Journal: Pharmaceuticals

Article Title: Sparstolonin B Suppresses Proliferation and Modulates Toll-like Receptor Signaling and Inflammatory Pathways in Human Colorectal Cancer Cells

doi: 10.3390/ph18030300

Figure Lengend Snippet: Evaluation of the TLR2-TLR4 signaling pathway in HCT-116 cells. Cells were treated with DMSO (1 μL/mL), SsnB (25 μM), and/or PMA (10 nM) for 12 h in all experiments. Images were acquired with a 40× objective. ( A ) PCR analysis of TLR2. Data show the mean ± SD and n = 4 different measurements. Statistical analysis was performed with the Kruskal–Wallis test and the difference between the groups was determined using Tukey’s test. *, p < 0.05 vs. SsnB. ( B ) PCR analysis of TLR4. Data show the mean ± SD and n = 4 different measurements. Statistical analysis was performed with the Kruskal-Wallis test and the difference between the groups was determined using Tukey’s test. *, p < 0.01 vs. SsnB. ( C ) Protein levels of Toll-like Receptor 2 (TLR2). Data show the mean ± SD and n = 6 different measurements. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparisons. *, p < 0.001 vs. all other groups. ( D ) Protein levels of TLR4. Data show the mean ± SD and n = 6 different measurements. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparisons. *, p < 0.001 vs. all other groups. ( E ) Myeloid differentiation primary response protein (MyD88), phospho-ERK, and phospho-NF-kB were immunofluorescence stained in HCT-116 cells. ( F ) MyD88 fluorescence staining was quantified using ImageJ software. Values are the mean ± SD and n = 8. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparisons. *, p < 0.001, vs. all other groups. **, p < 0.001, vs. control and DMSO groups. ( G ) Protein levels of MyD88. Data show the mean ± SD and n = 6 different measurements. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparisons. *, p < 0.001 vs. all other groups. ( H ) Phospho-ERK fluorescence staining was quantified using ImageJ software. Values are the mean ± SD and n = 8. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparisons. *, p < 0.01, vs. all other groups. ( I ) Phospho-NF-kB fluorescence staining was quantified using ImageJ software. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparisons. *, p < 0.001 vs. all other groups. ( J ) Protein levels of tumor necrosis factor-alpha (TNF-α). Data show the mean ± SD and n = 6 different measurements. Statistical analysis was performed with the Kruskal–Wallis test and the difference between the groups was determined with Dunn’s multiple comparisons. *, p < 0.05 vs. control, DMSO, and SsnB. ( K ) Protein levels of interleukin 1Β (IL-1B). Data show the mean ± SD and n = 6 different measurements. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparisons. *, p < 0.05 vs. all other groups. ( L ) Protein levels of IL-6. Data show the mean ± SD and n = 6 different measurements. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparisons. *, p < 0.001 vs. all other groups.

Article Snippet: The primary antibodies used were PCNA rabbit polyclonal ab (1:100 dilution, #bs-0754R, Bioss Antibodies Inc., Woburn, MA, USA), phospho-ERK1/2 (Thr202/Tyr204) rabbit polyclonal ab (1:200 dilution, #AF1015, Affinity Biosciences, Changzhou, Jiangsu, China), phospho-NF-kB rabbit polyclonal ab (1:200 dilution, #AF2006, Affinity Biosciences, Changzhou, Jiangsu, China), MyD88 rabbit polyclonal ab (1:100 dilution, # BT-AP05682, BT Lab, Shanghai, China), and cleaved-caspase 3 rabbit polyclonal ab (1:100 dilution, #9664S, Cell Signaling Tech., Danvers, MA, USA).

Techniques: Immunofluorescence, Staining, Fluorescence, Software, Control