CUZD1 is a critical mediator of the JAK/STAT5 signaling pathway that controls mammary gland development during pregnancy
Fig 4
Cuzd1 controls the expression of a subset of EGF family ligands in mammary epithelial cells.
(A) Cuzd1 overexpressing cells exhibit increased proliferation. HC11-Cuzd1 and HC11-LacZ cells were cultured under serum-free conditions for 48 h and 10% FBS was added along with BrdU 24 h prior to cell harvest. BrdU incorporation was measured using an ELISA-based assay. Data are expressed as Absorbance at 370nm ± SEM from ≥3 biological replicates. (B) Expression of EGF family ligands in HC11-Cuzd1 and HC11-LacZ cells. HC11-Cuzd1 and HC11-LacZ cells were cultured for 48 h. qPCR was performed to analyze relative expression levels of Epgn, Ereg, Egf, Hbegf, Areg, Btc, Nrg1, Nrg2, Nrg3 and Nrg4 mRNAs. Data are represented as relative gene expression ± SEM from ≥3 biological replicates. (C) Expression of EGF family ligands in Cuzd1-silenced HC11 cells. HC11 cells were transfected with siRNA (100nM) targeted against Cuzd1 or scrambled siRNA (control). Total RNA was prepared from HC11 cells 48 h after transfection and subjected to qPCR using gene-specific primers to assess the expression of Epgn, Ereg, Egf, Hbegf, Areg, Btc, Nrg1, Nrg2, Nrg3 and Nrg4 mRNAs. Data are represented as relative gene expression ± SEM from ≥3 biological replicates. (D) Proliferation of HC11-Cuzd1 cells upon ErbB perturbation. HC11-Cuzd1 cells were transfected with siRNA (50nM) targeted against ErbB1, ErbB2, ErbB3, ErbB4 or non-targeting siRNA (control). 48 h post transfection, the siRNA transfection mixture was removed and replaced with fresh growth medium and BrdU was administered 24 h prior to cell harvest. BrdU incorporation was measured using an ELISA-based assay. Data are expressed as Absorbance at 370nm ± SEM from ≥3 biological replicates. (E) Proliferation of HC11 cells with Cuzd1 knockdown and ligand supplementation. HC11 cells were transfected with siRNA (100nM) targeted against Cuzd1 or a non-targeting siRNA (control). 48h post-transfection, HC11 cells were supplemented with EPGN, NRG1, or a vehicle control and BrdU was added. BrdU incorporation was measured after 24h using an ELISA-based BrdU assay and resulting color reaction was measured using a plate reader at 370nm. Data are expressed as Absorbance at 370nm ± SEM from ≥3 biological replicates.