pet28a vector  (Addgene inc)

Journal: Science Advances

Article Title: Jagged1 regulates extracellular matrix deposition and remodeling in triple-negative breast cancer

doi: 10.1126/sciadv.aea9562

Figure Lengend Snippet: ( A ) MatrisomeDB classifications of proteins either up-regulated or down-regulated in MDA-MB-231 Jag1WT and MEF coculture spheroids compared to Jag1KO cocultures. n = 4 biological replicates. ( B ) Proteins differentially expressed by either MDA-MB-231 cells or MEFs in MEF + Jag1WT cocultures compared to MEF + Jag1KO cocultures. ( C ) Cross sections of MDA-MB-231 (magenta) and MEF (orange) coculture spheroids stained with the CNA35 collagen probe (green) and DAPI (blue). Scale bars, 200 μm. Western blots and dot blots of indicated proteins in coculture spheroids of MDA-MB-231 cells and ( D ) MEFs or ( E ) human TIFs. Quantifications of four independent experiments are shown to the right. Data are presented as the means ± SEM. P values are calculated by a two-tailed unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Spheroids were washed for 15 min in PBST and then incubated in CNA35 [20 ng/μl; pET28a-EGFP-CNA35 was a gift from M. Merkx; Addgene, plasmid no. 61603 ( )] and 600 nM 4′,6-diamidino-2-phenylindole (DAPI) in PBST overnight at +4°C with gentle shaking.

Techniques: Staining, Western Blot, Two Tailed Test

Journal: Science Advances

Article Title: Jagged1 regulates extracellular matrix deposition and remodeling in triple-negative breast cancer

doi: 10.1126/sciadv.aea9562

Figure Lengend Snippet: ( A ) Tumor masses of MDA-MB-231 Jag1WT or Jag1KO cell cocultures with MEF cells in the chick CAM xenograft model. P values are calculated by a two-tailed unpaired t test. n = 37 tumors for Jag1WT and n = 40 for Jag1KO. ( B ) Hematoxylin and eosin–stained tissue sections of CAM tumors in (A). Scale bars, 100 μm. CAM tumor cryosections stained for ( C ) collagen (CNA35; green in merge), FN (magenta in merge), actin (phalloidin; orange in merge), and nuclei (DAPI; blue in merge); ( D ) phosphorylated SMAD2 (Ser 465/467 )/SMAD3 (Ser 423/425 ) (p-SMAD); and ( E ) collagen (CNA35; green in merge), pan-cytokeratin (pan-cKRT; magenta in merge), αSMA (orange in merge), and nuclei (DAPI; blue in merge). Scale bars, 500 μm. ( F ) Quantifications of αSMA, p-SMAD, CNA35, and FN fluorescence integrated densities in CAM tumor cryosections. n = 4 tumors for αSMA and p-SMAD and n = 3 tumors for CNA35 and FN in each group. αSMA was quantified from glandular structures containing fibroblasts, and CNA35, FN, and p-SMAD were quantified from the whole-tumor area, excluding possible surrounding CAM. Data are presented as the means ± SEM. P values are calculated by a two-tailed unpaired t test. ( G ) mRNA expression level correlation with Jag1 mRNA expression in patients with breast cancer (TCGA, Cell 2015, n = 817) and ( H ) relapse-free survival of patients with low (black) or high (red) expression of ACTA2 , FN1 , COL1A2 , or COL3A1 in all breast cancer cases and in basal breast cancer separately in the Kaplan-Meier Plotter database .

Article Snippet: Spheroids were washed for 15 min in PBST and then incubated in CNA35 [20 ng/μl; pET28a-EGFP-CNA35 was a gift from M. Merkx; Addgene, plasmid no. 61603 ( )] and 600 nM 4′,6-diamidino-2-phenylindole (DAPI) in PBST overnight at +4°C with gentle shaking.

Techniques: Two Tailed Test, Staining, Fluorescence, Expressing