Journal: Oncotarget

Article Title: Intercellular transfer of small RNAs from astrocytes to lung tumor cells induces resistance to chemotherapy

doi: 10.18632/oncotarget.7273

Figure Lengend Snippet: The percentages of live and apoptotic cells were determined by FACS analysis (see Materials and Methods section). ( A ) Representative dot plot of FACS analysis of treated PC14 cells stained with annexin-V FITS and PI. PC14 singlet-cell events are distinguished from target cells by the CD340 specific marker. Numbers in the quadrants are the percentages of PC14 cells within each quadrant. ( B , C ) Quantitative results of FACS analysis. B. Percentage of live (low PI, low annexin V) PC14 cells cultured alone, with astrocytes, alone plus Taxol, with astrocytes in a contact-dependent manner (mixed co-culture) plus Taxol, and with astrocytes in a contact-independent manner (separated by a transwell membrane (TW)) plus Taxol. Results are expressed as percentages of untreated PC14 cells. C. Apoptotic (high annexin V, low PI) PC14 cells cultured alone, with astrocytes, alone plus Taxol, with astrocytes in a contact-dependent manner plus Taxol, and with astrocytes in a transwell (TW) plus Taxol. The results are expressed as percentage of total cells and are presented as means ± SEM. Analysis (One-way ANOVA) revealed significant differences in live cell percentages and apoptotic cells between annexin V-stained and PI-stained cells, P < 0.05. Post-hoc analysis was performed by Fisher's LSD: * P < 0.05, ** P = 0.01, **** P < 0.0001; n = 4.

Article Snippet: The cells were incubated at 37°C in a humidified 5% CO 2 incubator for 6 h, harvested, and stained for 15 min at RT with APC anti-human CD340 Ab to label PC14 cells and with anti-mouse GLAST-PE conjugated antibody (Miltenyi Biotec) to label astrocytes.

Techniques: Staining, Marker, Cell Culture, Co-Culture Assay, Membrane

Journal: Oncotarget

Article Title: Intercellular transfer of small RNAs from astrocytes to lung tumor cells induces resistance to chemotherapy

doi: 10.18632/oncotarget.7273

Figure Lengend Snippet: PC14 cells (CD340 positive) containing 22bpCy3 were assayed by FACS analysis (see Materials and Methods section). ( A ) Time course of 22bpCy3 transfer from astrocytes to PC14 cells. B−G. Representative dot plots of the results of FACS analysis after the different treatments, depicting total live-cell gate records. ( B ) PC14 cells (CD340 positive) alone. ( C ) 22bpCy3-transfected astrocytes (Cy3 positive) alone; positive for Cy3. D, E. PC14 cells co-cultured with astrocytes-22bpCy3 in a contact-dependent ( D ) or contact-independent (transwell, TW) manner ( E ) F, G. Astrocytes-22bpCy3 were co-cultured with PC14 cells that were untreated (control) ( F ) or treated with RNase A/T ( G ) ( H ) Quantitative results of FACS analysis, expressed as mean fluorescence intensity (MFI) of 22bpCy3 recorded in the PC14 cells and presented as means ± SEM. Analysis (One-way ANOVA) revealed significant differences in MFI of 22bpCy3 between the groups: P < 0.05. Post-hoc analysis by Fisher's LSD yielded * P < 0.05, ** P < 0.01, **** P < 0.0001; n = 5.

Article Snippet: The cells were incubated at 37°C in a humidified 5% CO 2 incubator for 6 h, harvested, and stained for 15 min at RT with APC anti-human CD340 Ab to label PC14 cells and with anti-mouse GLAST-PE conjugated antibody (Miltenyi Biotec) to label astrocytes.

Techniques: Transfection, Cell Culture, Control, Fluorescence

Journal: Oncotarget

Article Title: Intercellular transfer of small RNAs from astrocytes to lung tumor cells induces resistance to chemotherapy

doi: 10.18632/oncotarget.7273

Figure Lengend Snippet: Astrocytes-22bpCy3 were co-cultured with MDA-MB-231 cells for 6 h. MDA-MB-231 cells (CD340 positive) containing 22bpCy3 were assayed by FACS analysis (see Materials and Methods section). ( A − E ) Representative dot plots of the results of FACS analysis after the different treatments, depicting total live-cell gate records A. MDA-MB-231 cells (positive for CD340) alone. B. 22bpCy3-transfected astrocytes (Cy3-positive) alone. C, D. MDA-MB-231 cells co-cultured with astrocytes-22bpCy3 in a contact-dependent (C) or contact-independent (transwell, TW) manner (D). E. MDA-MB-231 cells co-cultured with astrocytes-22bpCy3 treated with RNase A/T. ( F ) Quantitative results of the FACS analysis, expressed as MFI of 22bpCy3 recorded in the MDA-MB-231 cells and presented as means ± SEM. Analysis (One-way ANOVA) revealed significant differences in MFI of 22bpCy3 between the groups: P < 0.01. Post-hoc analysis by Fisher's LSD yielded ** P < 0.01; n = 5.

Article Snippet: The cells were incubated at 37°C in a humidified 5% CO 2 incubator for 6 h, harvested, and stained for 15 min at RT with APC anti-human CD340 Ab to label PC14 cells and with anti-mouse GLAST-PE conjugated antibody (Miltenyi Biotec) to label astrocytes.

Techniques: Cell Culture, Transfection

Journal: Oncotarget

Article Title: Intercellular transfer of small RNAs from astrocytes to lung tumor cells induces resistance to chemotherapy

doi: 10.18632/oncotarget.7273

Figure Lengend Snippet: ( A , B ) The gap-junction inhibitor CBX inhibits transfer of 22bpCy3 from astrocytes to PC14 cells. PC14 cells (CD340 positive) containing 22bpCy3 were assayed by FACS analysis (see Materials and Methods section). A. Representative dot plots of the results of FACS analysis after the different treatments, depicting total live-cell gate records. B. Quantitative results of FACS analysis expressed as MFI of 22bpCy3 recorded in PC14 cells cultured with astrocytes-22bpCy3 that were treated with CBX (50 μM/150 μM) or untreated (control). CBX inhibited the transfer of endogenous sRNA from the astrocytes to the PC14 cells. C−F. Levels of mmu-miR-16* ( C ), mmu-miR-709 ( D ), mmu-miR-1195 ( E ) and endo-siRNA-1196 ( F ) were measured in PC14-GFP cells by qPCR (see Materials and Methods section). Results are presented as means ± SEM. Analysis (One-way ANOVA revealed significant differences in MFI of 22bpCy3 between the groups: P < 0.05. Post-hoc analysis by Fisher's LSD yielded * P < 0.05, **** P < 0.0001; n = 4.

Article Snippet: The cells were incubated at 37°C in a humidified 5% CO 2 incubator for 6 h, harvested, and stained for 15 min at RT with APC anti-human CD340 Ab to label PC14 cells and with anti-mouse GLAST-PE conjugated antibody (Miltenyi Biotec) to label astrocytes.

Techniques: Cell Culture, Control