Fig 2: NEK6 inhibits apoptosis through the activation of STAT3. (A) Immunoblot analysis of the phosphorylated STAT1 (p-STAT1), STAT1, phosphorylated STAT3 (p-STAT3), and STAT3 in RAW 264.7 cells stimulated with 10 µg/ml of gamma-irradiated M. tuberculosis. (B) Immunoblot analysis of the p-STAT1, STAT1, p-STAT3, and STAT3 in RAW 264.7 cells and BMDMs transfected with Myc-NEK6. (C) BMDMs from WT and NEK6-deficient (Nek6-/-) mice were stimulated with 10 µg/ml of gamma-irradiated M. tuberculosis for 24 h. The expressions of p-STAT1, STAT1, p-STAT3, STAT3, and NEK6 were analyzed by Western blotting. (D) Immunoblot analysis of the p-STAT3 and STAT3 in lungs, spleens, and livers from WT and Nek6-/- mice at 7 dpi. (E and G) The expression levels of BCL-2, BCL-XL, BCL-W, and MCL-1 in 10 µg/ml of gamma-irradiated M. tuberculosis-stimulated BMDMs from WT and Nek6-/- mice were calculated by qRT-PCR at the indicated times (E) and by Western blotting (G). (F and H) BMDMs from Nek6-/- mice were pretreated with 20 ng/ml IL-6 for 4 h h, then cells were stimulated with 10 µg/ml of gamma-irradiated M. tuberculosis. The expression levels of BCL-2, BCL-XL, BCL-W, and MCL-1 were calculated by qRT-PCR (F) and Western blotting (H) at the indicated times. (I) BMDMs from WT and Nek6-/- mice were stimulated with 10 µg/ml of gamma-irradiated M. tuberculosis for 24 h, and the apoptosis rates were detected by flow cytometry. (J) BMDMs from Nek6-/- mice were pretreated with 20 ng/ml of IL-6 for 4 h, and then cells were stimulated with 10 µg/ml of gamma-irradiated M. tuberculosis for 24 h. The apoptosis rates were detected by flow cytometry. Statistical significance between groups was determined by two-tailed Student’s t test. All data are presented as the means ± SDs and were derived from three independent experiments. All blots are representative of three independent experiments. **, P < 0.01.

Fig 3: Effect of RFX on cell death in ER+ mammary gland cancer. (A) RFX mediated activation of mitochondrial associated protein signalling in mammary gland cells. Protein extracted from control (1), toxic control (MNU, 47 mg kg-1) (2), RFX (25 mg kg-1) (3) and RFX (50 mg kg-1) (4) were subjected to immunoblotting of pro-apoptotic (BAD) and anti-apoptotic (BCL-2 and BCL-xl), and inflammatory marker (NF-?ß). ß-Actin was used as internal loading control. Each experiment was performed in triplicate; (B) respective quantitative densitometry graphs of immunoblots of the proteins; (C) and (D) regulation of caspase-3 and caspase-8 by RFX in serum samples respectively. The activity of caspase was determined by commercial fluorescence based kits; and (E) ratio of BAD/BCL-2 subjected to MNU and RFX treatment. The data were represented as mean ± SD of three independent experimental protocols. Comparisons were made on the basis of the one-way ANOVA followed by Bonferroni multiple test. All groups were compared to the toxic control and control groups (*/ap < 0.05, **/bp < 0.01, ***/cp < 0.001).

Fig 4: (A). The upper panel shows one example of a sectioned biopsy with a haematoxylin background and Ki-67 stained (brown). Villus length (a) and crypt depth (b) were assessed only where tissue orientation was optimal and allowed identification of tip of villus, transition from villus to crypt and the crypt bottom. Villi enlargement factor was assessed using the gridline intersection method of the surface epithelium in relation to muscularis mucosae (the latter depicted in c). (B). Villus length, crypt depth and villi enlargement factor were on a similar magnitude following HCD or HFD. Ki-67 cells in the crypts were significantly more stained in relation to all cells after HCD compared to after HFD (p = 0.027). (C). The lower panel shows the apoptosis biomarkers Caspase-3 and Bcl-XL, which did not differ between diets. BAX was increased following HCD as compared to HFD (p = 0.034). However, the ratios of BAX/Bcl-XL were on a similar level independent of diet. Data are means + SEM; n = 11–15; * p = 0.05 Wilcoxon signed-rank test.