Journal: Virus research

Article Title: Amino acid starvation accelerates replication of Ibaraki virus.

doi: 10.1016/j.virusres.2018.10.008

Figure Lengend Snippet: Fig. 3. IBAV infection results in LC3-II accumulation without affecting mTORC1 activity. HmLu-1 cells were infected with IBAV and then treated with DMEM without FBS for 1 h (0–1 hpi) as shown in supplemental Fig. 1. The cells were collected at the in- dicated time points, (A, C) subjected to western blot ana- lysis, and (B, D) the protein expression was quantified by densitometry. Phospho-S6K (Thr389) reflects the activity of mTORC1 and the amount of LC3-II reflects the balance of generation and degradation of the autophagosome. Each bar indicates mean ± SEM of four independent ex- periments. ****, p < 0.0001.

Article Snippet: Proteins were transferred to a PVDF membrane (Immobilon-P, Merck Millipore), blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 2% skim milk, and then incubated with one of the following primary antibodies: mouse anti-IBAV antiserum (1:5000 dilution, prepared in our laboratory (Tsuruta et al., 2016)), mouse antiIBAV NS3 antiserum (1:5000; prepared in our laboratory (Urata et al., 2014)) rabbit anti-LC3 (1:1000; MBL), rabbit anti-phospho-S6K (Thr389; 1:1000; Cell Signaling Technology), rabbit anti-total S6K (1:1000; Cell Signaling Technology), and mouse anti-alpha tubulin (1:10,000; Sigma-Aldrich).

Techniques: Infection, Activity Assay, Western Blot, Lysis, Expressing

Journal: Endocrinology

Article Title: Simvastatin Inhibits Wnt/β-Catenin Pathway in Uterine Leiomyoma.

doi: 10.1210/endocr/bqab211

Figure Lengend Snippet: Figure 2. Simvastatin reduced β-catenin expression in human leiomyoma cells. Human leiomyoma cells (HuLM) and primary cells were treated with simvastatin (1uM) for 48 hours. A, Simvastatin significantly reduced the mRNA expression of CNTTB in HuLM cells. B-C, Simvastatin reduced the protein expression of total and nonphosphorylated β-catenin in both HuLM and primary cells as seen in Western blotting and immunohistochemistry. D, Nuclear protein isolation was performed to check nuclear β-catenin level, which is the site of action of the active form of β-catenin. Simvastatin reduced nuclear levels of β-catenin.

Article Snippet: The cells were then incubated overnight at 4 °C with D ow nloaded from https://academ ic.oup.com /endo/article/162/12/bqab211/6382454 by U FSC user on 25 April 2024 primary antibodies against anti-WNT4 (Thermo Scientific, #9H2L10, AB_2608445), anti-total-β catenin (Cell Signaling, #8480S, AB_11127855), anti-nonphosphorylated-β-catenin (Cell Signaling, #4270S, AB_1903918), anti-c-Myc (Cell Signaling, #D84C12, AB_1903938), diluted in antibody dilution buffer (1× PBS/1% BSA/0.3% Triton X-100) in 1:100 ratio.

Techniques: Expressing, Western Blot, Immunohistochemistry, Isolation

Journal: Endocrinology

Article Title: Simvastatin Inhibits Wnt/β-Catenin Pathway in Uterine Leiomyoma.

doi: 10.1210/endocr/bqab211

Figure Lengend Snippet: Figure 3. Simvastatin reduced c-Myc expression, a downstream signaling target of β-catenin, in human leiomyoma cells. Human leiomyoma cells (HuLM) and primary cells were treated with simvastatin (1uM) for 48 hours. A, Simvastatin significantly reduced the mRNA ex- pression of c-Myc in HuLM cells. B, Simvastatin reduced the protein expression of c-Myc in primary cells as seen in immunohistochemistry. The band was not seen when Western blotting was attempted.

Article Snippet: The cells were then incubated overnight at 4 °C with D ow nloaded from https://academ ic.oup.com /endo/article/162/12/bqab211/6382454 by U FSC user on 25 April 2024 primary antibodies against anti-WNT4 (Thermo Scientific, #9H2L10, AB_2608445), anti-total-β catenin (Cell Signaling, #8480S, AB_11127855), anti-nonphosphorylated-β-catenin (Cell Signaling, #4270S, AB_1903918), anti-c-Myc (Cell Signaling, #D84C12, AB_1903938), diluted in antibody dilution buffer (1× PBS/1% BSA/0.3% Triton X-100) in 1:100 ratio.

Techniques: Expressing, Immunohistochemistry, Western Blot

Journal: Endocrinology

Article Title: Simvastatin Inhibits Wnt/β-Catenin Pathway in Uterine Leiomyoma.

doi: 10.1210/endocr/bqab211

Figure Lengend Snippet: Figure 4. Simvastatin reduced estrogen- and progesterone-induced increase of Wnt/β-catenin activation in human leiomyoma cells. Immortalized human leiomyoma cells (HuLM) and primary cells were treated with simvastatin (1uM) for 48 hours. Estrogen (100nM) and progesterone (100nM) were added 24 hours prior to fixation. Estrogen and progesterone treatment increased the expression of Wnt4, total β-catenin, and nonphosphorylated β-catenin in HuLM and primary cells. Simvastatin reduced the protein-induced increase in expression of these proteins.

Article Snippet: The cells were then incubated overnight at 4 °C with D ow nloaded from https://academ ic.oup.com /endo/article/162/12/bqab211/6382454 by U FSC user on 25 April 2024 primary antibodies against anti-WNT4 (Thermo Scientific, #9H2L10, AB_2608445), anti-total-β catenin (Cell Signaling, #8480S, AB_11127855), anti-nonphosphorylated-β-catenin (Cell Signaling, #4270S, AB_1903918), anti-c-Myc (Cell Signaling, #D84C12, AB_1903938), diluted in antibody dilution buffer (1× PBS/1% BSA/0.3% Triton X-100) in 1:100 ratio.

Techniques: Activation Assay, Expressing

Journal: Endocrinology

Article Title: Simvastatin Inhibits Wnt/β-Catenin Pathway in Uterine Leiomyoma.

doi: 10.1210/endocr/bqab211

Figure Lengend Snippet: Figure 5. Simvastatin reduced Wnt agonist–induced Wnt/β-catenin activation in human leiomyoma cells. Immortalized human leiomyoma cells (HuLM) were treated with simvastatin (1uM) for 48 hours. Lithium chloride (LiCl, 20mM) or Wnt agonist (WA1, 1uM) were added 6 hours prior to fixation. As expected, LiCl and WA1 increased the expression of Wnt4, total β-catenin, and nonphosphorylated β-catenin in HuLM cells. Simvastatin reduced the protein-induced increase in expression of these proteins.

Article Snippet: The cells were then incubated overnight at 4 °C with D ow nloaded from https://academ ic.oup.com /endo/article/162/12/bqab211/6382454 by U FSC user on 25 April 2024 primary antibodies against anti-WNT4 (Thermo Scientific, #9H2L10, AB_2608445), anti-total-β catenin (Cell Signaling, #8480S, AB_11127855), anti-nonphosphorylated-β-catenin (Cell Signaling, #4270S, AB_1903918), anti-c-Myc (Cell Signaling, #D84C12, AB_1903938), diluted in antibody dilution buffer (1× PBS/1% BSA/0.3% Triton X-100) in 1:100 ratio.

Techniques: Activation Assay, Expressing

Journal: Endocrinology

Article Title: Simvastatin Inhibits Wnt/β-Catenin Pathway in Uterine Leiomyoma.

doi: 10.1210/endocr/bqab211

Figure Lengend Snippet: Figure 6. Simvastatin reduced nonphosphorylated β-catenin, the active form of β-catenin, in patients who received simvastatin for 3 months as part of a phase 2 randomized clinical trial. Human leiomyoma tissue samples were obtained from an ongoing, double-blind, phase 2 randomized control trial (NCT03400826). Patients with uterine leiomyomas were recruited for treatment with simvastatin (40 mg daily) or a placebo (starch) for a total of 12 weeks. Patients underwent a hysterectomy/myomectomy at the end of the treatment, and the leiomyoma samples were collected after surgery to evaluate the effects of simvastatin on leiomyoma tissue. The tissues were fixed with a 10% buffered formalin solution for 24 hours and kept in 70% ethanol at 4 °C until immunohistochemistry. Simvastatin significantly reduced the expression of nonphosphorylated β-catenin, the active form of β-catenin, in the patients who received the treatment.

Article Snippet: The cells were then incubated overnight at 4 °C with D ow nloaded from https://academ ic.oup.com /endo/article/162/12/bqab211/6382454 by U FSC user on 25 April 2024 primary antibodies against anti-WNT4 (Thermo Scientific, #9H2L10, AB_2608445), anti-total-β catenin (Cell Signaling, #8480S, AB_11127855), anti-nonphosphorylated-β-catenin (Cell Signaling, #4270S, AB_1903918), anti-c-Myc (Cell Signaling, #D84C12, AB_1903938), diluted in antibody dilution buffer (1× PBS/1% BSA/0.3% Triton X-100) in 1:100 ratio.

Techniques: Control, Starch, Immunohistochemistry, Expressing

Journal: Endocrinology

Article Title: Simvastatin Inhibits Wnt/β-Catenin Pathway in Uterine Leiomyoma.

doi: 10.1210/endocr/bqab211

Figure Lengend Snippet: Figure 7. Summary illustration highlighting the effect of simvastatin on the Wnt/β-catenin pathway in human uterine leiomyoma. Simvastatin treatment reduced the expression of Wnt4 and its co-receptor LRP5. It also reduced the levels of total β-catenin and its translocation to the nu- cleus where it acts as a transcriptional activator. Treatment also reduced the levels of downstream mediators c-Myc and cyclin D1. Abbreviations: FZD, frizzled; LRP, low-density lipoprotein receptor-related protein; Dvl, disheveled; GSK3, glycogen synthase kinase 3; CK1, casein kinase 1; TCF/LEF, T-cell factor/lymphoid enhancer factor.

Article Snippet: The cells were then incubated overnight at 4 °C with D ow nloaded from https://academ ic.oup.com /endo/article/162/12/bqab211/6382454 by U FSC user on 25 April 2024 primary antibodies against anti-WNT4 (Thermo Scientific, #9H2L10, AB_2608445), anti-total-β catenin (Cell Signaling, #8480S, AB_11127855), anti-nonphosphorylated-β-catenin (Cell Signaling, #4270S, AB_1903918), anti-c-Myc (Cell Signaling, #D84C12, AB_1903938), diluted in antibody dilution buffer (1× PBS/1% BSA/0.3% Triton X-100) in 1:100 ratio.

Techniques: Expressing, Translocation Assay