Fig 1: Establishment of differential NIH3T3jag1 clones and Notch3 reporter sensor. (A) Immunofluorescence images representing the membranous localization of Jag1 in NIH3T3 clones. (B,C) Flow cytometry panel representing the quantitation of cell surface expression of Jag1 through mean fluorescence intensity, which indicated a differential degree of Jag1 expression across the clones (n = 3). (D,E) Live cell bioluminescence imaging of SNFT cells after Jag-1 peptide treatment (D) and graphical representation of the same (n = 2) (E) showing increased Notch3-sensor promoter activity. (F,G) Several Notch3 targets (hes1, pbx1, notch3) showed upregulation after peptide-induced activation at the transcript level (n = 3). Further, the Western blot of NICD3 and hes1 after differential Jag1 induction in SNFT (G) (also see Figure S6A) and the densitometric quantification of the proteins (H) (housekeeping control: alpha-tubulin, n = 2) highlight that the NICD3 cleavage and its target protein expression increased differentially upon induction due to the pathway activation. (I,J) Activation/inhibition kinetics of live-cell bioluminescence image depicting that peptide-induced promoter activity attenuated after DAPT blockade (n = 2). * p = 0.05, ** p = 0.01, *** p = 0.001.