Journal: Cell reports

Article Title: STAT-independent functions of Janus kinases 1 and 2 are obligatory for the postnatal development of mammary epithelial ducts

doi: 10.1016/j.celrep.2025.116703

Figure Lengend Snippet: (A) Schematic outline of the transplantation of GFP-labeled mammary epithelium from JAK1/2 conditional knockout females (MMTV-Cre Jak1 fl/fl Jak2 fl/fl CAG-LSL-GFP) and heterozygous JAK1/2 knockout controls (MMTV-Cre Jak1 fl/wt Jak2 fl/wt CAG-LSL-GFP) into the contralateral epithelial-free fat pads of 3-week-old wild-type recipient females ( N = 6). (B) Carmine alum-stained mammary gland wholemounts of a recipient female 6 weeks after transplantation; bars, 1 mm. (C) Mammary gland wholemounts of a postpartum recipient female 18 weeks after transplantation; carmine alum-stained (upper panel) and corresponding GFP fluorescent image (lower panel) prior to fixation and staining; bars, 1 mm. (D) Immunoblot analysis of JAK1 and JAK2 expression in immortalized explanted mammary epithelial cells from heterozygous and homozygous JAK1/2 conditional knockout mice and wild-type control (upper panel), as well as unsorted and GFP-sorted immortalized epithelial cells from the mammary glands of MMTV-Cre Jak1 fl/fl Jak2 fl/fl CAG-LSL-GFP females in addition to MMTV-Cre Jak1 fl/wt Jak2 fl/wt CAG-LSL-GFP heterozygous control females (lower panel; two biological repeats per genotype). Beta-actin (ACTB) was used as a loading control. The densitometry results of immunoblots from 3 technical repeats of the 2 biological repeats are shown in and . (E) Fluorescence-activated sorting and enrichment scores of GFP-labeled mammary epithelial cells from JAK1/2 conditional double-knockout females and heterozygous knockout controls shown in (D).

Article Snippet: Protein bands were detected using the ECL chemiluminescence kit for immunoblot analysis [KwikQuant Ultra Digital-ECLTM Substrate Solution (Cat. #R1002)] according to the manufacturer’s instructions (Kindle Biosciences, LCC).

Techniques: Transplantation Assay, Labeling, Knock-Out, Staining, Western Blot, Expressing, Control, Fluorescence, Double Knockout

Journal: Cell reports

Article Title: STAT-independent functions of Janus kinases 1 and 2 are obligatory for the postnatal development of mammary epithelial ducts

doi: 10.1016/j.celrep.2025.116703

Figure Lengend Snippet: (A) Immunoblot analysis of the expression and activation of selected STAT proteins in mammary epithelial cells from STAT3 and STAT5a/b conditional triple-knockout mice (MMTV-Cre Stat3/5 fl/fl ; N = 4) and age-matched Stat3/5 fl/wt controls ( N = 2 and 4, respectively). E-cadherin (CDH1) and beta-actin (ACTB) were used as loading controls. PC, wild-type control epithelial cells treated with IL-4 served as a positive control for tyrosine-phosphorylated STAT6 (pY-STAT6). (B) Immunohistochemistry of tyrosine-phosphorylated STAT1 (pY-STAT1) on histologic sections of mammary glands from postpartum STAT3/5 triple-knockout and control females; bars, 20 μm. (C and D) Immunoblot analysis of active and total levels of STAT1 before and after the retroviralbased expression of Cre recombinase in three immortalized mammary epithelial cell lines (C) and three mouse embryonic fibroblast lines (D). Wild-type mammary epithelial cells and interferon gamma-treated wild-type mouse fibroblasts served as positive controls (C) for STAT5a and active STAT1, respectively. The densitometry results of the STAT proteins in (C) from 3 technical repeats of the 3 biological repeats are shown in . (E) Immunohistochemistry of active STAT1 (pY-STAT1) in alveolar cells of mammary glands of a postpartum WAP-Cre Stat3/5 fl/fl female and an age-matched control; bars, 20 μm.

Article Snippet: Protein bands were detected using the ECL chemiluminescence kit for immunoblot analysis [KwikQuant Ultra Digital-ECLTM Substrate Solution (Cat. #R1002)] according to the manufacturer’s instructions (Kindle Biosciences, LCC).

Techniques: Western Blot, Expressing, Activation Assay, Triple Knockout, Control, Positive Control, Immunohistochemistry

Journal: Cell reports

Article Title: STAT-independent functions of Janus kinases 1 and 2 are obligatory for the postnatal development of mammary epithelial ducts

doi: 10.1016/j.celrep.2025.116703

Figure Lengend Snippet: (A) Schematic of the knockout of STAT1, STAT3, STAT5a, and STAT5b in canonical JAK/STAT signaling cascades of the mammary gland. (B) Carmine alum-stained mammary gland wholemounts of nulliparous female mice with a targeted deletion of four Stat genes in the mammary epithelium MMTV-Cre Stat3/5 fl/fl Stat1 −/− ) and a STAT1 single-knockout littermate control; bars, 1 mm. (C) Immunoblot analysis of the seven known mammalian STAT proteins in mammary epithelial cells (MECs) from STAT1/3/5a/5b quadruple-knockout females ( N = 4) in comparison to age-matched STAT3/5a/5b triple-knockout mice ( N = 2). Other controls: C1 and C2, positive controls for active STAT3 and STAT5, mammary gland tissues from wild-type mice on day 1 of involution and lactation, respectively; C3, spleen as a positive control for STAT4; C4, wild-type MECs as a negative control for STAT4; C5 and C6 interferon-treated and untreated MECs as positive and negative controls for STAT2; C7 and C8, IL-4-treated and untreated wild-type MECs as positive and negative controls for active STAT6. GAPDH and ACTB served as loading controls.

Article Snippet: Protein bands were detected using the ECL chemiluminescence kit for immunoblot analysis [KwikQuant Ultra Digital-ECLTM Substrate Solution (Cat. #R1002)] according to the manufacturer’s instructions (Kindle Biosciences, LCC).

Techniques: Knock-Out, Staining, Control, Western Blot, Quadruple Knockout, Comparison, Triple Knockout, Positive Control, Negative Control

Journal: Cell reports

Article Title: STAT-independent functions of Janus kinases 1 and 2 are obligatory for the postnatal development of mammary epithelial ducts

doi: 10.1016/j.celrep.2025.116703

Figure Lengend Snippet: (A) Immunoblot analysis of STAT1 and JAK1 expression and activation in response to the pharmacological inhibition of JAK1 using 1 μM itacitinib in three mammary epithelial cell lines co-deficient in STAT3, STAT5a, and STAT5b. Beta-actin (ACTB) was used as a loading control. The densitometry results of immunoblots from 2 technical repeats of the 3 biological repeats are shown in . (B) Schematic of a quadruple knockout of STAT3, STAT5a, and STAT5b along with JAK1 to genetically ablate the compensatory activation of STAT1 in the STAT3/5a/5b triple-knockout mammary epithelium and to determine the STAT-independent contribution of JAK2 to mammary gland development. (C) Carmine alum-stained mammary gland whole-mounts of 4- and 6-week-old nulliparous females that are conditionally deficient inSTAT3, STAT5a/b, and JAK1 (MMTV-Cre Stat3/5 fl/fl Jak1 fl/fl ) and littermate controls without the MMTV-Cre transgene; bars, 1 mm. Dotted lines mark the invasive fronts of the terminal ends of ducts. (D) Immunoblot analysis of STAT1 expression and activation in response to the knockout of JAK1 in the mammary epithelium of STAT3/5a/5b triple-knockout females; 4 biological repeats of quadruple-knockout females compared to 2 wild-type controls and 2 STAT3/5a/5b triple-knockout mice. Beta-actin (ACTB) served as a loading control. The densitometry results of immunoblots from the 4 biological and 2 technical repeats are shown in .

Article Snippet: Protein bands were detected using the ECL chemiluminescence kit for immunoblot analysis [KwikQuant Ultra Digital-ECLTM Substrate Solution (Cat. #R1002)] according to the manufacturer’s instructions (Kindle Biosciences, LCC).

Techniques: Western Blot, Expressing, Activation Assay, Inhibition, Control, Quadruple Knockout, Triple Knockout, Staining, Knock-Out

Journal: Cell reports

Article Title: STAT-independent functions of Janus kinases 1 and 2 are obligatory for the postnatal development of mammary epithelial ducts

doi: 10.1016/j.celrep.2025.116703

Figure Lengend Snippet: (A and B) Immunoblot analysis of tyrosine-phosphorylated JAK1 and JAK2 and expression of selected STAT proteins in mammary epithelial cells from quadruple STAT1/3/5a/5b conditional knockout mice and wild-type controls that were treated with either oncostatin M (OSM) alone and human growth hormone (hGH) alone (A) or a combination of both (B); 2 biological repeats of experimental and control animals. GAPDH served as a loading control. (C) Immunoblot analysis to assess the activation of STAT6 and tyrosine phosphorylation of JAK1 in STAT1/3/5a/5b-deficient quadruple-knockout cells and controls following stimulation with IL-4. GAPDH was used as a loading control. PC, splenocytes as positive controls for the validated absence of STAT2 and STAT4 in the quadruple-knockout epithelial cells. (D) Summary of canonical and noncanonical signaling mechanisms by which JAK2, in cooperation with JAK1, drives the postnatal development of the mammary gland.

Article Snippet: Protein bands were detected using the ECL chemiluminescence kit for immunoblot analysis [KwikQuant Ultra Digital-ECLTM Substrate Solution (Cat. #R1002)] according to the manufacturer’s instructions (Kindle Biosciences, LCC).

Techniques: Western Blot, Expressing, Knock-Out, Control, Activation Assay, Phospho-proteomics, Quadruple Knockout