Fig. 4

Progranulin induces cancer stem cell propagation by dedifferentiation and increased proliferation of cancer stem cells. a FACS sorted GFP-positive and GFP-negative T47D GFP-Sox2 promotor reporter cells were analysed for mammosphere-forming capacity. Results are expressed as relative mammosphere number ± SD (n = 2). *p < 0.05 as calculated by a Student’s t test. b Immunofluorescence staining of CD24 (red), GFP (green), DAPI (blue) expression of GFP-Sox2 reporter cells. c (Left) Frequencies of GFP-positive cells were analysed using Operetta high-content imaging system. Frequencies of GFP-positive cells among all cells at 72 h were calculated after vehicle (PBS) (dark green) or 1 μg progranulin (light green) treatment (n = 3). *p < 0.05 as calculated by a Student’s t test. c Images illustrating the GFP expression in cells at starting time and after 72 h of progranulin treatment. d Percentage cell growth of GFP-Sox2 reporter cells at 72 h of treatment (n = 3). *p < 0.05 as calculated by a Student’s t test. e (Left bar plots) Nine fields in three 96-well (three fields each well) with unstained GFP-Sox2 reporter cells or nuclear-stained cells with NucBlue (right) where analysed over time to observe changes in GFP expression. Cells were considered as dedifferentiated cells if they turned GFP positive during 72 h of treatment. **p < 0.01 and ***p < 0.001 as calculated by a Student’s t test. e (Right images) Example of differentiated cells with both unstained (proliferative) and NucBlue-stained cells (low-proliferative). f Illustrative summary of the expansion process of T47D GFP-Sox2-positive reporter cells after progranulin treatment. CSC cancer stem cell