Fig 1: miR-103 induces EC death and decreases sprouting angiogenesis in vitro and in vivo.(a,b) Apoptosis, as measured by cleaved PARP and caspase-3, 48 h after ectopic expression of miR-103 (a) or (b) a caspase Glo- luminescence assay for active caspases 3 and 7 after ectopic expression of miR-103 in HUVECs 48 h after exposure to the indicated doses of radiation. One out of three independent experiments. Bars show mean+s.e.m. ** indicates P<0.01 by two-tailed Student's t-test. (c) 3D angiogenic sprouting assay with expression of miR-103. Right panel bars show mean+s.e.m. of lectin stained sprout areas from at least 10 beads per group. *P<0.05 by two-tailed Student's t-test. Scale bar, 100 µm. One of four independent experiments. (d) 3D angiogenic sprouting assay with expression of Anti-miR-103. Right panel bars show mean+s.e.m. of lectin stained sprout areas from at least 10 beads per group. *P<0.05 by two-tailed Student's t-test. Scale bar, 100 µm. One of three independent experiments. (e) Quantification of angiogenesis in mouse Matrigel plugs after i.v injection of control or miR-103 inhibitor followed by 10 Gy local radiation (N=4–6 plugs per group, one out of the three independent experiments). Bars show mean+s.e.m. * indicates P<0.05 by two-tailed Student's t-test. (f) Whole mount images of mouse neonatal P12 retina stained with anti-CD31 5d post intraocular injection with miR-103. Right panel bars show mean+s.e.m. of CD31 area from n=5 retinas. *P<0.05 by two-tailed Student's t-test. Scale bar, 200 µm.
Fig 2: RAE1 knockdown induces apoptosis and prevents cell cycle progression in colorectal cancer (CRC) cells. A, Gene set enrichment analysis (GSEA) using The Cancer Genome Atlas (TCGA) dataset. FDR, false discovery rate; NES, normalized enrichment score. B, Protein expression using western blot analysis for RAE1, cleaved PARP, and MCM2 in RAE1-knockdown cells and control cells. ß-actin was used as the loading control for relative protein quantification. The normalized intensities of each protein are shown. C, Protein expression using western blot analysis for RAE1 and cleaved PARP in the rescued cells. D, Left, Apoptosis assays of CACO2 (upper) and SW480 (lower) cell lines transfected with the indicated siRNA. Right, Quantification of the cell distributions in apoptosis (Annexin V-FITC+). **P < .01; ***P < .005. E, Cell cycle analysis by flow cytometry after re–supplementation with FBS in control cells and RAE1-knockdown cells. *P < .05; **P < .01; ***P < .005. F, Western blot analysis of RAE1 and p21 in control and RAE1-knockdown cells
Fig 3: Effects of antagomiR-34a inhibitor on ccRCC cell proliferation and apoptosis in vivo. (A) Tumor volumes of xenografts were measured at indicated time points and represented by a line graph. **P (Student’s t-test) < 0.01, ***P < 0.001. Data are presented as mean ± SD (n = 3). (B) The excised tumors were photographed. (C) Percentages of Ki67-positive cells in xenografts were analyzed by IHC staining and presented as bar charts. **P (Student’s t-test) < 0.05. Data are presented as mean ± SD (n = 3). (C–E) Apoptosis of xenograft ACHN cells following different treatments was analyzed by TUNEL assay and presented as bar charts. *P (Student’s t-test) < 0.05. Data are presented as mean ± SD (n = 3). Scale bar, 50 µm. (F) The expression of p53, DAPK, PARP, and cleaved PARP (CL-PARP) in xenograft tumors was analyzed by immunoblot analysis. (Doxo, doxorubicin; miR-34a inhi, miR-34a inhibitor).
Fig 4: Apoptosis is promoted by (ACF) in triple-negative breast cancer cell lines in a time and dose-dependent manner. (A) Microscopic images (magnification, ×100) of MDA-MB-231 and HS578T cells. (B) Effects of ACF on the viability of MDA-MB-231 and HS578T cells. The cells were treated with various concentrations (0, 0.5, 1, 2 and 10 µM) of ACF for 6, 24 and 48 h, and cell viability was determined using the sulforhodamine B assay. (C) MDA-MB-231 and HS578T cells were treated with 0 or 5 µM ACF for 24 h. Caspase-3/7 activity was analyzed using Muse Caspase-3/7 kit, as described in the Materials and methods. A total of 4 populations of cells were distinguished: Live [caspase-3/7(-)/7-AAD(-)], apoptotic [caspase-3/7(+)/7-AAD(-)], apoptotic/dead cells [caspase-3/7(+)/7-AAD(+)], and necrotic [caspase-3/7(-)/7-AAD(+)]. (D) MDA-MB-231 and HS578T cells were treated with 0 or 5 µM ACF for 24 h. Cell death was detected as the percentage of Annexin V and/or 7-AAD-positive cells. The results are expressed as the percentage of surviving cells over control cells. Each value is reported as the mean ± standard deviation and is representative of results obtained from three independent experiments. *P<0.05 compared with non-treated cells. (E) MDA-MB-231 and HS578T cells were treated with 0 or 5 µM ACF for 24 h and then western blot analysis for cleaved PARP expression was performed. ß-actin served as the loading control. ACF, acriflavine; 7-AAD, 7-amino-actinomycin D.
Fig 5: IRE1a kinase activation by CSTMP induces UPS of ?F508-CFTR.(A and B) Overexpression of IRE1a kinase activates UPS of ?F508-CFTR. HEK293 cells expressing ?F508-CFTR were cotransfected with plasmids encoding WT or mutant IRE1a (1 µg/ml, 48 hours). Overexpression of WT and RNase-dead (N906A) IRE1a, but not kinase-dead (K599A) IRE1a, activates UPS of ?F508-CFTR. Representative surface biotinylation assays are presented in (A), and the results of multiple experiments (n = 3) are summarized in (B). (C and D) Effects of the IRE1a kinase activator CSTMP on ASK1 phosphorylation and cell death signals. HEK293 cells were treated with 3 to 100 µM CSTMP for 48 hours. Activation of cell death signals were analyzed by cleavage of PARP and caspase 3 (arrowhead). Representative immunoblots are shown in (C), and the results of multiple experiments are summarized in (D) (n = 5). CSTMP at a concentration of 10 µM activated ASK1 but not cell death signals (red arrow). (E and F) CSTMP induces UPS of ?F508-CFTR. Surface biotinylation assays were performed in HEK293 cells expressing ?F508-CFTR. Cells were treated with CSTMP (10 µM) for the indicated time periods. Representative immunoblots are presented in (E), and a summary of multiple experiments is depicted in (F) (n = 3). Cell surface–specific labeling of proteins was confirmed by the presence of the plasma membrane protein Na- and K-dependent ATPase (Na,K-ATPase) and the absence of the cytosolic protein aldolase A in the biotinylated fraction. (G and H) Comparison of surface expression levels of WT-CFTR and the CSTMP-rescued ?F508-CFTR. HEK293 cells transfected with WT- and ?F508-CFTR were incubated with or without CSTMP (10 µM) for 24 hours. Representative surface biotinylation results are shown in (G), and the quantification results of multiple experiments (n = 3) are summarized in (H). The cell surface levels of CFTR (biotin) were normalized to its total protein (lysate) levels as detailed in Materials and Methods. Bar and line graphs data are shown as the means ± SEM. **P < 0.01.
Supplier Page from Abcam for Apoptosis Western Blot Cocktail (pro/p17-caspase-3, cleaved PARP1, muscle actin)