Journal: Science translational medicine

Article Title: Restoring glucose uptake rescues neutrophil dysfunction and protects against systemic fungal infection in mouse models of kidney disease.

doi: 10.1126/scitranslmed.aay5691

Figure Lengend Snippet: Fig. 5. Aberrant activation of GSK3 in neutrophils in uremia. C57Bl/6 BM neutrophils + AAI or control or AAII serum were analyzed for pAKTSer473 expression by (A) FACS and immunoblot at 45 min. Histogram and images repre- sent of one of four and three experiments, respectively. Neutrophils ± pan PI3K inhibitor (at the indicated concentra- tions) ± C. albicans were assessed for (B) fungicidal activity and (C) ROS generation. (D) C57Bl/6 WT mice (n = 6) were treated with LY294002 and evaluated for fungal burden at 48 hours p.i. Neutrophils + AAI or control or AAII serum were analyzed for (E and G) pGSK3Ser9 expression by immunoblot and FACS, (F) pGSK3Ser21, (H) pp70S6KThr389, and (I) p4E-BP1Thr37/46 by immunoblot at 45 min. Immunoblot images represent one of three experiments. WT mice (n = 6) were either treated with Torin1 or left untreated. Mice were evaluated for (J) fungal burdens and (K) in vivo glucose uptake by the neutrophils in the kidneys. In vitro ROS (norm) = MFI of DHR in C. albicans–stimulated/DHR MFI-unstimulated cells. Pooled data from three to four experiments for (A) to (C) and (E) to (I) and two experiments for (D), (J), and (K) expressed as means ± SD. Statistical analyses by Student’s t test (C, D, and J) and one-way ANOVA (A, B, G, and K).

Article Snippet: GSK3 inhibitor Mice were treated (intraperitoneal injection) with LiCl (40 mg/kg per day) or PBS control starting at day 0 (relative to infection) and daily for next 2 days (for fungal load) or 4 days (for survival) p.i. (75). mTOR inhibitor Mice were intraperitoneally injected with Torin1 (Selleckchem) (20 mg/kg per day) or vehicle control [20% N-methyl-2-pyrrolidone + 50% polyethylene glycol 400 (PEG 400)] starting at day 0 (relative to infection) and daily for next 2 days p.i. (76).

Techniques: Activation Assay, Control, Expressing, Western Blot, Activity Assay, In Vivo, In Vitro

Journal: Science translational medicine

Article Title: Restoring glucose uptake rescues neutrophil dysfunction and protects against systemic fungal infection in mouse models of kidney disease.

doi: 10.1126/scitranslmed.aay5691

Figure Lengend Snippet: Fig. 6. GSK3 inhibitors rescue neutrophil dysfunction in uremia. (A) C57Bl/6 BM neutrophils + AAI or control serum ± GM-CSF (10 ng/ml) ± C. albicans were evaluated for ROS production at 180 min. In vitro ROS (norm) = MFI of DHR in C. albicans stimulated divided by unstimulated cells. Neutrophils + AAI or control serum ± LiCl were assessed for (B) pGSK3Ser9 levels, (C) Glut1 expression, and (D) glucose uptake by the neutrophils. (E) Basal glucose uptake by neutrophils was measured after treatment with the GSK3 inhibitor SB415286. (F) Antifungal activity of neutrophils + AAI or control serum ± LiCl or SB415286 + C. albicans was measured at 180 min. WT mice (n = 4 to 5) were either treated with LiCl or left untreated. (G) Fungal burdens and (H) glucose uptake by neutrophils in the kidneys were assessed at 48 hours p.i. GSK3 expression was measured in (I) purified neutrophils from BM and (J) neutrophils in the spleen of PMNGSK3 mice by immunoblot and FACS analyses, respectively. (K) PMNGSK3 and control mice (n = 5 to 8) were subjected to AAN. A cohort of PMNGSK3 or control mice (n = 5 to 6) received PBS. Mice were infected with C. albicans and fungal loads were quantified at 48 hours p.i. The histograms (B and J) and images (C and I) represent one of two to four experiments. For (C), lanes were run on the same gel but were nonconsecutive (splicing indicated by black dashed line). The data are pooled from three experiments for (A), (B), and (D) to (F) and two experiments for (G), (H), and (K), and expressed as means ± SD. Statistical analyses by Student’s t test (G and H) and one-way ANOVA (A, B, D, E, F, and K).

Article Snippet: GSK3 inhibitor Mice were treated (intraperitoneal injection) with LiCl (40 mg/kg per day) or PBS control starting at day 0 (relative to infection) and daily for next 2 days (for fungal load) or 4 days (for survival) p.i. (75). mTOR inhibitor Mice were intraperitoneally injected with Torin1 (Selleckchem) (20 mg/kg per day) or vehicle control [20% N-methyl-2-pyrrolidone + 50% polyethylene glycol 400 (PEG 400)] starting at day 0 (relative to infection) and daily for next 2 days p.i. (76).

Techniques: Control, In Vitro, Expressing, Activity Assay, Purification, Western Blot, Infection

Journal: Cancer Research

Article Title: Inhibition of Phosphatidylinositol 3-Kinase Destabilizes Mycn Protein and Blocks Malignant Progression in Neuroblastoma

doi: 10.1158/0008-5472.can-05-2769

Figure Lengend Snippet: Figure 5. Inhibition of PI3K causes increased phosphorylation and decreased levels of Mycn protein. A, Kelly cells were treated with 20 Amol/L LY294002 for 24 hours in 10% serum, with lactacystin (10 Amol/L) added at 3 hours. Immunoblots show increased levels of pMycn and decreased levels of pAkt. B, serum-starved SH-SY5Y cells were treated for 6 hours with vehicle (V; DMSO), LY294002, or wortmannin in the presence of IGF-I and lactacystin. IGF-1 led to increased levels of pAkt. LY294002 and wortmannin blocked pAkt, resulting in increased levels of pMycn and decreased levels of total Mycn. C, chemical inhibition of GSK3h (SH-SY5Y cells, GSK inhibitor II) led to an increase in the level of total Mycn protein. D, siRNA-mediated inhibition of GSK3h leads to reduced levels of pMycn and increased levels of total Mycn, consistent with GSK3h as the kinase that directly phosphorylates and destabilizes Mycn. Kelly cells growing in serum were transiently transfected with siRNA directed against GSK3h or a scrambled siRNA control (S). Levels of GSK3h, pGSK3h, Mycn, and pMycn were assessed at 24 hours.

Article Snippet: Lactacystin and GSK inhibitor II were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Transfection, Control

Journal: Molecular Oncology

Article Title: NUAK1 governs centrosome replication in pancreatic cancer via MYPT1 / PP1β and GSK3β ‐dependent regulation of PLK4

doi: 10.1002/1878-0261.13425

Figure Lengend Snippet: NUAK1 regulation of PLK4 is mediated by GSK3β. (A) Quantification of centrosome number by γ‐tubulin IF staining in synchronised Mia PaCa‐2 cells transfected with MYPT1, or non‐targeting (siC), siRNAs. Fifty cells scored per condition per experiment. Mean ± SEM of three independent experiments shown. One‐way ANOVA with post hoc t ‐test. (B) Immunoblot of total PLK4 in Mia PaCa‐2 cells transfected with MYPT1, versus non‐targeting (siCtrl), siRNA. Representative of three independent experiments. (C) Immunoblot of Ser9 phospho‐GSK3β in Mia PaCa‐2 cells transfected with NUAK1, versus non‐targeting, siRNA. Representative of three independent experiments. (D) Immunoblot of Ser9 phospho‐GSK3β in Mia PaCa‐2 cells treated with 10 μ m HTH‐01‐015 or DMSO vehicle for 4 h. Representative of three independent experiments. (E) Immunoblot of total PLK4 in Mia PaCa‐2 cells transiently transfected with constitutively active (S9A) or kinase‐dead (K85R) mutant GSK3β expression vector, compared with empty vector (EV). Representative of three independent experiments. (F) Immunoblots for endogenously expressed PLK4, NUAK1, and GSK3β in lysates and anti‐PLK4 immunoprecipitates, or IgG control IPs, of untreated asynchronous Mia PaCa‐2 cells. Representative of two independent experiments. (G) Immunoblot of total PLK4 in Mia PaCA‐2 cells pre‐treated for 1 h with 3 μ m CHIR‐99021 (GSK3i) or DMSO vehicle, followed by 10 μ m HTH‐01‐015 ± 3 μ m CHIR‐99021 for 1 h immediately prior to harvest. Representative of three independent experiments. (H) Quantification of centrosome number by γ‐tubulin IF in mitotic Mia PaCa‐2 cells treated with CHIR‐99021 (GSK3i), HTH‐01‐015 (HTH), both, or vehicle ctrl, from time of release from thymidine block. Cells were fixed for analysis at 10.5 h post‐release. Fifty cells were scored per treatment condition per experiment. Mean ± SEM of three independent experiments shown. One‐way ANOVA with post hoc Tukey's test. (I) Quantification of incidence of nuclear aberrations in Mia PaCa‐2 cells treated with CHIR‐99021 (GSK3i), HTH‐01‐015 (HTH), both, or vehicle ctrl, from time of release from thymidine block. HTH‐01‐015 and ctrl values are the same as Fig. as experiment was performed simultaneously. Cells were fixed for analysis at 13 h post‐release. One hundred cells were scored per treatment group per experiment. Mean ± SEM of three independent experiments shown. One‐way ANOVA with post hoc Tukey's test. For all panels, P value; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells in log phase growth were treated with the indicated concentrations of 5 or 10 μ m of NUAK1 inhibitor (HTH‐01‐015, Tocris, Bristol, UK), GSK3 inhibitor 3 μ m – (CHIR99021, Tocris), PLK4 inhibitor 100 n m (Centrinone, MedChem Express, Monmouth Junction, NJ, USA) throughout the study.

Techniques: Staining, Transfection, Western Blot, Mutagenesis, Expressing, Plasmid Preparation, Control, Blocking Assay