Journal: Cell Death & Disease

Article Title: Amlexanox, a selective inhibitor of IKBKE, generates anti-tumoral effects by disrupting the Hippo pathway in human glioblastoma cell lines

doi: 10.1038/cddis.2017.396

Figure Lengend Snippet: The Hippo pathway was involved in the proliferative regulation of glioma cells mediated by IKBKE. ( a ) U87 and U251 cells were transfected with control shRNA or IKBKE shRNA for 48 h, and the effects of IKBKE knockdown on the protein expression of the Hippo pathway were determined by western blot. ( b ) Glioma cells were transfected with IKBKE-expressing plasmid for 48 h, and the effects of IKBKE up-regulation on the protein expression were determined by western blot as in a . ( c and d ) Expression of YAP1 in the cytoplasmic and nuclear fractions was examined by western blot. ( e ) IKBKE-knockdown U87 and U251 cells were stained with DAPI and the antibody against YAP1 (scale bar=20 μ m)

Article Snippet: Antibodies against IKBKE, YAP1, p-YAP1 (Ser127), c-Myc and Ubiquitin were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Control, shRNA, Knockdown, Expressing, Western Blot, Plasmid Preparation, Staining

Journal: Cell Death & Disease

Article Title: Amlexanox, a selective inhibitor of IKBKE, generates anti-tumoral effects by disrupting the Hippo pathway in human glioblastoma cell lines

doi: 10.1038/cddis.2017.396

Figure Lengend Snippet: IKBKE interacts with and degrades LATS2. ( a ) Expression of LATS1/2 in IKBKE knockdown and control cells was examined at transcript levels. ( b ) The endogenous interaction between IKBKE and LATS1/2 in U87 cells was analyzed by immunoprecipitation. ( c ) LATS2 expression in IKBKE-knockdown U87 cells and control cells was measured after treatment with or without 20 μ M MG132 for 12 h. ( d ) Decreased LATS2 ubiquitylation level by IKBKE knockdown in U87 cells. ( e ) Measurement of LATS2 in cell lysates harvested at 0, 2, 4, 8 and 12 h after the addition of CHX (100 μ M) to arrest protein synthesis. ( f ) Schematic diagram of the mechanism of amlexanox-mediated antitumor activity by downregulation of IKBKE in GBM. IKBKE directly binds to and negatively regulates LATS1/2, which promotes YAP1 cytoplasmic retention and subsequent degradation

Article Snippet: Antibodies against IKBKE, YAP1, p-YAP1 (Ser127), c-Myc and Ubiquitin were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Knockdown, Control, Immunoprecipitation, Activity Assay

Journal: Cell Death & Disease

Article Title: Amlexanox, a selective inhibitor of IKBKE, generates anti-tumoral effects by disrupting the Hippo pathway in human glioblastoma cell lines

doi: 10.1038/cddis.2017.396

Figure Lengend Snippet: Amlexanox exhibited significant antitumor effects against subcutaneous tumors in vivo . ( a ) Images of nude mice and tumors from the DMSO and amlexanox treatment groups. ( b ) The tumor volumes and ( c ) weights, and ( d ) the body weights of mice were evaluated using the in vivo proliferation assay. ( e ) Representative images of the HE and immunohistochemical staining for IKBKE and the proteins of the Hippo pathway in tumor sections (× 200 magnification). IKBKE protein was effectively inhibited by amlexanox treatment. Besides, the expressions of LATS2 and p-YAP1(Ser127) increased, whereas YAP1, Axl, c-Myc, Cyr61, MMP-2 and MMP-9 expression levels were simultaneously decreased in amlexanox-treated group relative to the DMSO-treated group. Data are shown as the mean±S.D. * P <0.05, ** P <0.01, compared to the control ( n =6)

Article Snippet: Antibodies against IKBKE, YAP1, p-YAP1 (Ser127), c-Myc and Ubiquitin were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: In Vivo, Proliferation Assay, Immunohistochemical staining, Staining, Expressing, Control

Journal: Cell Death & Disease

Article Title: Amlexanox, a selective inhibitor of IKBKE, generates anti-tumoral effects by disrupting the Hippo pathway in human glioblastoma cell lines

doi: 10.1038/cddis.2017.396

Figure Lengend Snippet: Antitumor effects of amlexanox in a U87 orthotopic intracranial model. The mice were treated with intraperitoneal injection with DMSO or amlexanox (100 mg/kg) daily. The treatment started from the 7th day after implantation and lasted for ~21 days. ( a ) Representative images of bioluminescence of mice on days 7, 14, and 28 after implantation. ( b ) Quantitative analysis of these bioluminescence images for the DMSO and amlexanox treatment groups. ( c ) The overall survival of mice in the DMSO and amlexanox treatment groups. There was a substantial survival benefit for the amlexanox-treated mice. ( d ) Representative images of the HE and immunohistochemical staining for IKBKE and the Hippo pathway proteins in tumor sections (× 200 magnification). IKBKE protein was effectively inhibited by amlexanox treatment. Besides, the expressions of LATS2 and p-YAP1(Ser127) increased, whereas YAP1, Axl, c-Myc, Cyr61, MMP-2 and MMP-9 expression levels were simultaneously decreased in amlexanox-treated group relative to the DMSO-treated group. Data are shown as the mean±S.D. * P <0.05, ** P <0.01 compared to the control ( n =6)

Article Snippet: Antibodies against IKBKE, YAP1, p-YAP1 (Ser127), c-Myc and Ubiquitin were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Injection, Immunohistochemical staining, Staining, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Urolithin A attenuates hexavalent chromium-induced small intestinal injury by modulating PP2A/Hippo/YAP1 pathway

doi: 10.1016/j.jbc.2024.107669

Figure Lengend Snippet: The inv olvement of Hippo/YAP1 signaling pathway in the intestinal injury induced by Cr(VI). A , representative panoramic images of immunofluorescence staining with an antibody against YAP1 ( green ) in small intestinal sections from WT and HE mice treated with 20 mg/l Cr(VI) (40× and 200× magnification). The nuclei ( blue ) were stained with 4,6-diamino-2-phenyl indole (DAPI). B , YAP1 expression was quantified and expressed as relative fluorescence intensity normalized by to DAPI density. C , protein levels of indicated protein in small intestinal tissues were determined by immunoblotting analysis. D , representative images shown for small intestinal sections stained with p-YAP1(Ser127) antibody (200× magnification). E , quantification was shown for the fold change of indicated protein level relative to WT control mice. F , quantification of p-YAP1(Ser127)-stained area was expressed as percentage of small intestinal fields occupied (n = 3). ∗ p < 0.05, compared with WT control mice. # p < 0.05, compared with HE control mice. Cr(VI), hexavalent chromium; HE, heterozygous; PP2A, protein phosphatase 2A.

Article Snippet: The following antibodies were used: rabbit anti-PP2A Aα, anti-LATS1, anti-p-YAP1 (Ser109), p-YAP1 (Ser127) (Cell Signaling Technology), mouse anti-TAZ, rabbit anti-YAP1 (Proteintech group), and rabbit anti-PP2A Aβ (Bioworld).

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Urolithin A attenuates hexavalent chromium-induced small intestinal injury by modulating PP2A/Hippo/YAP1 pathway

doi: 10.1016/j.jbc.2024.107669

Figure Lengend Snippet: Hippo/YAP1 signaling pathway mediated Cr(VI)-induced intestinal injury by regulating cell proliferation and apoptosis. Representative images of staining with ( A ) Ki-67 and ( C ) TUNEL in small intestinal sections WT and HE mice upon 20 mg/l Cr(VI) treatment (200× magnification). The nuclei ( blue ) were stained with 4,6-diamino-2-phenyl indole. The numbers of ( B ) Ki-67–positive cells and ( D ) apoptotic cells were quantified and expressed as the average number of positive cells every five visual filed per mouse. Spearman correlation analysis was performed to analyze the association between YAP1 expression and the number of ( E ) Ki-67–positive cells or ( F ) apoptotic cells, respectively. ∗ p < 0.05, compared with WT control mice. # p < 0.05, compared with HE control mice. Cr(VI), hexavalent chromium; HE, heterozygous; TUNEL, terminal-deoxynucleotidyl transferase-mediated nick end labeling.

Article Snippet: The following antibodies were used: rabbit anti-PP2A Aα, anti-LATS1, anti-p-YAP1 (Ser109), p-YAP1 (Ser127) (Cell Signaling Technology), mouse anti-TAZ, rabbit anti-YAP1 (Proteintech group), and rabbit anti-PP2A Aβ (Bioworld).

Techniques: Staining, TUNEL Assay, Expressing, Control, End Labeling

Journal: The Journal of Biological Chemistry

Article Title: Urolithin A attenuates hexavalent chromium-induced small intestinal injury by modulating PP2A/Hippo/YAP1 pathway

doi: 10.1016/j.jbc.2024.107669

Figure Lengend Snippet: Urolithin A attenuated Cr(VI)-induced small intestinal injury by regulating Hippo/YAP1 signaling pathway. Mice were treated with 20 mg/l Cr(VI) in the presence or absence of urolithin A (20 mg/kg body weight) for 28 days (n = 10). A , representative panoramic images were shown for small intestinal sections stained with H&E (10× or 100× magnification). B , the length of intestinal villi (n = 3) and ( C ) depth of intestinal crypt (n = 3) were quantified by ImageJ. D , representative images were shown in small intestinal tissues stained with AB-PAS (200× magnification). E , AB-PAS staining was performed to count the number of goblet cells (n = 3). F , representative images of immunofluorescence staining with an antibody against occludin ( green ) in small intestinal tissues. The nuclei ( blue ) were stained with 4,6-diamino-2-phenyl indole. G , occludin expression was quantified and expressed as fluorescence intensity relative to control group (n = 3). H , the intestinal permeability was determined by the concentration of FITC-dextran in serum. I , the alkaline phosphatase (ALP) level was normalized to intestinal protein and expressed as unit/g protein. ∗ p < 0.05, compared with control mice. # p < 0.05, compared with urolithin A mice. AB-PAS, alcian blue/periodic acid-Schiff; Cr(VI), hexavalent chromium.

Article Snippet: The following antibodies were used: rabbit anti-PP2A Aα, anti-LATS1, anti-p-YAP1 (Ser109), p-YAP1 (Ser127) (Cell Signaling Technology), mouse anti-TAZ, rabbit anti-YAP1 (Proteintech group), and rabbit anti-PP2A Aβ (Bioworld).

Techniques: Staining, Immunofluorescence, Expressing, Fluorescence, Control, Permeability, Concentration Assay