Journal: iScience

Article Title: Oxygen tension-dependent variability in the cancer cell kinome impacts signaling pathways and response to targeted therapies

doi: 10.1016/j.isci.2024.110068

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Article Snippet: Rabbit monoclonal anti-phospho-p44/42 MAPK (T202/Y204) , Cell Signaling Technology , Cat# 9101; RRID:AB_331646.

Techniques: Control, Recombinant, Gentle, Red Blood Cell Lysis, Protease Inhibitor, cDNA Synthesis, BrdU Cell Proliferation Assay, Caspase-3 Assay, RNA Sequencing, Mass Spectrometry, Transgenic Assay, TaqMan Assay, Software, Modification, Flow Cytometry, Real-time Polymerase Chain Reaction, Isolation

Journal: bioRxiv

Article Title: Estradiol modulates neuronal network hyperexcitability in select NDD risk genes

doi: 10.64898/2026.02.18.706588

Figure Lengend Snippet: A. Schematic of experimental workflow to assess impact of estradiol on baseline gene expression in scn1lab -/- larvae. B-C . Comparison of number of Gene Ontology (GO) pathways that are originally upregulated in HOM+DMSO and downregulated by estradiol ( B ) or originally downregulated in HOM+DMSO and upregulated by estradiol ( C ). Selected reversed pathways are highlighted in the bar graphs, with bars indicating -log10(FDR). Asterisk for the p-adjusted values: * <0.05,**<0.01, ***<0.001. D. Schematic of experimental workflow to assess impact of estradiol on baseline brain activity in HOM larvae. Dissected larval brains are stained with phosphorylated ERK (pERK) as a marker of active neurons and total ERK (tERK) as a pan-neuronal marker. Images undergo voxel-by-voxel division of pERK by tERK to obtain an activity ratio. Activity is mapped onto the standard zebrafish brain atlas and quantified based on a 4-region or 149-region atlas. E. Voxel-wise Z score normalized pERK/tERK values representing brain activity differences in scn1lab Δ44/Δ44 + DMSO or estradiol compared to DMSO-treated scn1lab +/+ larvae and the difference (subtraction) between the two conditions. Rescued regions from the 149-region atlas are shown. For specific rescued regions, refer to SI Fig. 7. Images are a representative slice of the axial (top) and sagittal (bottom) view. Scale bar represents Z score (red/yellow, increased in mutant; cyan/purple, decreased in mutant). F. Bar graphs showing regional differences in pERK/tERK brain activity in the rhombencephalon, diencephalon, mesencephalon, and telencephalon. G. Left: Schematic of experimental workflow to assess impact of estradiol on induced seizure activity in scn1lab -/- larvae. Right: Pentylenetetrazole (PTZ)-induced seizure activity, with and without 24 h exposure to 20 𝜇M estradiol, is measured in scn1lab -/- larval zebrafish mutants using video-tracking. Representative activity tracings of control and scn1lab -/- larvae at 5dpf before and after exposure to DMSO vehicle or 20uM estradiol and 10mM PTZ or water. Ribbon shows +/- SEM. H. Average activity 30 min before and after the addition of 10mM PTZ or water to control and scn1lab -/- larvae before and after exposure to DMSO vehicle or 20uM estradiol. scn1lab -/- + E2 + PTZ larvae show significantly decreased activity compared to scn1lab -/- + vehicle + PTZ larvae (Unpaired t-test with Welch’s correction on post values, ****p<0.0001; n=24/group). I. Interaction models assessing genotype-x-estradiol-x-PTZ effect, comparing post- vs pre-PTZ measurements. Dissection of the mutant effect (HOM vs control), estradiol effect (Estradiol vs DMSO), and PTZ effect (PTZ vs water) (i). Three-way interaction model to estimate the PTZ effect among genotype-x-estradiol groups of larvae (ii). J. Left: Schematic of experimental workflow to assess impact of estradiol on neuronal busting activity in CRISPR-engineered SCN2A +/- iGLUTs . Right: Cellular and network bursting activity, with and without exposure to acute 100nM estradiol, is measured in SCN2A +/- NGN2-iGLUTs using multielectrode array. Bar graphs show mean number of cellular bursts and network bursts in controls and SCN2A +/- iGLUTs. Unpaired t-test, cellular bursts *p=0.0415, network bursts SCN2A +/- spontaneous vs SCN2A +/- E2 *p=0.0127, SCN2A +/- spontaneous vs WT spontaneous *p=0.0450.

Article Snippet: The following antibodies and dilutions were used: pERK (Cell Signaling #4370, RRID: AB_2315112) and tERK (Cell Signaling #4696, RRID:AB_390780), 1:500.

Techniques: Gene Expression, Comparison, Activity Assay, Staining, Marker, Mutagenesis, Control, Dissection, CRISPR

Journal: bioRxiv

Article Title: Deep Invaginations of Nuclear Envelope Coordinate Spatial Organization of Chromatin in Epithelium

doi: 10.64898/2026.03.10.710762

Figure Lengend Snippet: (A) The volcano plot of the differentially expressed genes (DEGs) in mature 7 d grown epithelium in comparison to developing 2 d grown epithelium. MAPK-signaling -associated genes highlighted in red. (B) Ingenuity pathway analysis (IPA) pathway generator -derived map presenting affected nuclear mechanotransduction and NE -associated components and their downstream actors. Red and green indicate up- and down-regulation, respectively. Purple lines indicate statistically significantly affected components (Abs(log2FC)) >1 and pAdj <0.05). (C) ClusterProfiler KEGG pathway over-representation “MAPK signaling pathway” analysis of the 7 d -grown mature epithelium showing significantly affected biological processes in the 7-d grown mature epithelium in comparison to the 2 d-grown epithelium. (D) IPA pathway generator -derived map showing affected components of MAPK-signaling. Red indicates up-regulated, and green indicates down-regulated. Purple lines indicate statistically significantly affected components (Abs(log2FC)) >1 and pAdj <0.05). (E) STRING analysis of MAPK-associated DEGs in the 7 d grown epithelium, indicating functional enrichment in three clusters within interconnected networks. (F) Transcription factor (TF) motif analysis of transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) of 2 d and 7 d grown cells indicates global decreased accessibility of TF motifs, highlighting significant suppression of MAPK/ERK-associated motifs in the mature epithelium. Box-and-whiskers plots presenting (G) intracellular normalized fluorescence intensity distribution, and (H) nucleo-cytoplasmic ratio of activated phosphorylated ERK1/2 (phospho-ERK, p44/42 MAPK) at 2, 4, and 7 d post-seeding. One-way ANOVA with Dunnett’s multiple comparisons test indicates statistical significance, n=three independent biological replicates. Box-and-whisker plots represent the 25 th –75 th percentiles as boxes, the median as a line within the box, and whiskers indicating the minimum and maximum values.

Article Snippet: Active MAPK was detected by using a mouse monoclonal Ab identifying the phosphorylated p44/42 MAPK (1:400, ERK1/2, Thr202/Tyr204 [E10], #9106, Cell Signaling Technology, MA, USA).

Techniques: Comparison, Derivative Assay, Functional Assay, Next-Generation Sequencing, Fluorescence, Whisker Assay

Journal: International Journal of Molecular Sciences

Article Title: Alpha Ketoglutarate Exerts In Vitro Anti-Osteosarcoma Effects through Inhibition of Cell Proliferation, Induction of Apoptosis via the JNK and Caspase 9-Dependent Mechanism, and Suppression of TGF-β and VEGF Production and Metastatic Potential of Cells

doi: 10.3390/ijms21249406

Figure Lengend Snippet: Effect of AKG on phosphorylation of MAP kinases and the influence of the selective JNK inhibitor (SP600125) on AKG-induced apoptosis in Saos-2 cells. The cells were incubated without or with AKG for 6 h and 24 h, and phosphorylated and total ERK1/2, JNK and p38 levels were determined with the ELISA assay. Quantification of the amounts of phosphorylated to total MAP kinases ( A – C ). The cells were treated with 50 mM AKG without or with SP600125 (5 μM) and harvested after 72 h of treatment for apoptosis analysis. The representative dot plots indicate the percentage of An − /PI + necrotic cells (Q1), An + /PI + late apoptotic cells (Q2), An − /PI − viable cells (Q3), and An + /PI − early apoptotic cells (Q4) in the AKG or/and SP600125-treated Saos-2 cell cultures ( D ). Quantification of the amounts of phosphorylated to total JKN kinase ( E ) and histogram representation of the quantitative percentage of apoptotic (early + late apoptosis) cells ( F ) in the control, SP600125, AKG, and SP600125 + AKG-treated Saos-2 cell cultures. Data are expressed as means ± SD for three independent experiments. ** p < 0.01, *** p < 0.001 in comparison to the control; one-way ANOVA test.

Article Snippet: The quantification of the intracellular levels of total and phosphorylated JNK, ERK1/2, p38, and AKT kinases and the contents of cyclin D1 and p21 proteins in the treated cells was carried out using the PathScan ® ELISA kits: Total SAPK/JNK Sandwich ELISA Kit, Phospho-SAPK/JNK (Thr183/Tyr185) Sandwich ELISA Kit, Total p44/42 MAPK (Erk1/2) Sandwich ELISA Kit, Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit, Phospho-p38 MAPK (Thr180/Tyr182) Sandwich ELISA Kit, Total Cyclin D1 Sandwich ELISA Kit, Total p21 Waf1/Cip1 Sandwich ELISA Kit (Cell Signaling Technology Danvers, MA, USA), and p38 MAPK alpha ELISA Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions as described previously [ ].

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Development, Growth & Differentiation

Article Title: Characterization and expression of a maternal axolotl Cyclin B1 during oogenesis and early development

doi: 10.1111/j.1440-169x.2007.00934.x

Figure Lengend Snippet: Fig. 6. Schematic representation of comparison of Cyclin B1, P-Tyr15 Cdk1 and phospo(P)-MAPKinase between axolotl early embryo and electrically activated unfertilized eggs (UFE). From the laying or electric activation taken as t = 0 references, schematic diagrams summarize typical external changes at the animal pole of fertilized egg or UFE, respectively. Maturation spots and first polar bodies are easily observed from both cell types as a depigmented area and a small dark vesicle; to recall axolotl polyspermic fertilization, three sperm marks are depicted. During the first 1.5–2.0 h, second polar body (shown as small circles) is extruded in the area of the maturation spot that is progressively pigmented again. Furrow formation is registered at 6 h leading to a two-cell stage embryo, then every 1.5 h leading to four- or eight-cell stage embryos; in UFE, at 6 h as well, fragmentation can be observed which ends activated UFE evolution and is thought to mimic the first cleavage. Until the second polar body extrusion, similar Cyclin B1, P-Tyr15 Cdk1 and P-MAPK signals are registered from cytosolic extracts of either fertilized egg or UFE; from laying to laying, UFE P-Tyr15 Cdk1 and P- MAPK levels however, exhibit a variability that is mentioned using hatched areas in the respective curves. Then the two cell types show differences in their co-regulation patterns. Until 6 h, up to three accumulation cycles of both Cyclin B1 and P-Tyr15 Cdk1 were counted in activated UFE, but only one in one-cell stage embryo. Moreover, MAPK-activating phosphorylation was not observed in the two-cell stage or older cleaving embryo, but, in activated UFE, it peaked after Cyclin B1 or P-Tyr15 Cdk1 maxima. These different observations could reflect the properties of a maternal cell cycle oscillator present at the end of the oocyte maturation, as well as different regulatory capacities of the two axolotl cellular models.

Article Snippet: Immunoreactive proteins were visualized using different primary antibodies: polyclonal goat affinity-purified anti-Xenopus Cyclin B1 antibody (De Smet et al. 2002); mouse monoclonal anti-Buffo Cyclin B1 antibody (Sakamoto et al. 1998); monoclonal antiCdk1 A17 antibody (gift from T. Hunt); polyclonal rabbit antiphospho-Tyr15 Cdk1 (Cell Signaling Technology, Beverly, MA, USA); monoclonal mouse antiphosphoMAP Kinase (P-MAPK) E10 antibody (Cell Signaling Technology).

Techniques: Comparison, Activation Assay, Phospho-proteomics