Fig 1: Figure 2: GSCs and DGCs grown as adherent cultures retain expression of their unique markers.A. Real-time qRT-PCR analysis showing FMOD mRNA upregulation in DGCs over GSCs. B. Western blotting showing FMOD protein overexpression, both in the total cellular proteome (left) and the secretome (right) of DGCs vs. GSCs. C. Real-time qRT-PCR analysis showing downregulated expression of four reprogramming factors in DGCs compared with GSCs. D. Expression of SSEA1, FMOD, CD133, and GFAP in GSCs vs. DGCs in adherent conditions. E. Flow cytometry analysis showing and enrichment of CD133-positive cells in the GSC cultures compared with DGC cultures.
Fig 2: Conditional silencing of fibromodulin (FMOD) in differentiated glioma cells (DGCs) formed de novo by glioma stem-like cell (GSC)-initiated tumors inhibits tumor growth.(A) Schema depicts the timeline of the intracranial orthotopic mouse glioma model using murine glioma stem cells (AGR53-GSC) in C57BL/6 mice (n = 10 per group). Mice were injected with AGR53-GSCs (1 × 105 cells per animal) transduced with either miRNT (nontargeting) or miRFMOD lentiviruses. Tumors were allowed to grow till day 13 and then miRNT or miRFMOD (and mCherry) were induced by doxycycline (100 µl of 1 mg/ml per animal) intraperitoneal injections at indicated times. Note that in vitro characterization shows that the highest knockdown of FMOD was obtained on the seventh day after doxycycline administration. First, in vivo imaging for mCherry expression depicting tumor size was done on day 21 post-injection, followed by imaging at regular intervals (as noted by the orange marks). (B) In vivo fluorescence (mCherry) imaging of mice injected with either AGR53-GSC/miRNT or AGR53-GSC/miRFMOD cells as per the timeline shown in (A). (C) The radiance efficiency for each time point in the two groups of animals as indicated was plotted. (D) Kaplan–Meier graph showing the survival of mice bearing tumors formed by AGR53-GSC/miRNT (Dox+) and AGR53-GSC/miRFMOD (Dox+) cells. (E) Hematoxylin and eosin staining shows a larger tumor (depicted by dark blue color due to extremely high cell density) in mice brain injected with AGR53-GSC/miRNT (Dox+) cells (top) compared to that of AGR53-GSC/miRFMOD (Dox+) cells (bottom). Magnification = ×0.8. (F) Confocal microscopy analysis showing FMOD expression in brains of mice injected with AGR53-GSC/miRNT (Dox+) and AGR53-GSC/miRFMOD (Dox+) cells. Red indicates FMOD, and blue indicates H33342 (stains nuclei). The merged images are shown for representation. Magnification = ×20, scale bar = 50 µm. (G) Brain sections showing areas of fluorescence (GFP, mCherry, and DAPI) for both AGR53-GSC/miRNT (Dox+) (left panel) and AGR53-GSC/miRFMOD (Dox+) (right panel) groups of animals. Note that the AGR53 cell line stably expresses GFP while mCherry expression is induced upon doxycycline addition. On day 13, prior to the administration of doxycycline, both AGR53-GSC/miRNT (left) and AGR53-GSC/miRFMOD (right) do not have any mCherry expression but have almost similar GFP expression. However, over time after the onset of doxycycline administration, both mCherry and GFP expression decreased in the miRFMOD group but not in the miRNT group. Merged images show an overlap of GFP and mCherry-positive tumor areas. Magnification = ×20, scale bar = 50 µm. For panel (C), the p-value was calculated by unpaired t-test with Welch’s correction, and for (D), the p-value was calculated by log-rank test. p-Value <0.05 was considered significant with *, **, and *** representing p-values <0.05, 0.01, and 0.001, respectively. Figure 5—source data 1.Source data used to generate Figure 5B. Figure 5—source data 2.Source data used to generate Figure 5C. Figure 5—source data 3.Source data used to generate Figure 5D. Figure 5—source data 4.Source data used to generate Figure 5E. Figure 5—source data 5.Source data used to generate Figure 5F. Figure 5—source data 6.Source data used to generate Figure 5G.
Fig 3: Differentiated glioma cell (DGC)-secreted fibromodulin (FMOD) is essential for tumor growth initiated by glioma stem-like cells (GSCs) in vivo in a co-implantation experiment.(A) Diagram of the inducible shFMOD lentiviral construct. (B) Schema depicts the GSC-DGC co-implantation experiment in C57BL/6 mice (n = 5 per group). Mice were injected subcutaneously with a combination of DBT-Luc-GSCs and DBT-Luc-DGCs transduced with either miRNT (nontargeting) or miRFMOD lentiviruses. To induce miRNT or miRFMOD (and mCherry), mice received doxycycline (100 µl of 1 mg/ml per animal) as intraperitoneal injections at indicated times. The control groups were only injected with DBT-Luc-GSCs or DBT-Luc-DGCs and did not receive doxycycline. (C) In vivo imaging of the injected mice shows tumor growth over time by bioluminescence and mCherry fluorescence, according to the timeline shown in (B). (D) Quantification of the total radiance. The different colors represent the different groups of animals. Significant differences between each of the groups were calculated using ANOVA. The p-values for days 28 and 32 are shown. A detailed comparison of the p-values between different groups is provided in Supplementary file 2. Figure 2—source data 1.Source data used to generate Figure 2C. Figure 2—source data 2.Source data used to generate Figure 2D.
Fig 4: Growth of human glioma stem-like cell (GSC)-initiated tumors requires secreted fibromodulin (FMOD).(A) Flow cytometry analysis shows the relative levels of CD133-positive cells in MGG8-GSCs compared to MGG8-DGCs. (B) Kaplan–Meier graph shows the survival of mice (n = 10 per group) injected intracranially with MGG8-GSC/shNT or MGG8-GSC/shFMOD cells (1 × 105 cells per animal). (C) Hematoxylin and eosin staining shows a larger tumor (depicted by dark blue color due to high cell density) in mice brain injected with MGG8-GSC/shNT cells (top) compared to that of MGG8-GSC/shFMOD cells (bottom). Magnification = ×0.8. (D) Confocal microscopy analysis shows FMOD expression in brains of mice injected with MGG8-GSC/shNT (top panel) and MGG8-GSC/shFMOD (bottom panel) cells. Red indicates FMOD, and blue indicates H33342 (stains nuclei). Magnification = ×20, scale bar = 50 µm. Statistical significance for panel (B) was calculated using the log-rank test. p-Value <0.05 is considered significant. Figure 6—source data 1.Source data used to generate Figure 6A. Figure 6—source data 2.Source data used to generate Figure 6B. Figure 6—source data 3.Source data used to generate Figure 6C. Figure 6—source data 4.Source data used to generate Figure 6D.
Fig 5: Integrin/FAK/Src/JAG1-dependent Notch pathway activation in endothelial cells mediates fibromodulin (FMOD)-induced angiogenesis.(A) Heatmap showing differentially regulated (log2 fold change >/<0.2) proteins in ST1 cells treated with vehicle or rhFMOD (400 nM) for 10, 30, and 60 min, assessed by reverse phase protein array (RPPA). Red and green depict upregulated and downregulated proteins, respectively. The red arrows indicate HES1 and pFAK proteins. (B) RT-qPCR analysis shows HES1 transcript levels in ST1 cells treated with rhFMOD (400 nM) with or without gamma-secretase inhibitor (GSI; 10 µM). (C) Western blotting shows HES1 protein levels in ST1 cells treated with rhFMOD (400 nM) with or without GSI (10 µM). (D) Quantification of the number of networks formed by ST1 cells treated with rhFMOD (400 nM) with or without GSI (10 µM). (E) Western blotting shows phospho-FAK levels in ST1 cells treated with rhFMOD (400 nM) with or without RGD peptide (10 µM). (F) Quantification of the number of networks formed by ST1 cells pretreated with indicated small-molecule inhibitors (PP2 [10 µM], PP3 [10 µM], and PF573228 [FAK inhibitor; 10 µM], H1152 [ROCK1 inhibitor; 0.5 mM], and Rac1 inhibitor [10 µM]) followed by incubation with conditioned medium (CM) of LN229/Vector or LN229/FMOD cells. (G) RT-qPCR analysis shows HES1 transcript levels in ST1 cells treated with rhFMOD (400 nM) with or without RGD peptide (10 µM). (H) Western blotting shows HES1 protein levels in ST1 cells treated with rhFMOD (400 nM) with or without RGD peptide (10 µM). (I) Western blotting shows JAG1 protein levels in ST1/shNT and ST1/shJAG1 cells. (J) RT-qPCR analysis shows HES1 transcript levels in ST1/shNT and ST1/shJAG1 cells treated with rhFMOD (400 nM). (K) Western blotting shows HES1 protein levels in ST1/shNT and ST1/shJAG1 cells treated with rhFMOD (400 nM). (L) Quantification of the number of networks formed by ST1/shNT and ST1/shJAG1 cells treated with bovine serum albumin (BSA) or rhFMOD (400 nM). For panels (B), (D), (F), (G), (J), and (L), n=3, and the p-values were calculated by unpaired t-test with Welch’s correction are indicated. p-Value <0.05 was considered significant with *, **, and *** representing p-values <0.05, 0.01, and 0.001, respectively. Figure 4—source data 1.Source data used to generate Figure 4A. Figure 4—source data 2.Source data used to generate Figure 4B. Figure 4—source data 3.Source data used to generate Figure 4C, E, H, I. Figure 4—source data 4.Source data used to generate Figure 4D. Figure 4—source data 5.Source data used to generate Figure 4F. Figure 4—source data 6.Source data used to generate Figure 4G. Figure 4—source data 7.Source data used to generate Figure 4J. Figure 4—source data 8.Source data used to generate Figure 4K. Figure 4—source data 9.Source data used to generate Figure 4L.
Supplier Page from OriGene Technologies for Fibromodulin (FMOD) (NM_002023) Human Over-expression Lysate