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. 2017 May 1;26(9):656–677. doi: 10.1089/scd.2016.0262

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Copyright 2017, Mary Ann Liebert, Inc.

PMC Copyright notice

FIG. 7. — Effect of miR-181a on SOX2 and SALL4 expression, YAP activity, and sensitivity to IM. (A) Analysis of expression of POU5F1, SOX2, CITED2, and SALL4 in K562-STI-R cells expressing the control sponge or the miR-181a sponge inhibitor. Normalized expression relative to ACTB, S18, and GAPDH is presented as fold difference. Triplicate analyses representing three independent experiments are shown. *P < 0.05 (B) Analyses of CD45, CD44, CXCR4, VLA-4, CD24, NOTCH1, and CD34 expression by FACS in K562-STI-R expressing the control sponge or the miR-181a sponge inhibitor. Representative data from three independent analyses are shown. (C) Western blot analysis was performed on the whole cell lysates prepared from K562-STI-R cells expressing the control sponge or the miR-181a sponge inhibitor. Levels of Lats1, Mob, and YAP along with phosphorylation of YAP on Ser127 and 397 were evaluated. Data from two independent electroporation reactions using control (ctrl sp: 1, 2) and miR-181a sponge (miR-181a sp: 1, 2) are presented. The results are representative of three independent experiments. Immunofluorescent analysis was performed to examine the cellular distribution of YAP (D) and F-actin (E). K562-STI-R cells were electroporated with GFP-tagged control sponge or miR-181a sponge. Anti-YAP followed by secondary antibody conjugated with AlexaFluor 555 and phalloidin conjugated with AlexaFluor 555 were used to visualize YAP and F-Actin, respectively (red), and DAPI (blue) was used to stain the nucleus. Confocal images were acquired with a Nikon C1si confocal. Z series sections were collected at 0.2 μm with a 60 × PlanApo objective. (F) RT-PCR analysis of AREG, BIRC5, and CTGF expression in K562-STI-R cells expressing the control sponge or the miR-181a sponge inhibitor. Normalized expression relative to ACTB and GAPDH is presented as fold difference. The triplicate analyses are representative of data from three independent experiments. *P < 0.05 (G) FACS analyses of the percentage of apoptotic cells in a population of K562-STI-R cells expressing control or miR-181a sponge inhibitor subjected to the incremental concentrations of IM. Cells 16 h after electroporation with vectors encoding the control sponge or the miR-181a sponge inhibitor were cultured for 24 h in the presence of IM. The percent of Annexin V-FITC-positive cells was determined. Representative data from three independent analyses are shown *P < 0.05.

FIG. 7. — Effect of miR-181a on SOX2 and SALL4 expression, YAP activity, and sensitivity to IM. (A) Analysis of expression of POU5F1, SOX2, CITED2, and SALL4 in K562-STI-R cells expressing the control sponge or the miR-181a sponge inhibitor. Normalized expression relative to ACTB, S18, and GAPDH is presented as fold difference. Triplicate analyses representing three independent experiments are shown. *P < 0.05 (B) Analyses of CD45, CD44, CXCR4, VLA-4, CD24, NOTCH1, and CD34 expression by FACS in K562-STI-R expressing the control sponge or the miR-181a sponge inhibitor. Representative data from three independent analyses are shown. (C) Western blot analysis was performed on the whole cell lysates prepared from K562-STI-R cells expressing the control sponge or the miR-181a sponge inhibitor. Levels of Lats1, Mob, and YAP along with phosphorylation of YAP on Ser127 and 397 were evaluated. Data from two independent electroporation reactions using control (ctrl sp: 1, 2) and miR-181a sponge (miR-181a sp: 1, 2) are presented. The results are representative of three independent experiments. Immunofluorescent analysis was performed to examine the cellular distribution of YAP (D) and F-actin (E). K562-STI-R cells were electroporated with GFP-tagged control sponge or miR-181a sponge. Anti-YAP followed by secondary antibody conjugated with AlexaFluor 555 and phalloidin conjugated with AlexaFluor 555 were used to visualize YAP and F-Actin, respectively (red), and DAPI (blue) was used to stain the nucleus. Confocal images were acquired with a Nikon C1si confocal. Z series sections were collected at 0.2 μm with a 60 × PlanApo objective. (F) RT-PCR analysis of AREG, BIRC5, and CTGF expression in K562-STI-R cells expressing the control sponge or the miR-181a sponge inhibitor. Normalized expression relative to ACTB and GAPDH is presented as fold difference. The triplicate analyses are representative of data from three independent experiments. *P < 0.05 (G) FACS analyses of the percentage of apoptotic cells in a population of K562-STI-R cells expressing control or miR-181a sponge inhibitor subjected to the incremental concentrations of IM. Cells 16 h after electroporation with vectors encoding the control sponge or the miR-181a sponge inhibitor were cultured for 24 h in the presence of IM. The percent of Annexin V-FITC-positive cells was determined. Representative data from three independent analyses are shown *P < 0.05.