Fig. 3

Par3 and aPKCλ control carcinogen-induced inflammatory signaling but not early immune cell recruitment. a Quantification of immunofluorescence analysis as previously described (2B) for p-p65 (S468) (Cell Signaling Techn. #3039) on paraffin sections of mice 24 h after DMBA treatment (% of DAPI positive cell nuclei; mean ± SEM; *p < 0.05, **p < 0.01; one-way ANOVA/Tukey’s post-hoc test). b Quantification of western blot analysis for total p65 protein in epidermal lysates of mice 24 h after DMBA treatment (mean ± SEM; *p < 0.05, **p < 0.01; one-way ANOVA/Tukey’s post-hoc test). Sample preparation and western blot analysis was performed as previously described [17]. c Quantification of immunofluorescence analysis as previously described (2B) for p-STAT3 (Y705) (Cell Signaling Techn. #9145) on paraffin sections of short-term DMBA/TPA treated mice (% of DAPI positive cell nuclei; mean + SEM; **p < 0.01, ***p < 0.001; P58: n(Cre ctrl) = 5, n(Par3eKO) = 4, n(aPKCλeKO) = 3, n(edKO) = 5; 24 h post-DMBA: n(Cre ctrl) = 3, n(Par3eKO) = 4, n(aPKCλeKO) = 4, n(edKO) = 3; short-term DMBA/TPA: n(Cre ctrl) = 5, n(Par3eKO) = 5, n(aPKCλeKO) = 4, n(edKO) = 4; one-way ANOVA/Tukey’s post-hoc). For short-term DMBA/TPA treatments, at P58 mice were treated with a single dose of DMBA (40 μg in 200 μl acetone, Sigma) and three applications of TPA (200 μl of 10⁻⁴M TPA/acetone) at P65, 68 and 72, and killed 2 days after the last TPA treatment. d Quantification of CD45-positive cells/mm dermis of untreated mice as described in 2B (mean ± SEM; ***p < 0.001; one-way ANOVA/Tukey’s post-hoc test). e Quantification of CD45-positive cells/ mm epidermis of untreated mice (mean ± SEM; *p < 0.05, **p < 0.01; one-way ANOVA/Tukey’s post-hoc test). f Quantification of CD3-positive cells/ mm dermis of untreated mice (mean ± SEM; one-way ANOVA; Tukey’s post-hoc test, *p < 0.05). g Quantification of CD3-positive cells/ mm epidermis of untreated mice (mean ± SEM; one-way ANOVA/Tukey’s post-hoc test, *p < 0.05). h Quantification of F4/80-positive cells/ mm dermis of untreated mice (mean ± SEM; **p < 0.01; ***p < 0.001; ***p < 0.001; one-way ANOVA; Tukey’s post-hoc test). i Quantification of CD45-positive cells/mm dermis of short-term DMBA/TPA treated mice (mean ± SEM; one-way ANOVA/Tukey’s post-hoc test). j Quantification of CD45-positive cells/ mm epidermis of short-term DMBA/TPA treated mice (mean ± SEM; one-way ANOVA/Tukey’s post-hoc test). k Quantification of CD3-positive cells/mm dermis of short term DMBA/TPA treated mice (mean±SEM, one-way ANOVA/Tukey’s post-hoc test). l Quantification of CD3-positive cells/mm epidermis of short-term DMBA/TPA treated mice (mean ± SEM, *p < 0.05, one-way ANOVA/Tukey’s post-hoc test). m Quantification of F4/80-positive cells/mm dermis of short term DMBA/TPA-treated mice (mean ± SEM, one-way ANOVA/Tukey’s post-hoc test). n Immunofluorescence analysis for CD45 (12-0451-82, Invitrogen) on paraffin sections of short-term DMBA/TPA-treated mice (scale bar = 50 µm). Immunofluorescence analysis was performed as described in 1E. Nuclei were counterstained with DAPI. o Immunofluorescence analysis for CD3 (AM11102PU-S, Acris) on paraffin sections of short-term DMBA/TPA-treated mice (scale bar: 50 µm). Immunofluorescence analysis was performed as previously described including additional antigen retrieval prior to blocking via incubation in 3% H₂O₂/0.5% KOH (Sigma-Aldrich) for 30 min at 37 °C. Nuclei were counterstained with DAPI. p Immunofluorescence analysis for F4/80 (MCA497PET, ABD serotec) as previously described (3D) on paraffin sections of short-term DMBA/TPA-treated mice (scale bar = 100 µm). Nuclei were counterstained with DAPI. See Suppl. Table 1 for antibody dilutions