Journal: iScience

Article Title: Primary cilia are required for the persistence of memory and stabilization of perineuronal nets

doi: 10.1016/j.isci.2021.102617

Figure Lengend Snippet: The role of the primary cilium in recent and remote memory (A) Experimental outline: AAV vector expressing control (GFP) or shIFT88 (IFT88) RNA was infused into CA1. Six weeks later mice were fear conditioned, and tested 4, 21 and 35 days later. (B) Effect of the primary cilium depletion from CA1 on memory. Mice infused into CA1 with control or shIFT88 AAV were fear conditioned, and tested at recent and remote time points. Data are represented as mean ± SEM (repeated measures two-way ANOVA, n = 10/group, effect of virus: F 2, 36 = 1.351, p = 0.0070; effect of day: F 1.917, 34.51 = 14.13, P<0.0001; post-hoc: ∗p = 0.0110 D35 GFP vs IFT88, ‡ p = 0.0251 IFT88 D35 vs D4, ## p = 0.0025 IFT88 D21 v D35). (C) Representative images demonstrating the depletion of primary cilia from CA1 region of DH. Size bar: 5 μm. (D) Quantification of the shIFT88 RNA effect. Data are represented as mean ± SEM. ∗∗∗∗ P < 0.0001 (unpaired t test, two-tailed; n = 8/group; t 1 4 = 8.466). (E) Effect of the primary cilium depletion from CA1 on remote memory. Mice infused into CA1 with control or shIFT88 AAV were fear conditioned, and tested at remote time point only. Data are represented as mean ± SEM (unpaired t test, two-tailed; n = 9 (GFP), n = 10 (IFT88)/group; t 1 7 = 7.674; ∗∗∗∗ P < 0.0001). (F) Effect of cyclopamine infusion into CA1 on recent and remote memory retrieval. Data are represented as mean ± SEM (two-way ANOVA, n = 9/group, effect of treatment F 1, 14 = 3.182 p = 0.0962; effect of day F 1, 14 = 1.755, p = 0.2065). See also A and S3B.

Article Snippet: The viral vectors carrying a construct coding for an shRNA targeting mouse IFT88 (IFT88: AAV-DJ/8-U6-GFP-m-IFT88-shRNA; Vector Biolabs), or control construct (GFP: pAAV2-CAG-GFP; Addgene) were bilaterally infused into the dorsal hippocampus (1.8 mm posterior, ±1.0 mm lateral and 2.0 mm ventral to bregma).

Techniques: Plasmid Preparation, Expressing, Control, Virus, Two Tailed Test

Journal: iScience

Article Title: Primary cilia are required for the persistence of memory and stabilization of perineuronal nets

doi: 10.1016/j.isci.2021.102617

Figure Lengend Snippet:

Article Snippet: The viral vectors carrying a construct coding for an shRNA targeting mouse IFT88 (IFT88: AAV-DJ/8-U6-GFP-m-IFT88-shRNA; Vector Biolabs), or control construct (GFP: pAAV2-CAG-GFP; Addgene) were bilaterally infused into the dorsal hippocampus (1.8 mm posterior, ±1.0 mm lateral and 2.0 mm ventral to bregma).

Techniques: Virus, Plasmid Preparation, Recombinant, Staining, Blocking Assay, Fluorsave, Isolation, Reverse Transcription, SYBR Green Assay, Sample Prep, Software, Real-time Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: GPR137-RAB8A activation promotes ovarian cancer development via the Hedgehog pathway

doi: 10.1186/s13046-025-03275-0

Figure Lengend Snippet: Identification of HH signaling as the downstream of RAB8A. A The protein levels of exogenous RAB8A (Flag) in RAB8A cells transfected with a Flag-tagged RAB8A (Flag-RAB8A, +) or an empty vector (Flag-RAB8A, -) for 24 h were detected by western blot. B The number of genes whose expression was altered by Flag-RAB8A-expression is summarized in the table. C Volcano scatter plot for the genes in (B). D GO analysis was conducted to compare the gene expression from RAB8A group and the control. E KEGG pathway enrichment analysis was conducted to compare the gene expression from RAB8A group and the control. F The protein expression of GLI1 and PTCH1 in HO8910 cells transfected with RAB8A (Flag-RAB8A, +) or an empty vector (Flag-RAB8A, -). G GLI luciferase (GLI-Luc) activities in HO8910 cells transfected with RAB8A or an empty vector (Con). H Nude mice with xenografts derived from HO8910 cells stably expressing RAB8A or control viruses. I Image showing the size of the representative tumor xenografts from two groups. J Weights of xenografts derived from HO8910 cells stably expressing RAB8A or control viruses at 21 d post inoculation. K The protein levels of GLI1 and RAB8A in tumors from HO8910 cells stably expressing RAB8A or control viruses. L The protein expression of PTCH1, GLI1, GLI2, GLI3-full length (GLI3F), GLI3-repressor (GLI3R), IFT88, KIF3A, and SuFu in SK-OV-3 cells transfected with RAB8A shRNA (shRAB8A, +) or scrambled shRNA (shRAB8A, -). M The protein expression of GLI1 and PTCH1 in A2780 cells transfected with RAB8A shRNA (shRAB8A, +) or scrambled shRNA (shRAB8A, -). N GLI luciferase (GLI-Luc) activities in SK-OV-3 cells transfected with RAB8A shRNA (shRNA RAB8A) or scrambled shRNA (shRNA Con). O GLI luciferase (GLI-Luc) activities in A2780 cells transfected with RAB8A shRNA (shRNA RAB8A) or scrambled shRNA (shRNA Con). P The mRNA levels of GLI1 and PTCH1 in SK-OV-3 cells transfected with RAB8A shRNA (shRNA-RAB8A) or scrambled shRNA (shRNA-Con). Q The mRNA levels of GLI1 and PTCH1 in A2780 cells transfected with RAB8A shRNA (shRNA-RAB8A) or scrambled shRNA (shRNA-Con). R Co-immunoprecipitation of endogenous SuFu and GLI1 in the presence of RAB8A shRNA (shRAB8A) in SK-OV-3 cells. IP: SuFu; WB: GLI1. IgG was used as a negative control. S Co-immunoprecipitation of endogenous SuFu and GLI1 in the presence of ectopic Flag-RAB8A in HO8910 cells. IP: SuFu; WB: GLI1. IgG was used as a negative control. T Nucleo-cytoplasmic separation assays in HO8910 cells transfected with Flag-RAB8A or an empty vector (Flag-RAB8A, -), and protein levels of GLI1 were detected by western blot. U GEPIA database displayed the correlation between RAB8A and PTCH1 . * p < 0.05; ** p < 0.01; error bar, SD

Article Snippet: Flag (M185-3, 1:10000) antibody was from MBL (Beijing, China), and antibodies for GLI1 (sc-20687, 1:1000), GLI3 (sc-74478, 1:1000), SuFu (sc-137014, 1:1000), IFT88 (sc-84318, 1:1000), KIF3A (sc-376680, 1:1000), MMP-2 (sc-13595, 1:1000), MMP-9 (sc-21733, 1:1000), GFP (sc-9996, 1:5000), EZRIN (sc-58758, 1:1000), GAPDH (sc-32233, 1:2000) and normal mouse IgG (sc-2025) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Gene Expression, Control, Luciferase, Derivative Assay, Stable Transfection, shRNA, Immunoprecipitation, Negative Control

Journal: Molecular Cancer Therapeutics

Article Title: Arginine Deiminase Resistance in Melanoma Cells Is Associated with Metabolic Reprogramming, Glucose Dependence, and Glutamine Addiction

doi: 10.1158/1535-7163.mct-13-0302

Figure Lengend Snippet: Figure 4. Analyses of protein expression in A2058 and its five ADIR cells. A, activated phosphorylation AKT (p-AKT) signaling. B, enhanced sensitivity of ADIR cells to the PI3K inhibitor (LY294002) and AKT (perifosine) as compared with their parental line. C, Western blotting analyses of mTOR signaling, AMPKa, and eIF1a. D, sensitivity of A2058 and its ADIR cells to mTOR inhibitor CCI-779. Cells were incubated with CCI-779 at concentrations ranging from 1 to 100 mmol/L for 72 hours and their viabilities were determined by MTT assay.

Article Snippet: Reagents were obtained from the following sources: ADI-PEG20 (specific activity, 5–10 IU/mg) from Polaris Pharmaceuticals, Inc.; LY294002, 2-deoxy-D-glucose (2- DG), 3-bromopyruvate (3-BP), 6-diazo-5-oxo-L-norleucine (DON), azaserine, deferoxamine mesylate (DFX), and CoCl2, from Sigma-Aldrich; perifosine from Cayman Chemical; temsirolimus (CCI-779) from Selleckchem.

Techniques: Expressing, Phospho-proteomics, Western Blot, Incubation, MTT Assay