Fig 2: Collagen and smooth muscle actin layer remain uninterrupted in Cav-1/STAT5a dKO mice mammary ducts following estrogen treatment. (A) Mammary glands of estrogen-treated WT, Cav-1 KO, and Cav-1/STAT5a dKO mice were stained with Masson’s trichrome to highlight the collagen (blue staining) lining the outside of the basement membrane of the ducts. Qualitatively, Cav-1 KO mammary glands stimulated with estrogen demonstrated a complete degradation of collagen surrounding the basement membrane, whereas WT ducts showed intact collagen deposition. Deletion of STAT5a from estrogen-treated Cav-1 KO mice restored collagen deposition to WT levels. For each experimental group, trichrome staining was performed in triplicate on mammary glands derived from 3 independent mice. (B) Mammary glands of estrogen-treated WT, Cav-1 KO, and Cav-1/STAT5a dKO mice were immunostained with an antibody recognizing alpha smooth muscle actin (SMA) to highlight the myoepithelial cells lining the inside of the basement membrane of the ducts. DAPI was used as a nuclear counterstain. The EVOS FL microscope was used to capture images at 40x objective with the DAPI and Texas Red light cubes (blue: DAPI immunostaining; red: SMA immunostaining). Qualitatively, the SMA layer surrounding estrogen-treated Cav-1 KO mammary ducts was disrupted (white arrows indicate breaks in the myoepithelial cells), but completely intact around WT ducts. Cav-1 KO mice lacking STAT5a expression maintained an intact SMA layer similar to WT mice. For each experimental group, immunofluorescence was performed in triplicate on mammary glands derived from 3 independent mice.

Fig 3: (A) Homeostasis between STAT5 and STAT3. While pSTAT5 levels declined upon termination of lactation, pSTAT3 levels increased. (B) Binding sites shared by STAT5 and STAT3 were identified by integrating STAT5A data at L1 and STAT3 data at I24. (C) Bar plot showing the reduction of STAT5A binding compared to the increase of STAT3 binding during involution. This indicates that STAT3 replaced STAT5, but not necessarily at the same binding sites. The venn diagrams illustrate the overlap of STAT5A and STAT3 enhancers for L1, I12 and I24. Due to the replacement of those two factors most overlaps could be detected at involution 12 h. (D) STAT3 binding to the three enhancer categories. Six percent (280) of class 1 STAT5A sites overlapped with STAT3. Thereby, STAT3 and H3K27ac peak height was reduced from L1 to I12, but not from I12 to I24. Twenty-five percent (1137) of class 2 enhancers co-bound STAT3, but by looking at the peak coverage during involution the H3K27ac decreased and STAT3 increased. The last group of common enhancers had 173 enhancers (55%) overlapping STAT5A and STAT3 enhancers and the coverage plot shows that the peak coverage of STAT3 increased and H3K27ac decreased slightly during involution. (E) Representative genes to demonstrate the STAT5/3 homeostasis. Glycam1 (class 1) displayed STAT3 co-binding at the promoter at L1, but no increase of STAT3 at the individual enhancers was observed. Wap (class 1) had no STAT3 co-binding at the third enhancer (E3). However, E1 and E2, which decommissioned at I24, co-bound STAT3 at I12 and even stronger at I24. The promoter showed continuous co-binding. The class 2 representative, Lalba, displayed constant co-binding at the promoter, and the enhancers showed a co-binding starting at involution 12 h, getting stronger at I24, when the STAT5A enhancers were absent. The resilient Bcl6 gene (class 3) showed continuous co-binding during involution.

Fig 4: SKLB-677 inhibited FLT3 activation and induced cell cycle arrest and apoptosis.(a) MV4-11 cells were treated with various concentrations of SKLB-677 or 0.1% DMSO for 1 h. The phosphorylation levels of FLT3 and downstream signal proteins were assessed using antibodies against total FLT3 (FLT3), phosphorylated Y589/591 FLT3 (p-FLT3), total STAT5 (STAT5), phosphorylated Y694 STAT5 (p-STAT5), total p44/42 MAPK (Erk1/2), and phosphorylated Y202/204 p44/42 MAPK (p-Erk1/2). β-actin was used as a loading control. (b) Primary PBMCs isolated from AML patients (FLT3-ITD positive) were treated with various concentrations of SKLB-677 or 0.1% DMSO for 6 h. The phosphorylation levels of FLT3 and downstream signal proteins were detected used the same method as that in panel a. (c) MV4-11 cells were treated with various concentrations of SKLB-677 or 0.1% DMSO for 24 h. Cell cycle analysis was performed using propidium iodide staining by flow cytometry. Data shown in the histogram are means ± SD from three independent experiments. (d) MV4-11 cells were cultured in the presence of SKLB-677 or 0.1% DMSO, followed by the detection of apoptosis by using Annexin-V and PI co-staining. The early and late apoptotic cells are shown in the right lower and right upper quadrant, respectively. The assays were performed in triplicate, and the data in the histogram represent average percentage of AnnexinV-positive cells ± SD. ns, not statistically significant, ***P < 0.005 vs. vehicle.

Fig 5: Transcriptional consequences of uSTAT5 depletion AGene set enrichment analysis (GSEA) of genes downregulated by STAT5 depletion shows a significant enrichment of genes expressed in common lymphoid progenitor (CLP) and in pre‐granulocyte and macrophage progenitor (pre‐GM).B, CGene ontology analysis (using DAVID) of genes upregulated (B) and downregulated (C) by uSTAT5 depletion. P‐values are raw P‐values from binominal test.DQuantitative RT–PCR was performed to validate uSTAT5 target genes. Expression was normalized to 18S rRNA. Histograms represent mean ± SD from three independent experiments. Two‐tailed Student's t‐test; *P < 0.05.