Fig. 3

Post-transcriptional silencing of Dectin-1 gene expression on mBMDCs brings down NLGP binding to the cells with functional consequences at cytokine production levels. a Western blotting to measure Dectin-1 protein suppression by RNAi in mBMDCs, compared to controls (left) (n = 3), as mean ± SD (right). b Extent of NLGP-FITC binding to mBMDCs on silencing Dectin-1 compared to controls, in confocal (left) (scale bar: 10 µm) and widefield (right) (scale bar: 20 µm) fluorescence micrographs. c MFIs from replicates of those fluorescence microscopy-based observations (n = 3), as mean ± SD (MFI and CTCF stand for mean fluorescence intensity and corrected total cell fluorescence respectively). d Agarose gel electrophoresis, quantifying IL-10 and IL-12A or IL-12p35 transcripts by RT-PCR, to assess effects of Dectin-1 depletion by RNAi vs control siRNA, on TCM and NLGP’s gradual influence on mBMDCs (TCM stands for tumor conditioning media). e Flow cytometric quantifications of intracellular IL-10 and IL-12p35 of same groups as staggered-offset histograms. f IL-10 and IL-12p35 secretions measured within 48-h culture supernatants of the same groups (n = 6) by ELISA, represented as mean ± SD. g Trend of cytokine production from the same mBMDC groups, through line diagrams, as transcription (red line) vs. intracellular expression (grey line) (above) and intracellular expression (grey line) vs. secretion (cyan/blue line) (below) for both IL-10 (left) and IL-12A or IL-12p35 (right) as mean ± SD from (d), (e) and (f). β-actin was taken as loading control in blots and gels. Numerical data of (a) were tested by one-way ANOVA (F = 169.1, df = 8), and those of (c) were tested by two-way ANOVA, both followed by Tukey’s multiple comparisons test. Numerical data of (f) were tested by one-way ANOVA (F = 67.74 and 102.9 respectively, df = 29 and 29 respectively), followed by Tukey’s multiple comparisons test. Gaussian distribution of data were verified before each test (See also Fig. S2a-d)