Journal: bioRxiv

Article Title: RIPK3 promotes neuronal survival by suppressing excitatory neurotransmission during CNS viral infection

doi: 10.1101/2024.04.26.591333

Figure Lengend Snippet: (A-I) Ripk3 -2xFV fl/fl Nestin Cre + primary neuron cultures were treated with B/B or ethanol vehicle in the presence of KN93, 666-15, or DMSO vehicle for 24h and then subjected to bulk RNA-sequencing. (A-C) Volcano plots showing differentially expressed genes (DEGs) in indicated comparisons. Data points in red are exhibit upregulated expression, while those in blue exhibit downregulated expression. Genes with an FDR < 0.05 were considered significant. (D) Selected significantly enriched IPA terms showing activation scores within each of the indicated comparisons. (E-I) Heatmaps showing selected significant DEGs associated with indicated pathways. Data are expressed as Z-scores of Log 2 Fold change values within each comparison. (J)Schematic showing treatment paradigm in which Ripk3 -2xFV fl/fl Syn1 Cre + mice were intracranially infected with ZIKV. On day 3 post infection, mice received ICV injection of B/B (or vehicle) combined with AIP, 666-15, or DMSO. Mice were then monitored for survival. (K)Survival analysis of mice in indicated treatment groups. N=5-8 animals/group. *p < 0.05, **p < 0.01. See also Figure S6 .

Article Snippet: The following chemical reagents were used in cell culture experiments: GYKI-52466 (1μM, Sigma, G119), MK801 (1μM, Sigma, M107), GSK 872 (1μM, Tocris, 6492), B/B Homodimerizer (200nM, Takara, 635058), L-Glutamic acid (100-1,000μM, Sigma, G1251), N-Methyl-D-aspartic acid (20μM-100μM, Sigma, M3262), KN93 (1μM, Sigma, K1385), myristoylated AIP (1μM, Tocris, 5959), 666-15 (1μM, Tocris, 5661), Actinomycin D (10 ng/ml, Sigma, A1410), Cycloheximide (1 μg/ml, Sigma, C7698), JSH-23 (50μM, Selleckchem, S7351), BAY 11–7085 (100μM, Tocris, 1743).

Techniques: RNA Sequencing, Expressing, Activation Assay, Comparison, Infection, Injection

Journal: Frontiers in Cell and Developmental Biology

Article Title: BCI, an inhibitor of the DUSP1 and DUSP6 dual specificity phosphatases, enhances P2X7 receptor expression in neuroblastoma cells

doi: 10.3389/fcell.2022.1049566

Figure Lengend Snippet: Inhibition of p38 prevents dephosphorylation of ERK1/2 by BCI in neuroblastoma cells. N2a cells were incubated in SFM for 1, 2, 4, 6, 8 or 24 h in the presence or absence (control) of BCI (10 μM). Total RNA was extracted from the cells and quantified. The expression of DUSP1 (A) and DUSP6 (B) mRNAs was determined and normalized to the DUSP1 or DUSP6 transcript levels in cells cultured in complete medium (time = 0, set as 100%). The results are shown as the mean ± SEM of five independent experiments performed in triplicate. * p ≤ .05, ** p ≤ .01, *** p ≤ .001 vs. control. (C) N2a cells cultured in SFM were treated with inhibitors of MEK1/2 (10 μM, U0126), p38 (10 μM, SB202190) or JNK (10 μM, SP600125) for 10 min before the addition of BCI (10 µM) or vehicle alone (control) for 1 h. Untreated cells cultured in SFM or complete medium (10% FBS) were also analyzed. The cell extracts were assayed in immunoblots to detect phospho-ERK1/2 and total ERK. (D) The histogram shows the phospho-ERK1/2 protein levels obtained by densitometry and normalization to the total ERK. The phospho-protein levels where normalized to those on untreated cells cultured in SFM, set as 100%. The values represent the mean ± SEM of five independent experiments performed in duplicate. ** p ≤ .01, *** p ≤ .001 vs. vehicle.

Article Snippet: The specific inhibitors of MAPKs and other proteins used were: SP600125 (JNK), SB202190 (p38), U0126 (MEK), 666-15 (CREB), SR11302 (AP-1), KJ Pyr 9 (Myc), cyclic Pifithrin α (p53) and VO-OH Pic (PTEN), all from Tocris Bioscience; BCI (DUSP1/DUSP6), okadaic acid (PP2A) and BBG (P2RX7) were supplied by Merck-Millipore; mithramycin A (Sp1) and actinomycin D were purchased from Sigma-Aldrich.

Techniques: Inhibition, De-Phosphorylation Assay, Incubation, Control, Expressing, Cell Culture, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidic Acid Is a Proinflammatory Stimulus of Renal Tubular Epithelial Cells

doi: 10.3390/ijms23137452

Figure Lengend Snippet: Pharmacologic dissection of LPA-induced cellular signaling pathways. HKC-8 cells were pretreated for 1 h with 666-15 (CREB1 inhibitor) 10 μM in ( A ), JSH23 (NFκΒ inhibitor) 100 μM in ( B ), PD98059 (MEK/ERK inhibitor) 50 μM in ( C ), or SP600125 (JNK inhibitor) 50 μM in ( D ) and then activated with LPA at a final concentration of 10 μΜ for 4 h. mRNA-expression levels of the indicated secreted factors were quantified with RT-qPCR. The Cq values of each gene were normalized against the Cq values of B2M . Statistical analysis was performed with unpaired t -test or Welch’s test in the case of normal data and with Mann–Whitney in the case of non-normal data. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See also .

Article Snippet: LPAR1/3 inhibitor (Ki16425, Cat. no: HY-13285, MedChemExpress, Monmouth Junction, NJ, USA) was added at 10 μM; LPAR2 inhibitor (H2L5186303, Cat. no: 10-1452, Focus Biomolecules, Plymouth Meeting, PA, USA) was added at 10 μM; CREB inhibitor (666-15, Cat. no: A616443, Toronto Research Chemicals, North York, ON, Canada) was added at 10 μM; JNK inhibitor (SP600125, Cat. no: 420119, Calbiochem, San Diego, CA, USA) was added at 50 μM; NFκB inhibitor (JSH-23, Cat. no: HY-13982, MedChemExpress, Monmouth Junction, NJ, USA) was added at 100 μM; and MEK/ERK inhibitor (PD98059, Cat. no: 513000, Calbiochem, San Diego, CA, USA) was added at 50 μM.

Techniques: Dissection, Protein-Protein interactions, Concentration Assay, Expressing, Quantitative RT-PCR, MANN-WHITNEY