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sc-12-0359_sm_SupplFigure1.pdf1248KFigure S1. CXCL12 protects hNPCs against ongoing camptothecin-induced apoptosis. hNPCs were treated with camptothecin (10 μM) for 1 hour and then CXCL12 (50 ng/ml) for 4 or 8 hours. Cultures were fixed or lysed and hNPC apoptosis was determined by TUNEL (A-F) or PARP cleavage assay (G, H). A-E) Representative pictures from TUNEL assay were shown. F) Quantification of TUNEL assay was determined as percentage of TUNEL positive cells against total cell number. Results are expressed as the mean ± SEM and are representative of 3 independent experiments using 3 different donors. *, P < 0.05, compared with camptothecin-treated group without CXCL12. G) Cleaved PARP protein levels were determined by Western blotting. H) Levels of cleaved PARP were normalized as a ratio of cleaved PARP to actin after densimetrical quantification of panel G and shown as fold change relative to control. Representative data were shown from 3 independent experiments using 3 different donors.
sc-12-0359_sm_SupplFigure2.tif1936KFigure S2. CXCL12 protects hNPCs from apoptosis induced by growth factor deprivation. A) hNPCs were deprived of growth factors for 48 hours with or without the treatment of CXCL12. Cultures were fixed or lysed and hNPC apoptosis was determined by TUNEL (A-F) or PARP cleavage assay (G, H). A-E) Representative pictures from TUNEL assay were shown. F) Quantification of TUNEL assay was determined as a percentage of TUNEL positive cells against total cell number. G) Cleaved PARP protein levels were determined by Western blotting. Actin was used as loading control. H) Levels of cleaved PARP were normalized as a ratio of cleaved PARP to actin after densimetrical quantification of panel G and shown as fold change relative to control. All results are expressed as mean ± SEM of triplicate samples and are representative of 3 independent experiments. ##, P < 0.01 compared with control; *, P < 0.05, **, P < 0.01, ***, P < 0.001 compared with growth factor deprivation group without CXCL12.
sc-12-0359_sm_SupplFigure3.tif1537KFigure S3. SiRNA silencing efficiency for CXCR7 and CXCR4. A, B) Forty eight hours after transfection, RNA was collected and expression of CXCR7 (A) and CXCR4 (B) were analyzed using Real-time RT PCR. Data were normalized to GAPDH and presented as fold change compared to control. C, D) Seventy-two hours after transfection, protein levels of CXCR7 (C) or CXCR4 (D) were determined by Western blotting.
sc-12-0359_sm_SupplFigure4.pdf687KFigure S4. Endosomal localization pattern of CXCR7 and CXCR4 in hNPCs. Colocalization of CXCR7 (A-C) and CXCR4 (D-F) with endosomal markers were determined by confocal microscopy. hNPCs were transfected with CXCR7-mcherry and CXCR4-EGFP for 24 hours, then cells were fixed and immunostained with monoclonal antibody against EEA1 (early endosome marker, middle panels A, D), Rab4 (recycling marker, middle panels B, E) or Rab11 (recycling marker, middle panels C, F). Right panels are merged pictures of left and middle panels. Images were acquired from a Zeiss LSM 510 META LASER scanning confocal microscope. Panels are representative of pictures taken from three separate donors.
sc-12-0359_sm_SupplFigure5.tif2198KFigure S5. MDC blocks endocytosis of fluorescent-labeled CXCL12. hNPCs were pretreated with DMSO, vehicle control (A) or MDC (B) for 1 hour and then were treated with CXCL12-Alex647 for 2 hours. A) Endocytosis of CXCL12 without MDC treatment. B) Lack of CXCL12 endocytosis after MDC treatment.
sc-12-0359_sm_SupplFigure6.pdf1079KFigure S6. CXCL12 induces CXCR4 and CXCR7 interaction after endocytosis. A) hNPCs were transfected with CXCR7-mcherry and CXCR4-EGFP. 24 hours later, cells were treated with CXCL12-ALEX647 (100 ng/ml) and triple-color confocal images were acquired in real time. Right panels are merged pictures of left and middle panels. B) hNPCs were treated with CXCL12 (100 ng/ml) for 2 and 6 hours. Cell lysates were collected and immunoprecipitated (IP) with monoclonal-antibody against CXCR4. CXCR7 protein levels in the lysates were determined by immunoblotting with rabbit- anti-CXCR7 antibody. Mouse IgG was used as control for immunoprecipitation. Immunoblotting of cell extracts before immunoprecipitation was used as control (Input). C) hNPCs were transfected with either CXCR7-mcherry or CXCR4-EGFP. 48 hours later, cells transfected with CXCR7-mcherry were immunostained with CXCR4, and cells transfected with CXCR4-GFP were immunostained with CXCR7. Images were acquired from a Zeiss LSM 510 META LASER scanning confocal microscope. Panels are representative of pictures taken from three separate donors.
sc-12-0359_sm_SupplFigure7.tif901KFigure S7. CXCL12 induced ERK1/2 activation in CXCR7-positive endosome. hNPCs were transfected with plasmid expressing CXCR7-mcherry and then treated with CXCL12 for 30 minutes. ERK1/2 phosphorylation and CXCR7 were visualized by immunocytochemistry. A) Phosphorylated ERK1/2 in CXCR7-positive endosome without CXCL12 treatment. B) Phosphorylated ERK1/2 in CXCR7-positive endosome with CXCL12 treatment.
sc-12-0359_sm_Supplvideo1.avi3982KVideo 1. CXCR7 and CXCR4 endocytotic trafficking with PBS treatment in TIRFm.
sc-12-0359_sm_Supplvideo2.avi6511KVideo 2. CXCR7 and CXCR4 endocytotic trafficking with CXCL12 treatment in TIRFm.

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