jak  (Cell Signaling Technology Inc)

Journal: CytoJournal

Article Title: Mapping the function of EF-hand domain-containing protein 2 and determining its clinical relevance in non-small-cell lung cancer through single-cell transcriptomics

doi: 10.25259/Cytojournal_29_2025

Figure Lengend Snippet: EFHD2 influences lung cancer cell proliferation, apoptosis, migration, invasion, and various signaling pathways. (a) WB analysis depicting EFHD2 protein expression in normal bronchial epithelial cells (BEAS-2B) and different lung cancer cell lines (A549, NCI-H1299, and HCC827). (b) Relative mRNA levels of EFHD2 in BEAS-2B and lung cancer cell lines, quantified through qRT-PCR. The data are presented as mean ± SD. (c) WB illustrating EFHD2 protein expression levels. (d) qRT-PCR analysis of EFHD2 mRNA expression. (e) CCK-8 assay assessing the impact of EFHD2 OE and KD on cell proliferation; data were presented as OD450 values over 72 h. (f) Flow cytometry analysis evaluating apoptosis rates. (g) Transwell migration and invasion assays. (h) Colony formation assay. (i) WB analysis of JAK and STAT protein expression, using GAPDH as a loading control. Data are expressed as mean ± SD. ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001 compared with the A549 group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the NCI-H1299 group. The scale bar represents 100 µm. EFHD2: EF-hand domain-containing protein 2, WB: Western blot, JAK: Janus Kinase, STAT: Signal transducers and activators of transcription, GAPDH: Glyceraldehyde 3-phosphate dehydrogenase, qRT-PCR: Quantitative real-time polymerase chain reaction, CCK-8: Cell counting kit 8, OE: Overexpression, KD: Knockdown, mRNA: Messenger RNA, OD450: Optical density at 450 nm, SD: Standard deviation.

Article Snippet: The membranes were then exposed overnight at 4°C to primary antibodies targeting EFHD2 (1:1000, ab24368, Abcam, China), JAK1 (1:1000, HY- P80196 , MedChemExpress, China), phospho-JAK (p-JAK) (1:1000, ab138005, Abcam, China), signal transducers and activators of transcription 3 (STAT3) (1:1000, ab68153, Abcam, China), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, ab8245, Abcam, China) (loading control).

Techniques: Migration, Protein-Protein interactions, Expressing, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Colony Assay, Control, Western Blot, Real-time Polymerase Chain Reaction, Cell Counting, Over Expression, Knockdown, Standard Deviation

Journal: CytoJournal

Article Title: Mapping the function of EF-hand domain-containing protein 2 and determining its clinical relevance in non-small-cell lung cancer through single-cell transcriptomics

doi: 10.25259/Cytojournal_29_2025

Figure Lengend Snippet: EFHD2 regulates cell proliferation, migration, invasion, apoptosis, and JAK/STAT signaling. (a) Co-IP identified that FLAG-EFHD2 interacted with Myc-JAK1 in A549+EFHD2 OE and NCI-H1299+EFHD2 KD cells. (b) CCK-8 detected cell proliferation. Inhibition of JAK decreased the proliferation of A549+EFHD2 OE cells, while activation of JAK increased the proliferation of NCI-H1299+EFHD2 KD cells. (c) Flow cytometry to detect apoptosis. Inhibition of JAK increased the apoptosis of A549+EFHD2 OE cells, whereas JAK agonist enhanced the apoptosis of NCI-H1299+EFHD2 KD cells. (d) Transwell migration and invasion. Inhibition of JAK reduced the migration and invasion of A549+EFHD2 OE cells, whereas activation of JAK promoted the migration and invasion of NCI-H1299+EFHD2 KD cells. (e) Colony formation assay. Inhibition of JAK decreased the colony formation of A549+EFHD2 OE cells, whereas activation of JAK enhanced the colony formation of NCI-H1299+EFHD2 KD cells. (f) Western blotting. JAK inhibition decreased the levels of p-JAK1 and p-STAT3 in A549+EFHD2 OE cells, whereas JAK activation increased the levels of p-JAK1 and p-STAT3 in NCI-H1299+EFHD2 KD cells. Data are expressed as mean ± SD. ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001 compared with the A549 group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the NCI-H1299 group. The scale bar represents 100 µm. JAK: Janus Kinase, STAT: Signal transducers and activators of transcription, Co-IP: Co-immunoprecipitation, EFHD2: EF-hand domain-containing protein 2, OE: Overexpression, KD: Knockdown, CCK-8: Cell counting kit 8, p-JAK: Phospho Janus Kinase, p-STAT: Phospho signal transducers and activators of transcription, SD: Standard deviation.

Article Snippet: The membranes were then exposed overnight at 4°C to primary antibodies targeting EFHD2 (1:1000, ab24368, Abcam, China), JAK1 (1:1000, HY- P80196 , MedChemExpress, China), phospho-JAK (p-JAK) (1:1000, ab138005, Abcam, China), signal transducers and activators of transcription 3 (STAT3) (1:1000, ab68153, Abcam, China), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, ab8245, Abcam, China) (loading control).

Techniques: Migration, Co-Immunoprecipitation Assay, CCK-8 Assay, Inhibition, Activation Assay, Flow Cytometry, Colony Assay, Western Blot, Immunoprecipitation, Over Expression, Knockdown, Cell Counting, Standard Deviation

Journal: bioRxiv

Article Title: PTPN1/2 inhibits alveolar macrophage-mediated control of lung metastasis

doi: 10.64898/2026.02.25.707995

Figure Lengend Snippet: A, B. Viability of 4T1 ( A ) and CMT167 ( B ) cells after a 3-day coculture with or without AMs, in the presence of AC484 (50 nM) and/or IFNγ at indicated concentrations (ng/mL). C, D. Comparison of cytokine levels in conditioned medium collected from AMs cocultured with 4T1 ( C ) or CMT167 ( D ) tumor cells, supplemented with AC484 or DMSO controls. Cross makes indicate values beyond the detection range. Cytokines in the 13-plex panel that did not reach the detection limit in either condition were not plotted. E . Viability of 4T1 cells after coculture with or without AMs, comparing direct cell-to-cell contact versus separation by a transwell insert. F . Representative histograms of p-STAT1 (Y701) in AMs from wild-type (WT) or IFNγ knockout (KO) mice after 48 h coculture with CMT167 cells. AMs cultured alone serve as controls. G . Analysis of the fold change in p-STAT1 geometric MFI normalized to non-cocultured AMs from F. H . Viability of CMT167 cells after coculture with or without AMs harvested from WT or IFNγ KO mice, in the presence or absence of AC484. I . Representative histograms of p-STAT1 (Y701) in AMs after 24 h coculture with 4T1 tumor cells, with AC484 (100 nM) and/or the JAK inhibitor ruxolitinib (3 μM). AMs cultured alone with DMSO served as controls. J . Analysis of the fold change in p-STAT1 geometric MFI, normalized to DMSO-treated, non-cocultured control AMs, from experiments in I. K . Viability of 4T1 cells after coculture with or without AMs, in the presence or absence of AC484 (100 nM) and/or ruxolitinib (3 μM). For all bar graphs, data represent individual animals (dots), with mean ± SEM (bars). Unpaired t-test for two-group comparisons; one-way ANOVA for more than two groups: ns, p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: In experiments involving the JAK inhibitor ruxolitinib, BAL cells were pre-incubated with ruxolitinib (3 μM, MedChemExpress) in complete RPMI for 2 h, washed, and then cocultured as described, with ruxolitinib maintained throughout the assay at 3 μM.

Techniques: Comparison, Knock-Out, Cell Culture, Control