Fig 2: JAG2 expression in intestinal tumors of ApcMin/+ mice and human CRC cell lines(A) RT-PCR analysis for NOTCH ligands in the normal intestine (N) and tumor (T) samples of APCMin/+ mice. GAPDH served as a loading control. (B) Quantitative real-time PCR (qRT-PCR) analysis for JAG2 expression in normal and tumor samples of APCMin/+ mice. JAG2 expression in 5 tumor samples was calculated as mean fold change relative to surrounding normal tissue using the 2−ΔΔCT method (p<0.001). (C) Western blot analysis for JAG2 in human CRC cell lines. X-ray film was exposed for a longer duration to detect JAG2 expression in all the cell lines tested. (D) HCT116 cells were co-transfected with NOTCH reporter plasmid (pHes1-luc), pRL-null plasmid, and empty vector or JAG2 expression plasmid. Twenty-four hours after transfection, the cells were treated with 10 μM DAPT for 24 h. Notch reporter activity was normalized to Renilla luciferase activity. These experiments are the representative images of more than 3 independent experimentations.

Fig 3: Silencing of JAG2 sensitizes HCT116 cells to doxorubicin and other chemotherapeutic agents(A) Control and JAG2-knockdown HCT116 cells were treated with 1 µM doxorubicin (DOX) for 48 hours. Cell viability was determined by trypan blue assay and expressed as a percentage of vehicle-treated cells. Bar graphs show mean ± SD of percent decrease of viable cells over DMSO-treated control (100%). (B) Control and JAG2-knockdown HCT116 cells were treated with 1 µM DOX for 48 hours. Total cell lysates were subjected to Western blot analysis for cleaved caspase-3. The Western blot is representative of 4 independent experiments (n=4). (C) Quantification of GAPDH-normalized cleaved caspase-3 over DOX-treated control cells. (D) Control and JAG2-knockdown HCT116 cells were treated with 1 µM DOX for 48 hours, and then harvested for analysis of cell cycle distribution using flow cytometry. The bar graph represents the distribution of the cells in different phases of the cell cycle. (n=3) (E) HCT116 cells were treated with 100 µM 5-fluorouracil (5-FU) or 10 µM oxaliplatin for 48 hours. Total cell lysates were subjected to Western blot analysis for cleaved caspase-3 and GAPDH. Representative blot of 3 independent experiments is shown (n=3).

Fig 4: Knockdown of JAG2 sensitizes p53-null HCT116 cells to doxorubicin(A) Control and JAG2-knockdown p53-null HCT116 cells were treated with 1 μM doxorubicin (DOX) for 48 hours. Cell viability was determined by trypan blue assay and expressed as a percentage of vehicle-treated cells. Bar graphs show mean ± SD of percent decrease of viable cells over DMSO-treated control (100%). (B) Control and JAG2-knockdown p53-null HCT116 cells were treated with 1 μM DOX for 48 hours. Total cell lysates were subjected to Western blot analysis for cleaved caspase-3. (C) The graph shows fold changes of GAPDH-normalized cleaved caspase-3 over DOX-treated control cells. (D) Western blot analysis of JAG2 expression in control (EGFP) and JAG2 over-expressing HT29 cells. X-ray film was exposed for a shorter duration to properly visualize JAG2 expression in JAG2 over-expressing HT-29 cells. (E) Control and JAG2 over-expressing HT29 cells were treated with 1 μM doxorubicin or 300 μM 5-FU for 48 hours for 24 hours. Total cell lysates were subjected to Western blot analysis for cleaved caspase-3. All Western blot images are representative images from at least 3 independent experiments. (F) Calcein AM/DAPI viability assay. Control and JAG2 over-expressing HT29 cells were treated with 1 μM doxorubicin or 300 μM 5-FU for 48 hours. Treated cells were stained with Calcein AM and DAPI for visualization of live and dead cells, respectively. Data are presented as the percent of live cells.

Fig 5: Expression of Notch pathway components in retinoblastoma primary tumors and cell lines(A) Nuclear Hes1 immunoreactivity was examined in 11 human retinoblastoma tumors by immunohistochemistry. The left panel shows strong diffuse immunoreactivity in tumor cells, with some negative dying cells present around a central region of necrosis (asterisk). The right side panel shows Hes1 positive tumor cells (asterisk) invading the optic nerve head. In the inset, spindled stromal cells in this region are negative for Hes1 (arrow). (B) Elevated protein levels of Jag2 and cleaved Notch1 receptor were found by Western blot in WERI Rb1 and Y79 lines, as compared to non-neoplastic adult retina. However, minimal Jag1 protein expression was observed in both retinoblastoma lines and in non-neoplastic adult retina; ß-Actin was used as a loading control. (C) mRNA levels of the Notch ligands Jag2 and DLL4 were much higher as compared to Jag1 and DLL1,3 in WERI Rb1 and Y79 lines, as found by qPCR. Normal adult retina although expressed high levels of DLL4 mRNA as compared to the other ligands. (D) Notch1 and 2 mRNA levels were more elevated than Notch3 in WERI Rb1 cells, whereas Notch1 was more expressed than Notch2 and 3 in Y79 cells. Notch1 and 2 mRNA levels were also highly expressed in the non-neoplastic adult retina, as determined by qPCR. (E) mRNA levels of the Notch target genes were determined by qPCR in WERI Rb1, Y79, and in non-neoplastic adult retina. Hey1 was the most highly expressed Notch target gene in both retinoblastoma lines, while Hes1 was the most represented Notch target gene in the non-neoplastic adult retina.