Journal: Molecular medicine reports

Article Title: Knockdown of biglycan expression by RNA interference inhibits the proliferation and invasion of, and induces apoptosis in, the HCT116 colon cancer cell line.

doi: 10.3892/mmr.2015.4383

Figure Lengend Snippet: Figure 1. Construction of the stable HCT116 cell line with biglycan down regulation. The (A) mRNA and (B) protein expression levels of biglycan in the shRNA‑biglycan/control‑transfected or non‑transfected cells was detected using reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. (C) The relative protein expression of big lycan was normalized against β‑actin. Each experiment was repeated three times and the data are expressed as the mean ± standard deviation (*P<0.05 or **P<0.01, compared with the shRNA‑control group). sh, short hairpin.

Article Snippet: The membrane was subsequently blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with the following diluted primary antibodies: Rabbit polyclonal anti-biglycan antibody (cat no. sc-33788; 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-caspase-3 antibody (cat no. wl01992a; 1:1,000), rabbit polyclonal anti-p27 antibody (cat no. wl01769; 1:1,000), rabbit polyclonal anti-cyclin A antibody (cat no. wl01753; 1:200), rabbit polyclonal anti-p21 antibody (cat no. wl0362; 1:100), rabbit polyclonal anti-cyclin D1 antibody (cat no. wl01435a; 1:100) (all from Wanleibio, Shenyang, China), rabbit polyclonal anti-p38 antibody (cat no. sc-7149; 1:100) and anti-phosphorylated-p38 antibody (cat no. sc-101758; 1:100) (both from Abcam, Cambridge, MA, USA) at 4 ̊C overnight.

Techniques: Expressing, Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Molecular medicine reports

Article Title: Knockdown of biglycan expression by RNA interference inhibits the proliferation and invasion of, and induces apoptosis in, the HCT116 colon cancer cell line.

doi: 10.3892/mmr.2015.4383

Figure Lengend Snippet: Figure 2. Downregulation of biglycan inhibits the proliferation of colon cancer cells and causes cell cycle arrest. (A) The effect of the downregulation of biglycan on HCT116 cell proliferation was measured using a cell counting kit‑8 assay, with each group containing six technical replicates. (B and C) The effect of the downregulation of biglycan on the HCT116 cell cycle distribution was analyzed by flow cytometry. (D) The protein expression levels of cyclin A, cyclin D1, p21 and p27 were detected by western blotting, and the results obtained from a representative experiment are shown. (E) The relative protein expres sion of these proteins were normalized against β‑actin. The data are expressed as the mean ± standard deviation (**P<0.01, compared with the shRNA‑control group). OD, optical density; sh, short hairpin.

Article Snippet: The membrane was subsequently blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with the following diluted primary antibodies: Rabbit polyclonal anti-biglycan antibody (cat no. sc-33788; 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-caspase-3 antibody (cat no. wl01992a; 1:1,000), rabbit polyclonal anti-p27 antibody (cat no. wl01769; 1:1,000), rabbit polyclonal anti-cyclin A antibody (cat no. wl01753; 1:200), rabbit polyclonal anti-p21 antibody (cat no. wl0362; 1:100), rabbit polyclonal anti-cyclin D1 antibody (cat no. wl01435a; 1:100) (all from Wanleibio, Shenyang, China), rabbit polyclonal anti-p38 antibody (cat no. sc-7149; 1:100) and anti-phosphorylated-p38 antibody (cat no. sc-101758; 1:100) (both from Abcam, Cambridge, MA, USA) at 4 ̊C overnight.

Techniques: CCK-8 Assay, Flow Cytometry, Expressing, Western Blot, Standard Deviation

Journal: Molecular medicine reports

Article Title: Knockdown of biglycan expression by RNA interference inhibits the proliferation and invasion of, and induces apoptosis in, the HCT116 colon cancer cell line.

doi: 10.3892/mmr.2015.4383

Figure Lengend Snippet: Figure 3. Downregulation of biglycan suppresses the migratory and invasive properties of colon cancer cells (magnification, x200). (A) The motility of shRNA‑biglycan/control or non‑transfected cells was determined using a scratch wound assay over a 24 h time period. (B) The migration rate was determined by the distance traveled by the cells to the front of the denuded area. (C) The invasion ability of cells in each group was measured using a Transwell assay. (D) The number of invasive cells were counted under an inverted microscope and cell numbers were plotted. The results obtained from a representative experi ment are shown. The data are expressed as the mean ± standard deviation (**P<0.01, compared with the shRNA‑control group).. sh, short hairpin.

Article Snippet: The membrane was subsequently blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with the following diluted primary antibodies: Rabbit polyclonal anti-biglycan antibody (cat no. sc-33788; 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-caspase-3 antibody (cat no. wl01992a; 1:1,000), rabbit polyclonal anti-p27 antibody (cat no. wl01769; 1:1,000), rabbit polyclonal anti-cyclin A antibody (cat no. wl01753; 1:200), rabbit polyclonal anti-p21 antibody (cat no. wl0362; 1:100), rabbit polyclonal anti-cyclin D1 antibody (cat no. wl01435a; 1:100) (all from Wanleibio, Shenyang, China), rabbit polyclonal anti-p38 antibody (cat no. sc-7149; 1:100) and anti-phosphorylated-p38 antibody (cat no. sc-101758; 1:100) (both from Abcam, Cambridge, MA, USA) at 4 ̊C overnight.

Techniques: Control, Scratch Wound Assay Assay, Migration, Transwell Assay, Inverted Microscopy, Standard Deviation

Journal: Molecular medicine reports

Article Title: Knockdown of biglycan expression by RNA interference inhibits the proliferation and invasion of, and induces apoptosis in, the HCT116 colon cancer cell line.

doi: 10.3892/mmr.2015.4383

Figure Lengend Snippet: Figure 4. Downregulation of biglycan activates the p38 signaling pathway and induces apoptosis in colon cancer cells. (A) The effect of biglycan downregula tion on the apoptosis of the HCT116 cells was detected using flow cytometry, and results are shown from a representative experiment. (B) The proportion of apoptotic cells in each group was determined. (C) The protein expression levels of caspase‑3 and p‑p38 were detected by western blotting, and the results obtained from a representative experiment are shown for each group. (D) The relative expression of these proteins was normalized against β‑actin. The data are expressed as the mean ± standard deviation (**P<0.01, compared with the shRNA‑control group; ##P<0.01, compared with the shRNA‑biglycan group). p‑, phosphorylated; sh, short hairpin.

Article Snippet: The membrane was subsequently blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with the following diluted primary antibodies: Rabbit polyclonal anti-biglycan antibody (cat no. sc-33788; 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-caspase-3 antibody (cat no. wl01992a; 1:1,000), rabbit polyclonal anti-p27 antibody (cat no. wl01769; 1:1,000), rabbit polyclonal anti-cyclin A antibody (cat no. wl01753; 1:200), rabbit polyclonal anti-p21 antibody (cat no. wl0362; 1:100), rabbit polyclonal anti-cyclin D1 antibody (cat no. wl01435a; 1:100) (all from Wanleibio, Shenyang, China), rabbit polyclonal anti-p38 antibody (cat no. sc-7149; 1:100) and anti-phosphorylated-p38 antibody (cat no. sc-101758; 1:100) (both from Abcam, Cambridge, MA, USA) at 4 ̊C overnight.

Techniques: Flow Cytometry, Expressing, Western Blot, Standard Deviation

Journal: Journal of the American Society of Nephrology

Article Title: AT1 Receptors in the Collecting Duct Directly Modulate the Concentration of Urine

doi: 10.1681/asn.2010101095

Figure Lengend Snippet: Figure 1. The increased level of AQP2 with thirsting was significantly attenuated in CD-KO mice. (A) AT1A receptor mRNA expression is reduced in the renal inner medulla of CD-KO mice compared with control mice as determined by real time PCR (1.00 0.09 versus 0.56 0.07; n 12; *P 0.01). No differences in AT1A receptor expression were detected in the cortex and the outer medulla of CD-KO and control mice. Representative confocal laser-scanning microscopy (20 and 40) of the inner medulla of Hoxb7-Cre

Article Snippet: The membranes were blocked in block- ing buffer (5% dry milk, and 0.1% Tween 20 in PBS) for 1 hour at room temperature and then incubated with primary polyclonal rabbit anti-AQP2 antibody (1:2000) (Novus Biologicals [NB110- 74682], Littleton, CO), and mouse anti- -actin (1:1000) (Sigma- Aldrich) overnight.

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Confocal Laser Scanning Microscopy

Journal: Journal of the American Society of Nephrology

Article Title: AT1 Receptors in the Collecting Duct Directly Modulate the Concentration of Urine

doi: 10.1681/asn.2010101095

Figure Lengend Snippet: Figure 3. Increases of AQP2 with thirsting are significantly attenuated in CD-KO mice compared with controls. Representative immunoblots and densitometric analysis of aquaporin-2 (AQP2) in renal outer and inner medulla in CD-KO and control mice at baseline (control: n 4; CD-KO: n 4) and after 18 hours of water deprivation (control: n 4; CD-KO: n 5). In water-deprived control mice, outer medullary AQP2 expression levels were significantly increased compared with baseline controls and water-deprived CD-KO mice. (A) Outer medullary AQP2 abundance tended to be increased in water-deprived CD-KO mice compared with baseline. (B) Inner medullary AQP2 expression levels were similar between CD-KO and control mice at baseline. After 18 hours of water deprivation, inner medulla AQP2 expression levels increased significantly in both groups. AQP2 expression levels were significantly lower in the inner medulla of CD-KO mice compared with control mice. For the densitometric analysis, AQP2 expression levels were normalized to the corresponding -actin expression and further evaluated by calculating the fold induction relative to the AQP2/-actin ratio observed in the sample from mouse #1 (baseline control). The sample from mouse #1 is present as a reference on all Western blots to allow comparison of all samples. †P 0.01 versus baseline control; #P 0.05 versus baseline CD-KO; *P 0.05 versus water deprivation control. The data are presented as the means SEM.

Article Snippet: The membranes were blocked in block- ing buffer (5% dry milk, and 0.1% Tween 20 in PBS) for 1 hour at room temperature and then incubated with primary polyclonal rabbit anti-AQP2 antibody (1:2000) (Novus Biologicals [NB110- 74682], Littleton, CO), and mouse anti- -actin (1:1000) (Sigma- Aldrich) overnight.

Techniques: Western Blot, Control, Expressing, Comparison

Journal: Journal of the American Society of Nephrology

Article Title: AT1 Receptors in the Collecting Duct Directly Modulate the Concentration of Urine

doi: 10.1681/asn.2010101095

Figure Lengend Snippet: Figure 4. Normal cellular localization of AQP2 in CD-KO mice. Representative confocal laser-scanning microscopy (64) of the inner medulla from CD-KO and control mice at baseline and after 18 hours of water deprivation. Under baseline conditions, aquaporin-2 (AQP2) (red) is diffusely localized around the apical membrane of the collecting duct in control (A and E) and CD-KO mice (B and F). After 18 hours of water deprivation, intense AQP2 expression strictly defined to the apical membrane of principle cells were detected in both control (C and G) and CD-KO (D and H) mice (C and D). However, similar to AQP2 protein expression levels (Figure 3), the extent of apical AQP2 labeling after 18 hours of water deprivation appeared to be stronger in control mice compared with CD-KO mice. DAPI, 4,6,diamidino-2-phenylindole; WD, water deprivation.

Article Snippet: The membranes were blocked in block- ing buffer (5% dry milk, and 0.1% Tween 20 in PBS) for 1 hour at room temperature and then incubated with primary polyclonal rabbit anti-AQP2 antibody (1:2000) (Novus Biologicals [NB110- 74682], Littleton, CO), and mouse anti- -actin (1:1000) (Sigma- Aldrich) overnight.

Techniques: Confocal Laser Scanning Microscopy, Control, Membrane, Expressing, Labeling

Journal: Journal of the American Society of Nephrology

Article Title: AT1 Receptors in the Collecting Duct Directly Modulate the Concentration of Urine

doi: 10.1681/asn.2010101095

Figure Lengend Snippet: Figure 5. AT1A receptor mRNA expression is reduced in the renal inner medulla of CD-KO (AQP2-Cre) mice compared with control mice as determined by real time PCR (1 0.08 versus 0.49 0.09; *P 0.05; n 4 to 8). (A) No differences in AT1A receptor expression were detected in the cortex of CD-KO (AQP2-Cre) and control mice (cortex 1.00 0.1285 versus 0.89 0.11; P 0.56; n 4 to 8). Basal urine osmolalities in CD-KO (AQP2-Cre) and control mice were similar (1120 41 versus 1114 18 mOsmol/ kg; n 8). After 18 hours of water deprivation, urine osmolality increased significantly in CD-KO (AQP2-Cre) and control mice (*P 0.001). (B) Urine osmolalities remained significantly lower in CD-KO (AQP2-Cre) mice compared with control mice (3081 90 versus 3535 147 mOsmol/kg; #P 0.05; n 9). The data are presented as the means SEM.

Article Snippet: The membranes were blocked in block- ing buffer (5% dry milk, and 0.1% Tween 20 in PBS) for 1 hour at room temperature and then incubated with primary polyclonal rabbit anti-AQP2 antibody (1:2000) (Novus Biologicals [NB110- 74682], Littleton, CO), and mouse anti- -actin (1:1000) (Sigma- Aldrich) overnight.

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction