crohn's disease associated adherent-invasive Escherichia coli infection.

doi: 10.1099/mic.0.001470

Figure Lengend Snippet: Fig. 8. Quantification of intracellular LF82 burden post-inhibitor treatment using imaging flow cytometry. RAW 264.7 cells infected with LF82::rpsMGFP were treated with 1 µM ST034307, 1 µM GSK2636771, 1 µM necrosulfonamide, 100 nM trametinib or 1 µM clomipramine for 24 hpi (a), 48 hpi (b) and 72 hpi (c). Infected cells treated with DMSO were used as a control. Intracellular LF82::rpsMGFP was counted via IFC. The spot count profile separated cells into those with no bacteria, cells containing 1–5 bacteria, or cells containing over five bacteria. The sub-populations of a graph represent the mean of three biological repeats. Error bars represent SEM. The number of portions of sub-populations represents the mean of three biological repeats.

Article Snippet: After washing, cells were provided with fresh RPMI media (3 % FBS, 50 μg ml−1 gentamicin) containing indicated concentrations of different chemical inhibitors (ST034307 [SML2109, Sigma- Aldrich], GSK2636771 [HY- 15245, MedChemExpress], necrosulfonamide [HY- 100573, MedChemExpress], trametinib [HY- 10999, MedChemExpress], and clomipramine [17321- 11- 6, Sigma- Aldrich]).

Techniques: Imaging, Flow Cytometry, Infection, Control, Bacteria

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Integrin αvβ3 Induces HSP90 Inhibitor Resistance via FAK Activation in KRAS -Mutant Non–Small Cell Lung Cancer

doi: 10.4143/crt.2021.651

Figure Lengend Snippet: Establishment of acquired AUY922 resistance in NSCLC cells harboring a KRAS mutation. (A) Antitumor effects of AUY922 alone against three KRAS -mutated NSCLC cell lines. NSCLC cells were treated with different concentrations of AUY922 for 72 hours. The results are expressed as a mean±SD of three independent experiments. (B) Results of western blotting analysis showing the endogenous protein levels in two KRAS -mutated NSCLC cell lines, A549 and H1944, after AUY922 treatment. (C) Anti-cancer effect of lapatinib in the A549 and H1944 cells, and their acquired AUY922-resistant counterparts, A549R and H1944R. Cell proliferation was measured using a CellTiter-Glo Luminescent Cell Viability Assay. The average results±SD of three independent experiments are shown. (D) Western blotting analysis of MEK, pMEK, pERK, ERK, AKT, HSP90, and PARP expression following incubation with increasing doses of AUY922 in the A549, A549R, H1944, and H1944R cell lines. β-Actin was included as a loading control (R, resistant cells). ERK, extracellular-signal-regulated kinase; HSP90, heat shock protein-90; MEK, mitogen-activated protein kinase; NSCLC, non–small cell lung cancer; PARP, poly(ADP-ribose) polymerase; SD, standard deviation.

Article Snippet: The human KRAS -mutated NSCLC cell lines, H23, H358, H647, H1944, and A549, were purchased from the American Type Culture Collection (Manassas, VA) and grown at 37°C in 5% CO 2 using RPMI-1640 (Welgene Inc., Gyeonsan, Korea) containing 10% fetal bovine serum (FBS) and 1x penicillin-streptomycin solutions from Welgene Inc. AUY922 (HSP90 inhibitor, luminespib), cilengitide trifluoroacetate (inhibitor of the αvβ3 receptor), and TAE226 (focal adhesion kinase [FAK] inhibitor) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Mutagenesis, Western Blot, Cell Viability Assay, Expressing, Incubation, Control, Standard Deviation

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Integrin αvβ3 Induces HSP90 Inhibitor Resistance via FAK Activation in KRAS -Mutant Non–Small Cell Lung Cancer

doi: 10.4143/crt.2021.651

Figure Lengend Snippet: FAK activation via ITGAvB3 induces AUY922 resistance in KRAS -mutated NSCLC cells. (A) Western blot analysis of the ITGB3 and ITGAv proteins in the acquired AUY922-resistant NSCLC cell lines (A549R and H1944R). (B) Real-time PCR analysis of miR-150 and miR-142 expression in the acquired AUY922-resistant NSCLC cell lines. (C) Western blotting analysis of ITGB3, pFAK, pRSK, RSK, pERK, AKT, pAKT, pMEK, and HSP90 expression in A549, A549R, H1944, and H1944R cell lines following incubation with 1 μM of AUY922. β-Actin was included as a loading control. FAK, focal adhesion kinase; ITGAv, integrin Av; ITGB3, integrin β3; NSCLC, non–small cell lung cancer; PCR, polymerase chain reaction.

Article Snippet: The human KRAS -mutated NSCLC cell lines, H23, H358, H647, H1944, and A549, were purchased from the American Type Culture Collection (Manassas, VA) and grown at 37°C in 5% CO 2 using RPMI-1640 (Welgene Inc., Gyeonsan, Korea) containing 10% fetal bovine serum (FBS) and 1x penicillin-streptomycin solutions from Welgene Inc. AUY922 (HSP90 inhibitor, luminespib), cilengitide trifluoroacetate (inhibitor of the αvβ3 receptor), and TAE226 (focal adhesion kinase [FAK] inhibitor) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Activation Assay, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Incubation, Control, Polymerase Chain Reaction

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Integrin αvβ3 Induces HSP90 Inhibitor Resistance via FAK Activation in KRAS -Mutant Non–Small Cell Lung Cancer

doi: 10.4143/crt.2021.651

Figure Lengend Snippet: Overexpression of ITGAv and ITGB3 induces AUY922 resistance in KRAS -mutated non–small cell lung cancer (NSCLC) cells. (A) Western blot analysis of ITGB3, ITGAv, and pFAK proteins in stably ITGAv and ITGB3 expressing NSCLC cell lines. (B, C) Effect of ITGAv and ITGB3 on AUY922 sensitivity of two KRAS -mutant NSCLC cell lines, A549/ITGAvB3 and H1944/ITGAvB3. CellTiter-Glo Luminescent Cell Viability Assays were performed 72 hours after drug treatment. Data represent the mean±SD of triplicate measurements relative to untreated cells. (D) IC 50 values are reported in the table. (E) Anti-apoptotic effect of TAE226 in A549/ITGAvB3 and H1944/ITGAvB3 cell lines. CellTiter-Glo Luminescent Cell Viability Assays were performed 72 hours after drug treatment. (F) Western blot analysis of ITGB3, ITGAv, and pFAK proteins in stably ITGAv and ITGB3 knock-downed AUY922-resistant NSCLC cell lines. FAK, focal adhesion kinase; ITGAv, integrin Av; ITGB3, integrin β3; NSCLC, non–small cell lung cancer; SD, standard deviation.

Article Snippet: The human KRAS -mutated NSCLC cell lines, H23, H358, H647, H1944, and A549, were purchased from the American Type Culture Collection (Manassas, VA) and grown at 37°C in 5% CO 2 using RPMI-1640 (Welgene Inc., Gyeonsan, Korea) containing 10% fetal bovine serum (FBS) and 1x penicillin-streptomycin solutions from Welgene Inc. AUY922 (HSP90 inhibitor, luminespib), cilengitide trifluoroacetate (inhibitor of the αvβ3 receptor), and TAE226 (focal adhesion kinase [FAK] inhibitor) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Over Expression, Western Blot, Stable Transfection, Expressing, Mutagenesis, Standard Deviation

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Integrin αvβ3 Induces HSP90 Inhibitor Resistance via FAK Activation in KRAS -Mutant Non–Small Cell Lung Cancer

doi: 10.4143/crt.2021.651

Figure Lengend Snippet: TSPAN7 contributes to the epithelial-mesenchymal transition of acquired AUY922-resistant cells. (A) Western blot analysis of the epithelial-mesenchymal transition signaling proteins, TSPAN7, E-cadherin, and N-cadherin, in the two AUY922 resistant NSCLC cell lines, A549R and H1944R. (B) Phase-contrast images of the A549R and H1944R cells revealing notable morphological changes (×200). (C, D) Migration and invasion assay of the A549R and H1944R cells. These NSCLC cell lines were serum-starved for 24 hours and cell migration and invasion assays were conducted at 48 hours. Cell migration and invasion indexes were measured as described in the Methods. Data are representative of three independent experiments. *p < 0.05. (E) Western blot analysis of A549R and H1944R cells at 72 hours after TSPAN7 siRNA transfection. β-Actin was used as a loading control. (F) TSPAN7 siRNA could not rescue the AUY922 sensitivity in A549R and H1944R cells. Two days after transfection, two cell lines were treated with AUY922 for 3 days, followed by cell viability assay. The data represent triplicate experiments. NSCLC, non–small cell lung cancer; TSPAN, tetraspanin 7.

Article Snippet: The human KRAS -mutated NSCLC cell lines, H23, H358, H647, H1944, and A549, were purchased from the American Type Culture Collection (Manassas, VA) and grown at 37°C in 5% CO 2 using RPMI-1640 (Welgene Inc., Gyeonsan, Korea) containing 10% fetal bovine serum (FBS) and 1x penicillin-streptomycin solutions from Welgene Inc. AUY922 (HSP90 inhibitor, luminespib), cilengitide trifluoroacetate (inhibitor of the αvβ3 receptor), and TAE226 (focal adhesion kinase [FAK] inhibitor) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Western Blot, Migration, Invasion Assay, Transfection, Control, Viability Assay

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Integrin αvβ3 Induces HSP90 Inhibitor Resistance via FAK Activation in KRAS -Mutant Non–Small Cell Lung Cancer

doi: 10.4143/crt.2021.651

Figure Lengend Snippet: FAK activation via miR-150 and miR-142 downregulation induce AUY922 resistance. (A) Real-time PCR analysis of ectopic miR-150 expression in A549R/miR-150 and H1944/miR-150 stable cells. (B) Effect of ectopic miR-150 expression on ITGB3, pFAK, TSPAN7 and N-cadherin in miR-control and miR-150 transfected resistant cells, revealed by western blot analysis. (C, D) Ectopic miR-150 expression renders A549R and H1944R cells sensitive to AUY922. Cell viability assays were performed at 72 hours after AUY922 treatment of the indicated cells at different concentrations. Data represent the mean±SD of triplicate experiments relative to untreated cells. (E) Real-time PCR analysis of ectopic miR-142 expression in A549R/miR-142 and H1944/miR-142 stable cells. (F) Effect of ectopic miR-142 expression on ITGB3, pFAK in miR-control and miR-142 transfected resistant cells, revealed by western blot analysis. (G, H) Ectopic miR-142 expression renders A549R and H1944R cells sensitive to AUY922. Cell viability assays were performed at 72 hours after AUY922 treatment of the indicated cells at different concentrations. Data represent the mean±SD of triplicate experiments relative to untreated cells. FAK, focal adhesion kinase; ITGAv, integrin Av; ITGB3, integrin β3; PCR, polymerase chain reaction; SD, standard deviation; TSPAN, tetraspanin 7.

Article Snippet: The human KRAS -mutated NSCLC cell lines, H23, H358, H647, H1944, and A549, were purchased from the American Type Culture Collection (Manassas, VA) and grown at 37°C in 5% CO 2 using RPMI-1640 (Welgene Inc., Gyeonsan, Korea) containing 10% fetal bovine serum (FBS) and 1x penicillin-streptomycin solutions from Welgene Inc. AUY922 (HSP90 inhibitor, luminespib), cilengitide trifluoroacetate (inhibitor of the αvβ3 receptor), and TAE226 (focal adhesion kinase [FAK] inhibitor) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Control, Transfection, Western Blot, Polymerase Chain Reaction, Standard Deviation

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Integrin αvβ3 Induces HSP90 Inhibitor Resistance via FAK Activation in KRAS -Mutant Non–Small Cell Lung Cancer

doi: 10.4143/crt.2021.651

Figure Lengend Snippet: A FAK inhibitor, and not an integrin inhibitor, shows synergistic effects with AUY922 in AUY922-resistant cells. (A, B) Synergistic effect of a cilengitide and AUY922 combination. Cell viability assays were performed in A549R and H1944R cells treated with 1,000 nM of AUY922 plus increasing concentrations of cilengitide for 3 days. (C, D) Synergistic effect of a TAE226 and AUY922 combination. Cell viability assays were performed in A549R and H1944R cells treated with 1,000 nM of AUY922 plus increasing concentrations of TAE226 for 3 days. The confidence interval (CI) was calculated using CalcuSyn software. (E) Western blot analysis of pFAK in A549R cells after each combination treatment. (F) Twelve mice harboring subcutaneous xenograft tumors derived from A549/ITGAvB3 cells were randomized into four groups. After the tumor volume reached 100 mm 3 , AUY922 at 10 mg/kg was administered 3 days/wk, and TAE226 (25 mg/kg) was administered 5 days/wk for up to 21 days. The tumor size was assessed at least three times a week. (G) Western blotting analysis detected cleaved PARP in the mouse xenograft tumors. CI, combi nation indexes; Fa, fraction affected; FAK, focal adhesion kinase; PARP, poly(ADP-ribose) polymerase; SD, standard deviation.

Article Snippet: The human KRAS -mutated NSCLC cell lines, H23, H358, H647, H1944, and A549, were purchased from the American Type Culture Collection (Manassas, VA) and grown at 37°C in 5% CO 2 using RPMI-1640 (Welgene Inc., Gyeonsan, Korea) containing 10% fetal bovine serum (FBS) and 1x penicillin-streptomycin solutions from Welgene Inc. AUY922 (HSP90 inhibitor, luminespib), cilengitide trifluoroacetate (inhibitor of the αvβ3 receptor), and TAE226 (focal adhesion kinase [FAK] inhibitor) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Software, Western Blot, Derivative Assay, Standard Deviation

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Integrin αvβ3 Induces HSP90 Inhibitor Resistance via FAK Activation in KRAS -Mutant Non–Small Cell Lung Cancer

doi: 10.4143/crt.2021.651

Figure Lengend Snippet: Schemas illustrating the identified mechanisms of acquired resistance to AUY922. FAK, focal adhesion kinase; ITGAv, integrin Av; ITGB3, integrin β3; TSPAN, tetraspanin 7.

Article Snippet: The human KRAS -mutated NSCLC cell lines, H23, H358, H647, H1944, and A549, were purchased from the American Type Culture Collection (Manassas, VA) and grown at 37°C in 5% CO 2 using RPMI-1640 (Welgene Inc., Gyeonsan, Korea) containing 10% fetal bovine serum (FBS) and 1x penicillin-streptomycin solutions from Welgene Inc. AUY922 (HSP90 inhibitor, luminespib), cilengitide trifluoroacetate (inhibitor of the αvβ3 receptor), and TAE226 (focal adhesion kinase [FAK] inhibitor) were purchased from Selleck Chemicals (Houston, TX).

Techniques:

Journal: Cell host & microbe

Article Title: Pseudomonas aeruginosa effector ExoS inhibits ROS production in human neutrophils

doi: 10.1016/j.chom.2017.04.001

Figure Lengend Snippet: (A) Cell lysates from uninfected human neutrophils, or from neutrophils infected with wild type PAO1, or a ΔpscD mutant strain were probed by Western blot for Akt, P-Akt (Thr308), p40phox, P-p40phox (Thr154), and Ras. The experiments were repeated 3 times with similar results. (B) Cell lysates from uninfected human neutrophils, or neutrophils infected for 30 minutes with PAO1, ΔpscD, or with strains in which the ADP-ribosyltransferase activity was inactivated in ExoS only (exoS(A-)), ExoT only (exoT(A-)), or both (exoST(A-)). P-Akt (Thr308), Akt, P-p40phox (Thr154), p40phox, total Ras, GTP-bound Ras, and Grb2 (loading control) were detected by western blot. The experiments were repeated 5 times with similar results. (C) Model of Ras (gray) bound to the Ras-binding domain (light blue) of PI3K (dark blue) based on the structure PDB:1he8 (Pacold et al., 2000). Residue Arg 41 of Ras is highlighted red. (D) Purified ExoS was used to ADP-ribosylate HA-tagged versions of Ras, or Ras(R41K), in vitro and subsequently mixed with purified PI3Kγ. The interaction between Ras and PI3Kγ was probed by immunoprecipitating Ras using an anti-HA-tag antibody. PI3Kγ, as well as unmodified and ADP-ribosylated Ras were detected by western blot. The experiments were repeated 3 times with similar results. Input and output levels of PI3Kγ were determined by densitometry. The input/output ratio for the untreated control sample was set to 100% and compared to the corresponding ExoS-treatment condition (mean and standard deviation of three independent replicates are noted below each lane). Results were compared by 1-way ANOVA with Bonferroni correction (**** p<0.0001). See also Figures S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-Akt rabbit polyclonal Cell Signaling Technology 9272 Phospho-Akt rabbit polyclonal Cell Signaling Technology 9275 p40 phox Santa Cruz Biotechnology Sc-48388 Phospho-p40 phox rabbit polyclonal Cell Signaling Technology 4311 Ras rabbit polyclonal Cell Signaling Technology 3965 GRB2 rabbit polyclonal Cell Signaling Technology 3972 Streptavidin APC eBioscience 17-4317-82 Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor® 488 Life Technologies A-11006 Bacterial Strains PAO1F (wild-type PAO1) ( Bleves et al., 2005 ) RP1831 PAO1F ΔpscD ( Sun et al., 2012 ) RP1903 PAO1F ΔexoS ( Sun et al., 2012 ) RP1883 PAO1F ΔexoT ( Sun et al., 2012 ) RP1945 PAO1F ΔexoST ( Sun et al., 2012 ) RP1947 PAO1F ΔexoS ΔexoT ΔexoY (Δ3TOX) ( Cisz et al., 2008 ) RP1949 PAO1F Δ popBD ( Sun et al., 2012 ) RP2750 PAO1F exoS (ADPR-) ( Sun et al., 2012 ) RP5481 PAO1F exoT (ADPR-) ( Sun et al., 2012 ) RP6202 PAO1F exoS (GAP-) exoT (GAP-) ( Sun et al., 2012 ) RP6203 PAO1F exoS (ADPR-) exoT (ADPR-) ( Sun et al., 2012 ) RP6205 pP25-GFPo constitutive GFP producing plasmid, CarbR ( Goodman et al., 2004 ) N/A BL21/pET28b-TAT-Ras This study RE9356 BL21/ pET28b-TAT-Ras(R41K) This study RE9357 Chemicals, Peptides, and Recombinant Proteins Luminol Sigma-Aldrich 123072 Superoxide dismutase from human erythrocytes Sigma-Aldrich S9636 Catalase from human erythrocytes Millipore 219008 Ficoll-Paque Plus (density 1.077 g/ml) GE Healthcare 17-1440-03 Dextran from Leuconostoc spp. (MR 450,00-650,000) Sigma-Aldrich 31392 1x RBC Lysis Buffer eBioscience 00-4333-57 100x Protease Inhibitor Cocktail Cell Signaling Technology 5871 10x Cell Lysis Buffer Cell Signaling Technology 9803 DAPI Solution 1mg/mL Pierce 62248 Human PI3K (p120γ), Active full length recombinant protein expressed in Sf9 cells SignalChem P29-10H NAD Sigma-Aldrich N1636 Human 14-3-3 Zeta, histidine tagged Sigma-Aldrich Z3402 Sulfo-NHS-LC-Biotin ThermoFisher 21327 PI3K inihibitor (AS-605240) Selleckchem S1410 PI3K inhibitor (GDC-0941) Selleckchem S1065 Critical Commercial Assays Active Ras Detection Kit Cell Signaling Technology 8821 Experimental Models: Cell Lines Human Peripheral Blood Neutrophils Case Western Reserve University: Hematopoietic Biorepository Core C57BL/6J mice Jackson Laboratory 000664 B6.129S- Cybb tm1Din /J (gp91 phox−/− ) mice Jackson Laboratory 002365 Software and Algorithms Metamorph Microscopy Automation and Image Analysis Software Molecular Devices Prizm GraphPad Software Open in a separate window Experimental Model and Subject Details

Techniques: Infection, Mutagenesis, Western Blot, Activity Assay, Control, Binding Assay, Residue, Purification, In Vitro, Standard Deviation

Journal: Cell host & microbe

Article Title: Pseudomonas aeruginosa effector ExoS inhibits ROS production in human neutrophils

doi: 10.1016/j.chom.2017.04.001

Figure Lengend Snippet: Experimental Model and Subject Details

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-Akt rabbit polyclonal Cell Signaling Technology 9272 Phospho-Akt rabbit polyclonal Cell Signaling Technology 9275 p40 phox Santa Cruz Biotechnology Sc-48388 Phospho-p40 phox rabbit polyclonal Cell Signaling Technology 4311 Ras rabbit polyclonal Cell Signaling Technology 3965 GRB2 rabbit polyclonal Cell Signaling Technology 3972 Streptavidin APC eBioscience 17-4317-82 Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor® 488 Life Technologies A-11006 Bacterial Strains PAO1F (wild-type PAO1) ( Bleves et al., 2005 ) RP1831 PAO1F ΔpscD ( Sun et al., 2012 ) RP1903 PAO1F ΔexoS ( Sun et al., 2012 ) RP1883 PAO1F ΔexoT ( Sun et al., 2012 ) RP1945 PAO1F ΔexoST ( Sun et al., 2012 ) RP1947 PAO1F ΔexoS ΔexoT ΔexoY (Δ3TOX) ( Cisz et al., 2008 ) RP1949 PAO1F Δ popBD ( Sun et al., 2012 ) RP2750 PAO1F exoS (ADPR-) ( Sun et al., 2012 ) RP5481 PAO1F exoT (ADPR-) ( Sun et al., 2012 ) RP6202 PAO1F exoS (GAP-) exoT (GAP-) ( Sun et al., 2012 ) RP6203 PAO1F exoS (ADPR-) exoT (ADPR-) ( Sun et al., 2012 ) RP6205 pP25-GFPo constitutive GFP producing plasmid, CarbR ( Goodman et al., 2004 ) N/A BL21/pET28b-TAT-Ras This study RE9356 BL21/ pET28b-TAT-Ras(R41K) This study RE9357 Chemicals, Peptides, and Recombinant Proteins Luminol Sigma-Aldrich 123072 Superoxide dismutase from human erythrocytes Sigma-Aldrich S9636 Catalase from human erythrocytes Millipore 219008 Ficoll-Paque Plus (density 1.077 g/ml) GE Healthcare 17-1440-03 Dextran from Leuconostoc spp. (MR 450,00-650,000) Sigma-Aldrich 31392 1x RBC Lysis Buffer eBioscience 00-4333-57 100x Protease Inhibitor Cocktail Cell Signaling Technology 5871 10x Cell Lysis Buffer Cell Signaling Technology 9803 DAPI Solution 1mg/mL Pierce 62248 Human PI3K (p120γ), Active full length recombinant protein expressed in Sf9 cells SignalChem P29-10H NAD Sigma-Aldrich N1636 Human 14-3-3 Zeta, histidine tagged Sigma-Aldrich Z3402 Sulfo-NHS-LC-Biotin ThermoFisher 21327 PI3K inihibitor (AS-605240) Selleckchem S1410 PI3K inhibitor (GDC-0941) Selleckchem S1065 Critical Commercial Assays Active Ras Detection Kit Cell Signaling Technology 8821 Experimental Models: Cell Lines Human Peripheral Blood Neutrophils Case Western Reserve University: Hematopoietic Biorepository Core C57BL/6J mice Jackson Laboratory 000664 B6.129S- Cybb tm1Din /J (gp91 phox−/− ) mice Jackson Laboratory 002365 Software and Algorithms Metamorph Microscopy Automation and Image Analysis Software Molecular Devices Prizm GraphPad Software Open in a separate window Experimental Model and Subject Details

Techniques: Plasmid Preparation, Recombinant, Red Blood Cell Lysis, Protease Inhibitor, Lysis, Western Blot, Software, Microscopy