Journal: Drug Design, Development and Therapy

Article Title: Effect of berberine on the renal tubular epithelial-to-mesenchymal transition by inhibition of the Notch/snail pathway in diabetic nephropathy model KKAy mice

doi: 10.2147/dddt.s124971

Figure Lengend Snippet: Figure 7 Berberine inhibits the expression of snail1. Notes: (A) Representative photographs of immunohistochemistry of snail1 in DN model KKAy mice. 400× magnification. (B) Comparison of MOD of snail1 protein in DN model KKAy mice. (C) Representative band of snail1 protein by Western blot in DN model KKAy Mice. (D) Comparison of the gray value of snail1 protein in DN model KKAy mice (n=3). (E) Representative photographs of in situ hybridization for snail1 in DN model KKAy mice. 400× magnification. (F) Comparison of MOD of snail1 mRNA in DN model KKAy mice. **P,0.01 compared with the NC and ##P,0.01 compared with the MG. Abbreviations: DN, diabetic nephropathy; MG, model group; MOD, mean optical density; NG, normal control group; TG, treatment group.

Article Snippet: Transmission electron microscopy (TEM) was performed using a JEM 100CX electron microscope (JEOL, Tokyo, Japan). immunohistochemistry Kidney tissues were fixed in 10% neutral formalin, embedded in paraffin, sliced (3 μm), dewaxed, washed three times with Drug Design, Development and Therapy 2017:11submit your manuscript | www.dovepress.com Dovepress Dovepress 1068 Yang et al phosphate-buffered saline (PBS) for 5 min each, incubated in 0.01% Triton for 8 min and in 3% hydrogen dioxide solution for 10 min, antigen repaired with citrate buffer solution in a microwave, blocked with 10% goat serum, and incubated with primary antibodies against jagged1 (1:100 dilution; Novus, Littleton, CO, USA), notch1 (1:100 dilution; Abcam, Cambridge, UK), hes1 (1:125 dilution; CST, Danvers, MA, USA), snail1 (1:100 dilution, Novus), E-Cad (1:100 dilution, Abcam), and α-SMA (1:200 dilution, Abcam) overnight at 4°C.

Techniques: Expressing, Immunohistochemistry, Comparison, Western Blot, In Situ Hybridization, Control

Journal: Drug Design, Development and Therapy

Article Title: Effect of berberine on the renal tubular epithelial-to-mesenchymal transition by inhibition of the Notch/snail pathway in diabetic nephropathy model KKAy mice

doi: 10.2147/dddt.s124971

Figure Lengend Snippet: Figure 10 BBR inhibits the HG-induced expression of snail1. Notes: (A) Representative band of snail1 protein by Western blot in mRTECs. (B) Comparison of the gray value of snail1 protein in mRTECs (n=3). (C) Comparison of the mRNA level of snail1 protein by reverse transcription polymerase chain reaction in mRTECs (n=3). **P,0.01 NG and ##P,0.01, #P,0.05 compared with the model group. Abbreviations: BBR, berberine; DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester; HG, high glucose; mRTECs, mouse renal tubular epithelial cells; NG, normal control group.

Article Snippet: Transmission electron microscopy (TEM) was performed using a JEM 100CX electron microscope (JEOL, Tokyo, Japan). immunohistochemistry Kidney tissues were fixed in 10% neutral formalin, embedded in paraffin, sliced (3 μm), dewaxed, washed three times with Drug Design, Development and Therapy 2017:11submit your manuscript | www.dovepress.com Dovepress Dovepress 1068 Yang et al phosphate-buffered saline (PBS) for 5 min each, incubated in 0.01% Triton for 8 min and in 3% hydrogen dioxide solution for 10 min, antigen repaired with citrate buffer solution in a microwave, blocked with 10% goat serum, and incubated with primary antibodies against jagged1 (1:100 dilution; Novus, Littleton, CO, USA), notch1 (1:100 dilution; Abcam, Cambridge, UK), hes1 (1:125 dilution; CST, Danvers, MA, USA), snail1 (1:100 dilution, Novus), E-Cad (1:100 dilution, Abcam), and α-SMA (1:200 dilution, Abcam) overnight at 4°C.

Techniques: Expressing, Western Blot, Comparison, Reverse Transcription, Polymerase Chain Reaction, Control

Journal: Cell adhesion & migration

Article Title: Atypical PKCs activate Vimentin to facilitate prostate cancer cell motility and invasion.

doi: 10.1080/19336918.2021.1882782

Figure Lengend Snippet: Figure 1. aPKC knockdown decreases prostate cancer cell migration and invasion. Figure 1(a) and 1(b) represent the effects of aPKC siRNA treatments (20 nM against scrambled siRNA) on PC-3 and DU-145 cell migration in wound healing assay and Figure 1c and 1d represent the effects of aPKC siRNA treatments on prostate cell invasion in Boyden chamber assay in the presence of basement extract (BME). In the wound healing assay, microscopic photographs (40×) of scratches on cells at the beginning (day 0) were compared with the photographs taken after 3 days. The effects of aPKC attenuation are shown compared to their controls. Experiments were performed for each cell line and representative photographs are shown (N = 3). Figure 1b bar graph represents a comparison of calculated percent wound closure for the photographs taken using ImageJ (NIH, Rockville, MD, USA). For the Boyden chamber assay (Figure 1b), invaded cells in the bottom surface of transwell insert were stained with 0.5% crystal violet and microscopic photographs were taken (100×). Subsequently, crystal violet was dissolved in 70% ethanol and absorbance was measured at 590 nm which is directly proportional to the number of invaded cells (Figure 1d). Figure 1e shows the effect of RNA interference (siRNA) of PKC-ι and PKC-ζ in prostate cancer cells (PC-3 and DU-145). PC-3 and DU-145 (1 × 105) cells were seeded in T25 flasks and after 24 h post seeding time, fresh medium was supplied and siRNA (20 nM for PKC-ι/ζ or 30 nM for SNAIL1 or PRRX1) treatments were conducted for 48 h using ‘siTran’ siRNA transfection reagent (TT300002, Origene Technologies, Inc.) according to the manufacturer’s recommended ratios. The expression of the protein levels of PKC-ι, PKC-ζ, E-cadherin and Vimentin are shown. Total protein (80 μg) was loaded into each well and β-actin was used as the internal control in each Western blot. Experiments were performed in each trial and representative bands are shown (N = 4). The blots are cropped from different gels and separated with a white space between them. Densitometry values for the Western blots are also shown (figure 1(f)). Figure 1(g) shows the mRNA levels of PKC-ι, PKC-ζ, E-cadherin and Vimentin for aPKC attenuation for respective samples based on quantitative real-time PCR (qPCR) (N = 3). All values are reported as the means ± SD. Statistical significance is indicated by an asterisk (*P≤<0.05).

Article Snippet: Enhanced chemiluminescence solution (34,080, Pierce Inc.). siRNA (human small interfering RNA) for PKC-ζ (SR321432), PKC-ι (SR321426), SNAIL1 (SR304489, OriGene Inc.) and PRRX1 (AM16708, Thermo Fisher Scientific).

Techniques: Knockdown, Migration, Wound Healing Assay, Boyden Chamber Assay, Comparison, Staining, Transfection, Expressing, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cell adhesion & migration

Article Title: Atypical PKCs activate Vimentin to facilitate prostate cancer cell motility and invasion.

doi: 10.1080/19336918.2021.1882782

Figure Lengend Snippet: Figure 2. Effects of RNA interference (siRNA) for aPKCs, transcription factors of PRRX1 and SNAIL1 on the expression of EMT markers in prostate cancer cells. Figure 2(a) and 2(b) show the expression of the protein levels for EMT markers for aPKC attenuation (Smad2/ 3, pSmad2/3 RhoA, Par6 and N-cadherin) and the transcription factors of SNAIL1 and PRRX1. Figure 2c and 2d show the expression of SNAIL1, PRRX1, total PKC-ι, phosphorylated PKC-ι (T555), total PKC-ζ, phosphorylated PKC-ζ (S410), E-cadherin and Vimentin for the siRNA knockdown of the expression of SNAIL1 for PC-3 and DU-145 cell lines. PC-3 and DU-145 (1 × 105) cells were seeded in T25 flasks and after 24 h post seeding time, fresh medium was supplied and siRNA (20 nM for PKC-ι/ζ or 30 nM for SNAIL1 or PRRX1) treatments were conducted for 48 h using ‘siTran’ siRNA transfection reagent. Total protein (80 μg) was loaded into each well and β- actin was used as the internal control in each Western blot. Representative densitometry values for the Western blots are shown (Figure 2(b) and 2(d)). Experiments were performed and representative bands are shown (N = 4). The blots are cropped from different gels and separated with a white space between them Fig. 2E shows the mRNA levels of SNAIL1, PRRX1, PKC-ι, PKC-ζ, E-cadherin and Vimentin for SNAIL1 and PRRX1 siRNA knockdown samples for PC-3 and DU-145 cells based on quantitative real-time PCR (qPCR) (N = 3). All values are reported as the means ± SD. Statistical significance is indicated by an asterisk (*P < 0.05).

Article Snippet: Enhanced chemiluminescence solution (34,080, Pierce Inc.). siRNA (human small interfering RNA) for PKC-ζ (SR321432), PKC-ι (SR321426), SNAIL1 (SR304489, OriGene Inc.) and PRRX1 (AM16708, Thermo Fisher Scientific).

Techniques: Expressing, Knockdown, Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-137 and miR-34a directly target Snail and inhibit EMT, invasion and sphere-forming ability of ovarian cancer cells

doi: 10.1186/s13046-016-0415-y

Figure Lengend Snippet: MiR-137 and miR-34a are downregulated in OC tissues and decreased expressions of miR-137 and miR-34a are associated with poor survival in OC patients. a Venn diagram showing the overlap of miRNAs that were predicted to bind to the Snail 3′-UTR by alternative algorithms (TargetScan, miRSystem and DIANA-MicroT-CDS). The 6 predicted miRNAs were common to these three algorithms. b , c qPCR analysis of miR-137 ( b ) and miR-34a ( c ) levels in 50 paired cancerous and normal tissue samples from OC patients. d , e Kaplan-Meier analysis of overall survival in 50 OC patients with high median ( n = 25) or low median ( n = 25) expression levels of miR-137 ( d ) or miR-34a ( e )

Article Snippet: The Snail 3′-UTR luciferase vector was purchased from OriGene.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-137 and miR-34a directly target Snail and inhibit EMT, invasion and sphere-forming ability of ovarian cancer cells

doi: 10.1186/s13046-016-0415-y

Figure Lengend Snippet: Snail is a direct target of miR-137 and miR-34a in OC cells. a Relative miR-34a expression in OC cell lines (SKOV-3 and ES-2) and normal ovarian epithelial NOEC cells. b , c ES-2 cells were transfected with reporter constructs containing either wild-type (WT) Snail , or Snail 3′-UTR with mutation (MUT), along with miR-137 mimic ( b ), miR-34a mimic ( c ), or negative control mimic (Neg mimic), respectively. Relative luciferase activity was measured. d , e qPCR analysis of Snail expression in OC cells after overexpression ( d ) or knockdown ( e ) of miR-137 and miR-34a. f Western blotting analysis of Snail expression in OC cells after overexpression or knockdown of miR-137 and miR-34a. g , h qPCR analysis of indicated mRNAs in OC cells after transient overexpression or knockdown of miR-137 ( g ) and miR-34a ( h ). i Relative mRNA expression of Snail in OC tissues and matched normal tissues. j Analysis of Snail mRNA expression using microarray (Oncomine) on normal ovary versus OC tissue. ** P < 0.01

Article Snippet: The Snail 3′-UTR luciferase vector was purchased from OriGene.

Techniques: Expressing, Transfection, Construct, Mutagenesis, Negative Control, Luciferase, Activity Assay, Over Expression, Western Blot, Microarray