Journal: Frontiers in cell and developmental biology

Article Title: Hormone-Responsive BMP Signaling Expands Myoepithelial Cell Lineages and Prevents Alveolar Precocity in Mammary Gland.

doi: 10.3389/fcell.2021.691050

Figure Lengend Snippet: FIGURE 1 | BMPR1a was upregulated by hormones during pregnancy. (A) Immunofluorescence staining for BMPR1a (red) and K14 (green) in 6-week-old wild-type mouse mammary gland ducts and terminal end buds. Arrows point to myoepithelial cells. n = 3 mice. Scale bar, 25 µm. (B) Western blotting for BMPR1a in isolated mammary gland epithelial cells at the indicated developmental stages (P14.5, pregnancy day 14.5; P18.5, pregnancy day 18.5; L2, lactation day 2). β-Actin was used as a loading control. (C) Immunofluorescence staining for BMPR1a in mammary glands of wild-type mice at the indicated time points (P14.5, pregnancy day 14.5). n = 3 mice per time point. Scale bar, 25 µm. (D) qRT-PCR analysis of Bmpr1a in FACS sorted myoepithelial (Myo) and luminal (Lu) cells from wild-type mice at (Continued)

Article Snippet: The following primary antibodies were used: BMPR1a (1:1000, 12702-1-AP, Proteintech), Sp1 (1:2000, 21962-1-AP, Proteintech), pSmad1/5 (1:1000, 9516, CST), β-Casein (1:500, sc-166530, Santa Cruz), Plin2 (1:1000, ab108323, Abcam), Stat5 (1:1000, ab68465, Abcam), pStat5 (1:1000, 9359, CST), K14 (1:1000, ab7800, Abcam), p63 (1:1000, ab124762, Abcam), Slug (1:1000, 9585, CST), K8 (1:1000, ab59400, Abcam), P-cadherin (1:1000, AF761, R&D), GAPDH (1:1000, 5174, CST), β-Actin (1:1000, 4970, CST), and β-Tubulin (1:5000, ab6046, Abcam).

Techniques: Staining, Western Blot, Isolation, Control, Quantitative RT-PCR

Journal: Frontiers in cell and developmental biology

Article Title: Hormone-Responsive BMP Signaling Expands Myoepithelial Cell Lineages and Prevents Alveolar Precocity in Mammary Gland.

doi: 10.3389/fcell.2021.691050

Figure Lengend Snippet: FIGURE 2 | BMPR1a deficiency resulted in enlarged alveoli during pregnancy. (A) Schematic map of the constructs used to generate inducible mammary epithelial BMPR1a cKO mice (cKO). (B) Western blotting for BMPR1a and pSmad1/5 in mammary epithelial cells isolated from control (Ctrl) and cKO mice at pregnancy day 14.5 (P14.5). β-Actin was used as a loading control. Statistical analysis the expression of BMPR1a/β-Actin and pSmad1/5/β-Actin. n = 3 mice. (C,D) Whole-mount staining of mammary glands from control and cKO mice at 6 weeks of age. The mice were treated with Dox from 4 weeks of age. Quantification of duct length beyond the lymph node. n ≥5 biological replicates. Scale bar, 1 mm. (E) Whole-mount staining of mammary glands from control and cKO mice at pregnancy day 12.5 (P12.5) (n = 3 per group), pregnancy day 14.5 (P14.5) (n = 4 per group) and pregnancy day 18.5 (P18.5) (n = 3 per group). High-magnification areas are shown in black boxes. Scale bar, 1 mm. (F) Hematoxylin and eosin staining of mammary glands at the indicated time points (P12.5, pregnancy day 12.5; P14.5, pregnancy day 14.5; P18.5, pregnancy day 18.5). n = 3 mice per time point. Scale bar, 100 µm. (G) Quantitation of alveoli density in control and cKO mice HE-stained mammary gland sections at pregnancy day 14.5. n = 3 mice per group. Data were presented as means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: The following primary antibodies were used: BMPR1a (1:1000, 12702-1-AP, Proteintech), Sp1 (1:2000, 21962-1-AP, Proteintech), pSmad1/5 (1:1000, 9516, CST), β-Casein (1:500, sc-166530, Santa Cruz), Plin2 (1:1000, ab108323, Abcam), Stat5 (1:1000, ab68465, Abcam), pStat5 (1:1000, 9359, CST), K14 (1:1000, ab7800, Abcam), p63 (1:1000, ab124762, Abcam), Slug (1:1000, 9585, CST), K8 (1:1000, ab59400, Abcam), P-cadherin (1:1000, AF761, R&D), GAPDH (1:1000, 5174, CST), β-Actin (1:1000, 4970, CST), and β-Tubulin (1:5000, ab6046, Abcam).

Techniques: Construct, Western Blot, Isolation, Control, Expressing, Staining, Quantitation Assay

Journal: Frontiers in cell and developmental biology

Article Title: Hormone-Responsive BMP Signaling Expands Myoepithelial Cell Lineages and Prevents Alveolar Precocity in Mammary Gland.

doi: 10.3389/fcell.2021.691050

Figure Lengend Snippet: FIGURE 3 | BMPR1a deficiency resulted in precocious alveolar differentiation during pregnancy. (A) Immunohistochemistry staining for WAP in control and cKO mammary glands at pregnancy day 14.5. n = 3 mice per group. Scale bar, 50 µm. (B) Immunohistochemistry staining and western blotting for β-Casein in control and cKO mammary glands at pregnancy day 14.5. n = 3 mice per group. β-Tubulin was used as a loading control. Scale bar, 50 µm. Statistical analysis the expression of β-Casein/β-Tubulin. n = 3 mice. (C) qRT-PCR analysis of Wap, Lalba and Csn2 in mammary epithelial cells isolated from control and cKO mice at pregnancy day 14.5. n = 4–6 biological replicates. (D) Immunofluorescence staining and western blotting for Plin2 in control and cKO mammary glands at pregnancy day 14.5. n = 3 mice per group. GAPDH was used as a loading control. Scale bar, 25 µm. Statistical analysis the expression of Plin2/GAPDH. n = 3 mice. (E) Heatmap for milk protein-related gene expression in control and cKO mammary glands at pregnancy day 14.5. n = 4 mice per group. (F) Immunohistochemistry staining and western blotting for Stat5 and pStat5 in control and cKO mammary glands at pregnancy day 14.5. n = 3 mice per group. β-Actin was used as a loading control. Scale bar, 50 µm. Statistical analysis the expression of pStat5/β-Actin. n = 3 mice. (G) Immunohistochemistry staining for Prlr in control and cKO mammary glands at pregnancy day 14.5. n = 3 mice per group. Scale bar, 50 µm. (H) qRT-PCR analysis of Elf5, Tnfsf11, Src, Socs-1, and Socs-2 in mammary epithelial cells isolated from control and cKO mice at pregnancy day 14.5. n ≥3 biological replicates. Data were presented as means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: The following primary antibodies were used: BMPR1a (1:1000, 12702-1-AP, Proteintech), Sp1 (1:2000, 21962-1-AP, Proteintech), pSmad1/5 (1:1000, 9516, CST), β-Casein (1:500, sc-166530, Santa Cruz), Plin2 (1:1000, ab108323, Abcam), Stat5 (1:1000, ab68465, Abcam), pStat5 (1:1000, 9359, CST), K14 (1:1000, ab7800, Abcam), p63 (1:1000, ab124762, Abcam), Slug (1:1000, 9585, CST), K8 (1:1000, ab59400, Abcam), P-cadherin (1:1000, AF761, R&D), GAPDH (1:1000, 5174, CST), β-Actin (1:1000, 4970, CST), and β-Tubulin (1:5000, ab6046, Abcam).

Techniques: Immunohistochemistry, Staining, Control, Western Blot, Expressing, Quantitative RT-PCR, Isolation, Gene Expression

Journal: Frontiers in cell and developmental biology

Article Title: Hormone-Responsive BMP Signaling Expands Myoepithelial Cell Lineages and Prevents Alveolar Precocity in Mammary Gland.

doi: 10.3389/fcell.2021.691050

Figure Lengend Snippet: FIGURE 4 | BMPR1a deficiency resulted in reduced myoepithelial lineages and compromised repopulating capacity of mammary stem cells. (A) FACS analysis of mammary gland epithelial cells from control and cKO mice at pregnancy day 14.5 using the Lin/CD24/CD29/CD61 surface marker set. n = 4 mice per group. (B) Quantification of Lin-CD24+CD29high (Myo), Lin-CD24+CD29low (Lu) and Lin-CD24+CD29lowCD61+ cell populations in control and cKO mice at pregnancy day 14.5. n = 4 biological replicates. (C) Immunofluorescence staining for K14 (red) and K8 (green) in control and cKO mammary glands at pregnancy day 14.5. n = 3 mice per group. Scale bar, 25 µm. (D) Western blotting for K14 in control and cKO mammary glands at pregnancy day 14.5. β-Actin was used as a loading control. Statistical analysis the expression of K14/β-Actin. n = 3 mice. (E) Immunofluorescence staining for K14 (red) and K8 (green) in HC11 mammary epithelial cells treated with BMP4 (50 ng/mL) for 24 h. n = 3 biological replicates. Scale bar, 25 µm. (F) Western blotting for K14 in HC11 cells treated with BMP4 (50 ng/mL) for 24 h. β-Actin was used as a loading control. Statistical analysis the expression of K14/β-Actin. n = 4 biological replicates. (G) Illustration of the mammary transplantation assays using K14-rtTA/teto-Cre/Bmpr1afl/fland control mouse mammary stem cells and the induction strategy. (H) Whole-mount staining of mammary glands from the mammary transplantation assays and quantification of the proportion of ductal trees. n = 5 mice per group. Scale bar, 1 mm. (I) Immunohistochemistry staining for Integrin β3 in control and cKO mammary glands at pregnancy day 14.5. Quantification of Integrin β3+ cells in alveoli. n = 3 mice per group. Scale bar, 50 µm. Data were presented as means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: The following primary antibodies were used: BMPR1a (1:1000, 12702-1-AP, Proteintech), Sp1 (1:2000, 21962-1-AP, Proteintech), pSmad1/5 (1:1000, 9516, CST), β-Casein (1:500, sc-166530, Santa Cruz), Plin2 (1:1000, ab108323, Abcam), Stat5 (1:1000, ab68465, Abcam), pStat5 (1:1000, 9359, CST), K14 (1:1000, ab7800, Abcam), p63 (1:1000, ab124762, Abcam), Slug (1:1000, 9585, CST), K8 (1:1000, ab59400, Abcam), P-cadherin (1:1000, AF761, R&D), GAPDH (1:1000, 5174, CST), β-Actin (1:1000, 4970, CST), and β-Tubulin (1:5000, ab6046, Abcam).

Techniques: Control, Marker, Staining, Western Blot, Expressing, Transplantation Assay, Immunohistochemistry

Journal: Frontiers in cell and developmental biology

Article Title: Hormone-Responsive BMP Signaling Expands Myoepithelial Cell Lineages and Prevents Alveolar Precocity in Mammary Gland.

doi: 10.3389/fcell.2021.691050

Figure Lengend Snippet: FIGURE 5 | BMPR1a regulated the expression of p63 and Slug through the pSmad1/5-Smad4 complex. (A) Heatmap for myoepithelial layer-related gene expression in control and cKO mammary glands at pregnancy day 14.5. n = 4 mice per group. (B) qRT-PCR analysis of Itgb3, Adamts18, Cdh2, Tspan8, Lgr5, Procr, and Cd44 in FACS-sorted myoepithelial cells from control and cKO mice at pregnancy day 14.5. n = 4–5 biological replicates. (C) Western blotting for p63 and Slug in wild-type mouse mammary glands at the indicated time points (P14.5, pregnancy day 14.5; P18.5, pregnancy day 18.5; L2, lactation day 2). β-Actin was used as a (Continued)

Article Snippet: The following primary antibodies were used: BMPR1a (1:1000, 12702-1-AP, Proteintech), Sp1 (1:2000, 21962-1-AP, Proteintech), pSmad1/5 (1:1000, 9516, CST), β-Casein (1:500, sc-166530, Santa Cruz), Plin2 (1:1000, ab108323, Abcam), Stat5 (1:1000, ab68465, Abcam), pStat5 (1:1000, 9359, CST), K14 (1:1000, ab7800, Abcam), p63 (1:1000, ab124762, Abcam), Slug (1:1000, 9585, CST), K8 (1:1000, ab59400, Abcam), P-cadherin (1:1000, AF761, R&D), GAPDH (1:1000, 5174, CST), β-Actin (1:1000, 4970, CST), and β-Tubulin (1:5000, ab6046, Abcam).

Techniques: Expressing, Gene Expression, Control, Quantitative RT-PCR, Western Blot