Journal: International Journal of Nanomedicine

Article Title: Second-generation aptamer-conjugated PSMA-targeted delivery system for prostate cancer therapy

doi: 10.2147/ijn.s23747

Figure Lengend Snippet: Figure 5 Bcl-2, Cyclin D1, and Wnt3a knock down following miRNA/PAMAM-PEG-APT complexes delivery to LNCaP cells. The cells were treated with miRNA/ PAMAM-PEG-APT (miRNA) and NC-miRNA/PAMAM-PEG-APT (NC-miRNA) complexes and processed for immunoblotting with anti-Bcl-2, anti-Cyclin D1, and anti-Wnt3a antibodies at 24, 48, and 72 hours post-transfection. β-actin was a loading control. Abbreviations: PAMAM, polyamidoamine; PEG, polyethylene glycol; APT, aptamer.

Article Snippet: The blotted membrane was immunostained with antibodies specific to anti-Bcl-2 (BioWorld, Visalia, CA), anti-Cyclin D1 (BioWorld) and anti-Wnt3a antibodies (R&D Systems Inc, Minneapolis, MN).

Techniques: Knockdown, Western Blot, Transfection, Control

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 1. miR‑214 is downregulated in liver cancer and targets Wnt3a. (A) Reverse transcription‑quantitative polymerase chain reaction was performed to examine the expression of miR‑214 in 24 paired human hepatocellular carcinoma and non‑tumor tissues. (B) Relative expression of miR‑214 in liver cancer cell lines and a normal liver cell line. **P<0.01; *P<0.05. (C) miR‑214 seed region sequence in the 3'UTR of Wnt3a. (D) Wnt3a protein expression as detected by immunohistochemistry. (E) Protein expression levels of Wnt3a were measured by western blot analysis in HepG2 cells transfected with miR‑214 or miR‑ctrl. (F) miR‑214 was co‑transfected with pmirGLO, pmirGLO‑Wnt3a‑3'‑UTR‑wt or pmirGLO‑Wnt3a‑3'‑UTR‑mut in HepG2 cells. Relative luciferase activity was measured after 48 h. *P<0.05 vs. control. miR, microRNA; mut/M, mutant; UTR, untranslated region; wt/W, wild‑type.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: Polymerase Chain Reaction, Expressing, Sequencing, Immunohistochemistry, Western Blot, Transfection, Luciferase, Activity Assay, Control, Mutagenesis

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 2. miR‑214 inhibits the proliferation of liver cancer cells. CCK8 assay was performed to detect the effects of miR‑214 on cell proliferation at 24, 48, and 72 h in (A) HepG2 and (B) Hep3B cells. CCK8 assay was performed to detect the effects of siWnt3a on cell proliferation at 24, 48 and 72 h in (C) HepG2 and (D) Hep3B cells. Wnt3a overexpression vector was co‑transfected with miR‑ctrl or miR‑214 into (E) HepG2 and (F) Hep3B cells, and cell proliferation was detected by CCK8 assay. *P<0.05; **P<0.01 vs. miR‑ctrl + Wnt3a‑ctrl. CCK8, Cell Counting kit‑8; ctrl, control; miR, microRNA; OD, optical density; si, small interfering RNA.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: CCK-8 Assay, Over Expression, Plasmid Preparation, Control, Small Interfering RNA

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 3. Overexpression of miR‑214 or Wnt3a silencing affects cell cycle progression. Cell cycle analysis of (A) HepG2 and (B) Hep3B cells following transfection with miR‑214 or miR‑ctrl for 48 h. Cell cycle analysis of (C) HepG2 and (D) Hep3B cells following transfection with siWnt3a or si‑ctrl for 48 h. *P<0.05. ctrl, control; miR, microRNA; si, small interfering RNA.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: Over Expression, Cell Cycle Assay, Transfection, Control, Small Interfering RNA

Journal: Biochemical and biophysical research communications

Article Title: Therapeutic potential of targeting PCLAF in endometrial cancer: Insights from Wnt/β-catenin and P53 regulatory mechanisms.

doi: 10.1016/j.bbrc.2025.152092

Figure Lengend Snippet: Fig. 6. The impact of PCLAF knockdown on proteins linked to the Wnt/β-catenin signaling pathway. (A) GSEA analysis of transcriptome sequencing revealed that PCLAF stimulates the Wnt/β-catenin pathway in EC. (B, C) WB analysis displayed that silencing PCLAF led to reduced levels of WNT3A, WNT5A, β-catenin, c-Myc, and MMP7. *p < 0.05, **p < 0.01, and ***p < 0.001, **p < 0.01, and ***p < 0.001.

Article Snippet: Then incubated with primary antibodies of appropriate dilution, all of which were goat anti-rabbit IgG: PCLAF (Abcam, USA); WNT3A, WNT5A, β-catenin (GeneTex, USA); c-Myc, MMP7, P–P53, P21, Cleaved caspase3 (Wanlei, China); CyclinD1, BAX, BCL2 (HuaBio, China); PMAIP1 (Boster, China); all of them were in 1:1000 dilution, while β-actin (Boster, China) was in 1:5000 dilution.

Techniques: Knockdown, Sequencing

Journal: Nature Chemical Biology

Article Title: Inhibitors of Hedgehog acyltransferase block Sonic Hedgehog signaling

doi: 10.1038/nchembio.1184

Figure Lengend Snippet: Figure 1 | RU-SKI 43 inhibits Hhat. (a) RU-SKIs inhibit Shh palmitoylation in vitro. Membranes from cells transfected with wild-type Hhat or the inactive HhatD339A mutant were preincubated with DMSO or 12.5 μM compound, then incubated with ShhN (wild type or ShhNC24A) and [125I]iodo-palmitoyl CoA (IC16), a radioiodinated analog of palmitoyl CoA. [125I]Iodo-palmitoyl incorporation was normalized to that measured for ShhN plus DMSO; error bars represent mean ± s.d. (n = 2–4). (b) RU-SKI 43 inhibition kinetics with purified Hhat. Each point represents mean ± s.d. (n = 2). (c,d) RU-SKIs inhibit Shh protein palmitoylation in cells. COS-1 cells expressing hemagglutinin (HA)-Hhat and Shh were treated with DMSO, 25 μM RU-SKIs for 16 h (c) or 0 μM, 10 μM or 20 μM RU-SKI 43 for 5 h (d) and labeled with [125I]iodo- palmitate. Shh-specific immunoprecipitates (IPs) were analyzed by SDS-PAGE and phosphorimaging (top panels in c and d) or western blotting (lower panel in c and middle panels in d). Total cell lysates (TCL) were analyzed by western blotting. Anti-Shh, Shh-specific antibody. (e) RU-SKI 43 shows specificity for Hhat. COS-1 cells expressing H-Ras N17, Fyn or c-Src were treated with RU-SKI 43 and labeled with the indicated fatty acids. (f) RU-SKI 43 does not inhibit Porcupine. Mouse L-Wnt3a cells were transfected with Flag-Porcupine or empty vector (EV), treated with DMSO, 10 μM RU-SKI 43 or 100 nM Wnt-C59 and labeled with [125I]iodo-pentadecanoate. Full, uncut gel images are presented in Supplementary Figures 9–12.

Article Snippet: Rabbit monoclonal anti-Shh (C9C5) antibody (2207; 1:1,000 for western blotting) and rabbit monoclonal anti-Wnt3a (C64F2) antibody (2721; 1:40 for immunoprecipitation) were purchased from Cell Signaling (Danvers, MA).

Techniques: In Vitro, Transfection, Mutagenesis, Incubation, Inhibition, Purification, Expressing, Labeling, SDS Page, Western Blot, Plasmid Preparation