Journal: Oncoimmunology

Article Title: Interferon alpha bioactivity critically depends on Scavenger receptor class B type I function

doi: 10.1080/2162402X.2016.1196309

Figure Lengend Snippet: ApoAI mimetic peptide L37pA potentiates the antiviral and cytotoxic activity of IFNα. (A) Cytopathic effect reduction assay in L929 cells pretreated overnight with 2-fold serial dilutions of IFNα starting from 125 U/mL with L37pA (200 µg/mL), L37mut (200 µg/mL) or with no peptide. Cells viability was quantified 24 h after EMCV infection. (Extra sum-of-squares F test *** p < 0.001). (B) Cytotoxicity assay in L929 cells incubated for 3 d with IFNα (1500 U/mL) and L37pA (200 µg/mL), L37mut or with no peptide (one way ANOVA followed by Dunnett's multiple comparison test. *** p < 0.001). (C) Quantitative real time PCR analysis of interferon-stimulated genes after 3 h of stimulation of L929 cells with IFNα (200 U/mL), L37pA (200 µg/mL) or the combination. Data are expressed as mean + SEM (one way ANOVA, followed by Dunnett's multiple comparison test. ** p < 0.01, *** p < 0.001).

Article Snippet: The following cells were purchased from the ATCC: L929 (ATCC CCL1), BJ (ATCC CRL2522), 3T3 (ATCC CCL163), RAW 264.7 cells (ATCC TIB-71), MC-38 and CT-26 (ATCC CRL2638).

Techniques: Activity Assay, Infection, Cytotoxicity Assay, Incubation, Real-time Polymerase Chain Reaction

Journal: Oncoimmunology

Article Title: Interferon alpha bioactivity critically depends on Scavenger receptor class B type I function

doi: 10.1080/2162402X.2016.1196309

Figure Lengend Snippet: Mechanisms of IFNα and L37pA synergy. We determined the expression of Cxcl11 and Fas as readout of the effect of IFNα plus L37pA using quantitative real time RT-PCR in L929 cells treated as follows: (A) Cells were stimulated with IFNα (200 U/mL) for 3 h alone or in combination with L37pA (200 µg/mL), high density lipoprotein (HDL) (5 µg/mL), apolipoprotein A-I (ApoA-I) (30 µg/mL) or serum amyloid A (SAA) (30 µg/mL). (B) Cells were stimulated with IFNα (200 U/mL) for 3 h in combination with L37PA (200 µg/mL), the Alzheimer amyloid β peptide (Aβ) (200 µg/mL), cathelicidin (LL37) (200 µg/mL), phenol-soluble modulin 1 (M1) (200 µg/mL) or LPS (160 µg/mL). (C) Cells were pretreated with neutralizing antibodies against TLR2 (5 µg/mL), TLR4 (40 µg/mL) or with the combination for 1 h. Then, cells were treated with IFNα (200 U/mL) alone or plus L37pA (200 µg/mL) for 3 h. (D) Cytotoxicity assay in L929 cells incubated for 3 d with IFNα (1500 U/mL) and L37pA (200 µg/mL), and pretreated with neutralizing antibodies against TLR2 (5 µg/mL), TLR4 (5 µg/mL) for 1 h. Data are expressed as mean + SEM (one way ANOVA, followed by Dunnett's multiple comparison test. ** p < 0.01, *** p < 0.001).

Article Snippet: The following cells were purchased from the ATCC: L929 (ATCC CCL1), BJ (ATCC CRL2522), 3T3 (ATCC CCL163), RAW 264.7 cells (ATCC TIB-71), MC-38 and CT-26 (ATCC CRL2638).

Techniques: Expressing, Quantitative RT-PCR, Cytotoxicity Assay, Incubation

Journal: Oncoimmunology

Article Title: Interferon alpha bioactivity critically depends on Scavenger receptor class B type I function

doi: 10.1080/2162402X.2016.1196309

Figure Lengend Snippet: Inhibition of SR-B1 impairs IFNα function. L929 cells were transfected with siRNA targeting SR-B1 (siSR-B1) or control scrambled siRNA (siCTL) for 48 h. (A) SR-B1 mRNA was determined by quantitative real time PCR. Data are expressed as mean + SEM. (t-test. *** p < 0.001). Total SR-B1 protein was detected by immunoblot and β-actin is shown as loading control. (B) The silenced cells were stimulated with IFNα (200 U/mL) for 2 h. Then, ISGs expression was determined using quantitative real time PCR. mRNA levels were normalized to Rplpo. (C) Quantitative real time PCR analysis of ISGs expression after 3 h of stimulation with IFNα (200 U/mL) in L929 cells pretreated for 1 h with BLT1 (15 µM), ITX-5061 (30 µM) and Glyburide (500 µM). (D) Flow cytometry analysis of the IFNα receptor 1 (IFNAR1) in L929 cells treated with BLT-1 (15 µM) for 3 h. Normalized geometric mean of three samples (left panel) and representative histogram (right panel). Data are expressed as mean + SEM (E) Phosphorylated STAT1 and total STAT1 protein determined by immunoblotting in L929 cells stimulated with IFNα (1500 U/mL) for 30 min after incubation of BLT-1 (15 µM) or DMSO for 1 h. (F) Cytopathic effect reduction assay in mouse L929 cells pretreated overnight with 2-fold serial dilutions of IFNα starting from 125 U/mL in the presence or absence of BLT-1 (15 µM). Cells viability was quantified 24 h after EMCV infection (Extra sum-of-squares F test *** p < 0.001). (G) Cytotoxicity assay in mouse L929 incubated for 3 d with IFNα (1500 U/mL) in the presence or absence of BLT-1 (15 µM). (t-test or one way ANOVA, followed by the Dunnett's Multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The following cells were purchased from the ATCC: L929 (ATCC CCL1), BJ (ATCC CRL2522), 3T3 (ATCC CCL163), RAW 264.7 cells (ATCC TIB-71), MC-38 and CT-26 (ATCC CRL2638).

Techniques: Inhibition, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Flow Cytometry, Incubation, Infection, Cytotoxicity Assay

Journal: Oncoimmunology

Article Title: Interferon alpha bioactivity critically depends on Scavenger receptor class B type I function

doi: 10.1080/2162402X.2016.1196309

Figure Lengend Snippet: SR-B1 is necessary for the clathrin-mediated endocytosis. (A) Quantitative real time PCR analysis of ISGs expression 3 h after stimulation with IFNα (200 U/mL) in L929 cell pretreated for 1 h with chlorpromazine (CPZ) (10 µM) or BLT1 (15 µM). Data are expressed as mean + SEM (t-test. * p < 0.05). (B) Flow cytometry analysis of the IFNα receptor 1 (IFNAR1) prior treatment with BLT-1 (15 µM) or CPZ (10 µM) for 3 h and flow cytometry analysis in L929 cells treated with BSA-FITC ( C ) or L37pA-FITC (D) for 1 h prior treatment with CPZ (10 µM) or BLT-1 (15 µM) for 30 min at 37°C or 4°C. The normalized geometric mean (GM) fluorescence (number of replicates = 3) and a representing image at 37°C is shown. Data are expressed as mean + SEM (one way ANOVA, followed by Dunnett's multiple comparison test. *** p < 0.001).

Article Snippet: The following cells were purchased from the ATCC: L929 (ATCC CCL1), BJ (ATCC CRL2522), 3T3 (ATCC CCL163), RAW 264.7 cells (ATCC TIB-71), MC-38 and CT-26 (ATCC CRL2638).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Fluorescence

Journal: Oncoimmunology

Article Title: Interferon alpha bioactivity critically depends on Scavenger receptor class B type I function

doi: 10.1080/2162402X.2016.1196309

Figure Lengend Snippet: BLT-1 increases NDV infection. (A) Representative flow cytometry dot plot 24 h after infection with an MOI of 1 of NDV expressing GFP in L929 cells pretreated 1 h with BLT-1 (15 µM), vehicle (DMSO) or left untreated (UT). The geometric mean (GM) of GFP expression and the percentage of infection (%) are expressed as mean + SEM (number of replicates = 3). (B) Quantitative real time PCR analysis of ISGs mRNA at 6 h, 12 h and 24 h postinfection at an MOI of 1 after treatment with BLT-1 (15 µM) for 1 h. Data are expressed as mean + SEM (number of replicates = 3). (t-test. * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The following cells were purchased from the ATCC: L929 (ATCC CCL1), BJ (ATCC CRL2522), 3T3 (ATCC CCL163), RAW 264.7 cells (ATCC TIB-71), MC-38 and CT-26 (ATCC CRL2638).

Techniques: Infection, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction

Journal: ACS Applied Bio Materials

Article Title: Development of Biomimetic Gelatin-Hydroxyapatite Composites Containing Doxycycline with Osteogenic Potential

doi: 10.1021/acsabm.5c00355

Figure Lengend Snippet: (A) Viability percentage of L929 fibroblasts treated with 5 mg/mL eluates containing composites HA-gelatin in different concentrations of doxycycline, at different experiment times, incubated for 24 h; (B) viability percentage of MC3T3 preosteoblasts treated with 5 mg/mL eluates containing composites HA-gelatin in different concentrations of doxycycline, at different experiment times, incubated for 24 h.

Article Snippet: Mouse fibroblasts L929 (ATCC CCL-1), and murine preosteoblasts MC3T3 (ATCC, CRL-2593) were used for tests.

Techniques: Incubation