Journal: PLoS ONE

Article Title: Soluble HMGB1 Is a Novel Adipokine Stimulating IL-6 Secretion through RAGE Receptor in SW872 Preadipocyte Cell Line: Contribution to Chronic Inflammation in Fat Tissue

doi: 10.1371/journal.pone.0076039

Figure Lengend Snippet: List of primers used in this study for RT-PCR on SW872 RNA.

Article Snippet: Primary and secondary antibodies were diluted in PBS-BSA and incubated at room temperature with agitation for 45 min. Primary antibodies were anti-TLR2 (clone T2.5, 25 µg.mL −1 , HyCult Biotechnology), anti-TLR4 directly conjugated with R-Phycoerythrin (clone HTA125, 25 µg.mL −1 HyCult Biotechnology) or anti-RAGE (polyclonal antibody, Chemicon international) at 1∶100 dilution.

Techniques:

Journal: PLoS ONE

Article Title: Soluble HMGB1 Is a Novel Adipokine Stimulating IL-6 Secretion through RAGE Receptor in SW872 Preadipocyte Cell Line: Contribution to Chronic Inflammation in Fat Tissue

doi: 10.1371/journal.pone.0076039

Figure Lengend Snippet: A) TLR2, TLR4, RAGE and HMGB1 mRNA expression was assessed by RT-PCR from total RNA obtained from SW872 cell after 12h in culture, with (+) and without (−) Reverse Transcriptase (RT). B) Cell surface expression of TLR2, TLR4 and RAGE were analyzed by FACS C) SW872 cells were treated with rHMGB1 1 µg.mL −1 for 5 h with control mouse IgG (CTL), TLR2 or TLR4 blocking antibody (n = 4). D) SW872 cells were treated as in (C) except that control goat IgG (CTL) or goat polyclonal anti-RAGE were used (n = 4). All values are expressed as means +/− SD and degrees of significance are indicated in the figure captions as follow, * p<0.05, ** p<0.01, *** p<0.001 and ns = not significant.

Article Snippet: Primary and secondary antibodies were diluted in PBS-BSA and incubated at room temperature with agitation for 45 min. Primary antibodies were anti-TLR2 (clone T2.5, 25 µg.mL −1 , HyCult Biotechnology), anti-TLR4 directly conjugated with R-Phycoerythrin (clone HTA125, 25 µg.mL −1 HyCult Biotechnology) or anti-RAGE (polyclonal antibody, Chemicon international) at 1∶100 dilution.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Blocking Assay

Journal: PLoS ONE

Article Title: miR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells

doi: 10.1371/journal.pone.0068296

Figure Lengend Snippet: ( A ) TLR2 expression levels were analyzed by real-time PCR following transfection with 25 nM miR-1225-5p mimic. Increasing the amount of miR-1225-5p within ARPCs resulted in a 1.3 fold reduction of TLR2 mRNA levels 24 hours after transfection. Expression data were normalized on the housekeeping gene β-actin. Data are representative of three independent experiments (means ± SEM), *p<0.01. ( B–C ) TLR2 protein expression after transfection with 50 nM miR-1225-5p mimic. A strong reduction of TLR2 in ARPC was found after 3 days from transfection with miR-1225-5p mimic, as shown by immunofluorescence staining. To-pro-3 counterstains nuclei (blue). Original view X63.

Article Snippet: The cells were blocked for 1 h (BSA in PBS, pH 7.4) and then incubated with a mouse anti-human TLR2 mAb (Hycult Biotechnology) and a mouse anti-human CD133 mAb (Miltenyi Biotec) overnight at 4°C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunofluorescence, Staining

Journal: Medical Oncology (Northwood, London, England)

Article Title: Evidence of a role for interleukin-6 in anoikis resistance in oral squamous cell carcinoma

doi: 10.1007/s12032-022-01664-5

Figure Lengend Snippet: Cytokine production in PGK, DOK and SCC-4 cells. PGK, DOK and SCC-4 were untreated or stimulated for 24 h with TLR2 agonists: Pam3CSK4 (0.2 μg/ml), Pam2CSK4 (0.2 μg/ml) or TLR4 agonist LPS (0.5 μg/ml). Levels of ( A ) IL-6, ( B ) IL-11, ( C ) IL-8 and ( D ) TNF-α in the culture supernatants were determined by ELISA. Results were plotted using Graphpad prism 8. Data shown are representative of at least three independent experiments. Values represent the mean ± S.E.M of three independent experiments. Statistical analysis was performed using one-way ANOVA with a post hoc Tukey’s test to compare mean values between untreated and treated groups within the cell line, * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Antibody information: Human anti-IL-6Ra (MAB227), Mouse monoclonal IgG 1 , clone 17506 (R&D systems); Human IL-6 (MAB2061), 1936 (R&D systems); Isotype Control (MAB004), mouse monoclonal IgG 2B , clone 20116 (R&D systems); Human TLR2 antibody (HM1054), mouse monoclonal IgG 1, Clone T2.5 (Hycult Biotech).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Medical Oncology (Northwood, London, England)

Article Title: Evidence of a role for interleukin-6 in anoikis resistance in oral squamous cell carcinoma

doi: 10.1007/s12032-022-01664-5

Figure Lengend Snippet: The effect of TLR2 neutralising antibody on IL-6 production in SCC-4 cells. ( A ) DOK and ( B ) SCC-4 cells were untreated or pre-treated with anti-TLR2 Ab (10 μg/ml) for 1 h, prior to stimulation with Pam2CSK4 (0.2 μg/ml) and Pam3CSK4 (0.2 μg/ml) for 24 h. Culture supernatants were analysed for IL-6 secretion by ELISA. Results were plotted using Graphpad prism 8. Values represent the mean ± S.E.M of three independent experiments. Statistical analysis was performed using one-way ANOVA with a post hoc Dunnett’s test to compare mean values among the groups, * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Antibody information: Human anti-IL-6Ra (MAB227), Mouse monoclonal IgG 1 , clone 17506 (R&D systems); Human IL-6 (MAB2061), 1936 (R&D systems); Isotype Control (MAB004), mouse monoclonal IgG 2B , clone 20116 (R&D systems); Human TLR2 antibody (HM1054), mouse monoclonal IgG 1, Clone T2.5 (Hycult Biotech).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Medical Oncology (Northwood, London, England)

Article Title: Evidence of a role for interleukin-6 in anoikis resistance in oral squamous cell carcinoma

doi: 10.1007/s12032-022-01664-5

Figure Lengend Snippet: TLR2 and TLR6 expression levels in SCC-4 cells and DOK cells. DOK and SCC-4 cells were left untreated or pre-treated with anti-TLR2 Ab (10 μg/ml) for 1 h, prior to stimulation with Pam3CSK4 (0.05 μg/ml) or Pam2CSK4 (0.05 μg/ml) for 24 h. Lysates were collected and samples were run on 12% SDS-PAGE gel. Western Blot were probed with anti-TLR2 and anti-TLR6 antibodies with anti-GAPDH used as a loading control. Experiment was done in triplicate. Densitometric analysis was performed using image lab software on TLR2, TLR6 and GAPDH blots. Sample blots for ( A ) DOK and ( B ) SCC-4 and collated densitometry data for ( C ) TLR2 and ( D ) TLR6 are presented. Results were plotted using Graphpad prism 8. Values represent the mean ± S.E.M of three independent experiments. Statistical analysis was performed using two-way ANOVA with a post hoc Sidak’s test to compare mean values among the groups, * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Antibody information: Human anti-IL-6Ra (MAB227), Mouse monoclonal IgG 1 , clone 17506 (R&D systems); Human IL-6 (MAB2061), 1936 (R&D systems); Isotype Control (MAB004), mouse monoclonal IgG 2B , clone 20116 (R&D systems); Human TLR2 antibody (HM1054), mouse monoclonal IgG 1, Clone T2.5 (Hycult Biotech).

Techniques: Expressing, SDS Page, Western Blot, Software

Journal: Medical Oncology (Northwood, London, England)

Article Title: Evidence of a role for interleukin-6 in anoikis resistance in oral squamous cell carcinoma

doi: 10.1007/s12032-022-01664-5

Figure Lengend Snippet: Survival rate in anoikis assays DOK and SCC-4 cells treated with rhIL-6, IL-6 neutralising antibody, IL-6Ra antibody, IL-6Ra + rhIL-6, Pam2CSK4, anti-TLR2 neutralising antibody + Pam2CSK4, and anti-TLR2 neutralising antibody + Pam2CSK4 for 24 h. Tissue culture plates were coated with 200 μl poly-(hydroxyethyl methacrylic) acid (poly-HEMA) thus inhibiting the ability of the cells to attach to the tissue culture plastic. ( A ) DOK and ( B ) SCC-4 cells were then seeded at 100,000 cells/well and allowed to attach. For those cells to be treated with 30 ng/ml rhIL-6, 10 μg/ml IL-6 neutralising antibody, 40 μg/ml IL-6Ra antibody, 40 μg/ml IL-6Ra + 30 ng/ml rhIL-6, 0.2 μg/ml Pam2CSK4, and 10 μg/ml anti-TLR2 neutralising antibody + 0.2 μg/ml Pam2CSK4, complete medium was used as control, as well as medium containing 10 μg/ml isotype IgG antibody. After 24 h, 50 μl of alamarBlue ® dye was added to each well and incubated again at 37 °C/5% CO 2 for 3.5 h. SCC-4 cells demonstrated significant resistance to anoikis (% survival) when treated with 30 ng/ml rhIL-6 and 0.2 μg/ml Pam2CSK4 compared to the control group. There is a100% survival of adherent cells in the uncoated plates. Fluorescence was measured using a SpectraMax Gemini plate reader at excitation wavelength 544 nm and emission wavelength 590 nm. Results were plotted using Graphpad prism 8. Results represent at least three experiments performed in duplicate (mean ± SEM ( n )). Statistical analysis was performed one-way ANOVA with a post hoc Tukey’s test to compare mean values among the groups, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: Antibody information: Human anti-IL-6Ra (MAB227), Mouse monoclonal IgG 1 , clone 17506 (R&D systems); Human IL-6 (MAB2061), 1936 (R&D systems); Isotype Control (MAB004), mouse monoclonal IgG 2B , clone 20116 (R&D systems); Human TLR2 antibody (HM1054), mouse monoclonal IgG 1, Clone T2.5 (Hycult Biotech).

Techniques: Incubation, Fluorescence