Fig 2: Effects of GHRH antagonists in human primary PA cell cultures. Cell viability, assessed by Alamar blue assay, in primary cells derived from GH-PAs (A,B) and ACTH-PAs (C,D). The cells were either untreated (c, control) or treated with 0.1 µm octreotide (OCT), 1 µm MIA-602 (A) or 1 µm MIA-690 (B), both alone or in combination with OCT, for the times indicated. Results, expressed as percent of control, are means ± SEM of three replicates. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. c; # p < 0.05 by unpaired Student’s t-test (n = 3 for A; n = 2 for B; n = 1 for C and D). For C and D, because of n = 1, no error bars are presented and no statistical analysis was performed. Expression profile of GHRH, SV1, and GHRH-R in GH-PAs (E) and ACTH-PAs (F) assessed by real-time PCR. Results represent absolute mRNA copy number adjusted by a normalization factor (NF), calculated from the expression levels of three control genes (GAPDH, ACTB, and HPRT) and are means ± SEM (n = 3 for E; n = 1 for F).

Fig 3: S100A7 expression inversely associated with TLR4 expression in breast tumor. (A) Bar diagram representing the level of LPS (endotoxin) in the mammary fat pad of each group. The data presented here are the mean ± SEM of three biological triplicate (n = 3). One-way ANOVA (Tukey's multiple comparisons test) was used to calculate the P-values. (*P < 0.05, **P < 0.01, and ***P < 0.001, ns indicates nonsignificant). (B) Expression of S100A7 and TLR4 was analyzed in tissue lysates harvested from each group by western blot. ß-Actin (ACTB) was used as a loading control. Bar diagrams represent the fold changes of S100A7 and TLR4 in three biological replicates of all the different groups. The data represented the mean ± SEM of three biological replicates (*P < 0.05, **P < 0.01, and ***P < 0.001, ns indicates nonsignificant). One-way ANOVA (Tukey's multiple comparisons test) was used to calculate the P-values. (C) Representative immunohistochemistry (IHC) images of same malignant breast tumor tissues stained with S100A7 and TLR4 antibodies. Scale bar = 300 µm. (D) Spearman’s correlation analysis of S100A7 and TLR4 protein expression in tissue microarrays (TMAs) containing malignant breast tumor tissues (n = 20) and their normal adjacent controls (n = 20). (E) Spearman’s correlation analysis of S100A7 and TLR4 protein expression only in malignant breast tumor tissues (n = 20). Two-tailed t-test was used to calculate the P-values.