Fig 1: CD114-positive (CD114+) cells have slower growth than CD114-negative (CD114−) and unsorted populations. Equal numbers of CD114+, CD114−, and parental cells were plated in wells of 96-well plates and monitored with continuous live cell imaging. Cell confluence was calculated every 6 h and fold increase in confluence was calculated versus confluence at the time of cell seeding. (A and B) Fold increase in confluence after 120 h for D283 (A) and Daoy (B) cells. (C) Longitudinal change in confluence for Daoy cells over time.
Fig 2: Changes in cell growth and CD114 surface expression after G-CSF exposure. (A) Daoy cells were sorted into CD114+, CD114−, and parental populations using FACS. Cells were exposed to vehicle (DMSO), 0.1 or 1 ng/mL G-CSF and at 200 h cell viability was determined by AlamarBlue assay. Differences in viability in CD114+ and parental cells were compared using Student’s t-tests. *P < .05. (B) Longitudinal growth by confluence compared to vehicle control in sorted cells exposed to 1 ng/mL G-CSF for up to 198 h. Cells were plated in equal cell numbers immediately after sorting. Cell confluence was calculated via the IncuCyte live cell imaging system. (C) Parental D283 cells were plated and then treated with either 0.04 µM carboplatin or vehicle for 48 h. Media was then exchanged and cells were incubated in fresh media with or without 10 ng/mL G-CSF for 24 h. All cells were harvested and analyzed by flow cytometry for the percentage of live cells expressing CD114, and differences in CD114 expression were compared using Student’s t-tests. **P < .01. P-value between untreated control and carboplatin + G-CSF is .006.
Fig 3: Efficacy of chemotherapy on sorted cell populations. Daoy cells (A) and SL00278 cells (B) were sorted by FACS into equal numbers of live CD114+, CD114−, and parental populations. Cells were then exposed to drug or vehicle (DMSO), and changes in confluence (A) and cell viability (B) were calculated over time (*P < 0.05, **P < 0.01). (C) Parental D283 cells were exposed to 0.17 µM methotrexate and parental D341 cells were exposed to 0.04 µM carboplatin for 48 h. Cells were harvested and analyzed by flow cytometry for CD114 expression, with differences between treated and untreated cells compared using Student’s t-tests. *P < .05. P = .02 for D283 and P = .12 for D341.
Fig 4: JAK–STAT pathway activity and response to ruxolitinib in medulloblastoma cells. Parental Daoy cells and Daoy cells transduced with CSF3R to overexpress CD114 (Daoy_CSF3R) were plated, treated with G-CSF (10 ng/mL for 15 min), and exposed to vehicle alone or increasing concentrations of ruxolitinib (RUX). Cells were harvested, separated by SDS-PAGE, and Western blots were performed for total and phosphorylated JAK1, JAK2, STAT3, and STAT5, with GAPDH used as a loading control.
Fig 5: Changes in mRNA expression of select genes in CD114+, CD114- and parental cells. Daoy (A) and HMB-5 (B) cells were sorted by FACS and RNA was immediately isolated. Quantitative PCR was performed using primers for CSF3R, NRP1, MSI1 (Musashi1), Twist1, SOX2, SOX9, and MYCN with GAPDH used as a control.
Supplier Page from Sino Biological, Inc. for Human GCSF Receptor / G-CSFR Gene ORF cDNA clone expression plasmid