Journal: Cell Death & Disease

Article Title: Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity

doi: 10.1038/s41419-024-06828-x

Figure Lengend Snippet: A , B Flow cytometry analysis (left) and MFI intensity (right) demonstrate the expression levels of p-STAT5, p-S6, p-AKT473 and p-Erk in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n ≥ 4). C Gene-set enrichment analysis of RNA-seq data shows the enrichment of hallmark-IL6-JAK-STAT3-signaling related genes in Mst1/2-deficient NK cells compared to WT NK cells. D Flow cytometry analysis (left) and MFI intensity (right) reveals the expression level of p-STAT3 (Ser727) in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n = 4). E ChIP-qPCR analysis demonstrated binding of p-STAT3 conserved motifs in the Tcf7 5′ regulatory region (−17.5 kb), while no binding was observed in a region without p-STAT3-binding motifs (+0.2 kb), serving as a negative control. The experiments were using sorted NK cells from WT mice with anti-p-STAT3 antibody or isotype-matched IgG. F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis. The representative plots (left) and summary data (right) show the expression level of p-STAT3 (F), TCF1 ( G ), total cellular ROS ( H ) and the percentage of 7AAD + NK cells ( I ) ( n = 4). J Schematic illustration of Mst1/2 regulate NK cell survival and function via controlling metabolic state and transcriptional activity. Data A , B and D – I are representative of three independent experiments with similar results.

Article Snippet: F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis.

Techniques: Flow Cytometry, Expressing, RNA Sequencing, ChIP-qPCR, Binding Assay, Negative Control, Activity Assay

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Oligonucleotides sequences used for quantifying miR-141 and mRNA of indicated genes

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques:

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: The correlation between miR-141 level and the expression profile of MxA and STAT3 in infected A549 cells. (A) Quantification of steady-state miR-141 in infected A549 cells with MOI of 0.5 and transfected with either pre-miR-141 or miR-141 inhibitor compared with noninfected cells (control) using qRT-PCR. (B) Relative gene expression of MxA and STAT3 in infected A549 cells transfected with either specific inhibitor against miR-141 or pre- miR-141 compared with control-transfected cells using qRT-PCR. Error bars indicate the STD of three independent experiments. Student two-tailed t -test used for statistical analysis, (*) indicates P-values ≤ 0.05, and (**) indicates P ≤ 0.01. (C) Flow cytometric assay quantifies the kinetic proteins expression profile of MxA (in blue dots) and STAT3 (in red dots) in infected and transfected A549 cells compared with control cells. (D) Western blot analysis reveals the protein expression level of MxA and STAT3 in infected and transfected cells compared to control cells, β-actin expression profile severed as an internal control

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Expressing, Infection, Transfection, Control, Quantitative RT-PCR, Gene Expression, Two Tailed Test, Flow Cytometry, Western Blot

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Quantification analysis of miR-141, MxA, and STAT3 in transfected and infected A549 cells

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Transfection, Infection, Expressing, Control

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Mass Cytometry Assessment of Cell Phenotypes and Signaling States in Human Whole Blood

doi: 10.1007/978-1-0716-2553-8_10

Figure Lengend Snippet: Whole blood phosphoflow panel 1

Article Snippet: 142 Nd cCasp3 D3E9 Fluidigm 3142004A 143 Nd CD19 HIB19 Biolegend 302202 144 Nd pPLCg2 [Y759] K86-689.37 Fluidigm 3144015A 145 Nd CD4 RPA-T4 Fluidigm 3145001B 146 Nd IgD IA6-2 Fluidigm 3146005B 147 Sm CD20 2H7 Fluidigm 3147001B 148 Nd IgA Polyclonal Fluidigm 3148007B 149 Sm CD25 2A3 Fluidigm 3149010B 150 Nd pStat5 [Y694] 47 Fluidigm 3150005A 151 Eu CD123 6H6 Fluidigm 3151001B 153 Eu pStat1 [Y701] 4a Fluidigm 3153005A 154 Sm CD45 HI30 Fluidigm 3154001B 155Gd CD27 L128 Fluidigm 3155001B 156 Gd p38 [T180/Y182] D3F9 Fluidigm 3156002A 157 Gd CD24 ML-5 Biolegend 311102 158 Gd pStat3 [Y705] 4/P-Stat3 Fluidigm 3158005A 159Tb CD11c Bu15 Fluidigm 3159001B 160Gd CD14 M5E2 Fluidigm 3160001B 161 Dy CD141(BDCA-3) AD5-14H12 Miltenyi 130-090-694 162 Dy CD66b 80H3 Fluidigm 3162023B 163 Dy CD56 NCAM16.2 Fluidigm 3163007B 164 Dy IkBa L35A5 Fluidigm 3164004A 165 Ho pCREB [S133] 87G3 Fluidigm 3165009A 166 Er CD16 B73.1 Biolegend 360702 167 Er CD38 HIT2 Fluidigm 3167001B 168 Er CD8 SK1 Fluidigm 3168002B 169 Tm CD45RA HI100 Fluidigm 3169008B 170 Er CD3 UCHT1 Fluidigm 3170001B 171 Yb pERK1/2 [T202/Y204] D13.14.4E Fluidigm 3171010A 172 Yb Anti-Ki-67 B56 Fluidigm 3172024B 174 Yb HLA-DR L243 Fluidigm 3174001B 175Lu CD7 CD7-6B7 Biolegend 343102 176 Yb CD127/IL-7Ra P48-48 Novus Bio MAB306-100 209Bi CD11b/Mac-1 ICRF44 Fluidigm 3209003B Open in a separate window 1 Open channels are not shown but include Pd channels, Cd channel, Pt channels, 89Y,152Sm and 173Yb Whole blood phosphoflow panel1.

Techniques:

Journal: Oncogene

Article Title: Stat3 contributes to keloid pathogenesis via promoting collagen production, cell proliferation and migration.

doi: 10.1038/sj.onc.1209531

Figure Lengend Snippet: Figure 1 Stat3 expression and activation are elevated in keloid skin tissues. (a) Hematoxylin–eosin stain of paraffin tissue sections of normal skin (NS1) and keloid scar (KS28). D, E and K indicate dermis, epidermis and keratin, respectively. (b) Cryosections of normal skin and keloid scar tissue were stained with either anti-rabbit immunoglobulinG (a-rabbit IgG) or polyclonal anti-Stat3 (C-20) (a- Stat3) and counterstained with 4,6-diamidino-2-phenylindole (DAPI). (c) Cryosections were probed with anti-mouse IgG (a-mouse IgG) or monoclonal anti-phosphoStat3 (pStat3) (a-pStat3) and counterstained with DAPI. Pictures were taken together with phase contrast and all scale bars represent 50 mM. (d) Equal amounts of tissue lysates from normal skin (NS) and keloid samples (KS) were subjected to Western blot analysis with anti-pY705 Stat3 antibody. The blot was stripped and reprobed with anti-pS727 Stat3, and subsequently with anti-Stat3 and anti-actin.

Article Snippet: Polyclonal anti-phosphoTyr705 Stat3, monoclonal antiphosphoSer727 Stat3, anti-pJak1 (Tyr1022/1023), anti-pJak2 (Tyr1007/1008), anti-phosphoSrc (Tyr416), anti-phosphoEGFR (Tyr845) and anti-Bcl-XL antibodies were purchased from Cell Signaling Technology, whereas monoclonal pStat3 (B7) (sc-8059), polyclonal Stat3 (C-20) (sc-482), monoclonal cyclin D1 (sc-20044), anti-mouse IgG (sc-2025) and anti-rabbit IgG (sc-2027) antibodies were acquired from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Activation Assay, Staining, Western Blot

Journal: Oncogene

Article Title: Stat3 contributes to keloid pathogenesis via promoting collagen production, cell proliferation and migration.

doi: 10.1038/sj.onc.1209531

Figure Lengend Snippet: Figure 2 Enhanced Stat3 phosphorylation and expression in keloid fibroblasts (KFs). (a) Western blot analyses of total cell lysates from normal fibroblasts (NFs) and KFs with equal amounts of protein, and antibodies as indicated. The graph on the upper right illustrates the pY705 Stat3 level normalized to Stat3 expression, whereas the graph on the lower right depicts an average of the normalized pY705 Stat37s.d. of the KFs and NFs presented. (b) Normal fibroblasts, NF5 and NF4, and KFs, KF48 and KF8, were cultured in serum-free media for 2 days before stimulating with 10% fetal bovine serum. Total cell lysates were harvested at day 1–5, and phosphorylations of Stat3 were analysed by Western blot after loading equal amounts of protein. (c) NF4 and KF48 fibroblasts were incubated in serum-free Dulbecco’s modified Eagles’s medium for 2 days before harvesting daily over 5 days to investigate the serum-independent phosphorylation of Stat3. Equal amounts of total cell lysates were subjected to Western blot analysis with antibodies as indicated. Arrowheads indicate the position of the protein bands.

Article Snippet: Polyclonal anti-phosphoTyr705 Stat3, monoclonal antiphosphoSer727 Stat3, anti-pJak1 (Tyr1022/1023), anti-pJak2 (Tyr1007/1008), anti-phosphoSrc (Tyr416), anti-phosphoEGFR (Tyr845) and anti-Bcl-XL antibodies were purchased from Cell Signaling Technology, whereas monoclonal pStat3 (B7) (sc-8059), polyclonal Stat3 (C-20) (sc-482), monoclonal cyclin D1 (sc-20044), anti-mouse IgG (sc-2025) and anti-rabbit IgG (sc-2027) antibodies were acquired from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Cell Culture, Incubation

Journal: Signal Transduction and Targeted Therapy

Article Title: Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

doi: 10.1038/s41392-026-02650-3

Figure Lengend Snippet: Determination of M1 vs. M2 polarization in iMac. a Pro-inflammatory M1 cytokines IL-1β, TNFα, IL-6, and IL-8 measured by ELISA. b Anti-inflammatory M2 cytokines IL-4, IL-10, IL-13, CCL5, CCL17, and CCL20 measured by ELISA. For a , b iMac were cultured in 6-well plates (3 × 10 6 cells/well). To challenge with tumor factors, iMac were co-cultured with 50% fresh supernatant from SK-MEL-28 cells every 24 h for 3 days. Supernatants were collected during the final 24 h for cytokine measurement. c Activation (phosphorylation) of NF-kB p65, STAT1, STAT3, and STAT6 in iMac was determined using Western blotting after conditioning with melanoma (SK-MEL-28) supernatant for 72 h. Protein band intensity was quantified using Odyssey version 3.0 software. After adjusting for loading using the β-actin band, data are presented as fold change to the iMac (Dox-) group, which is set as 1. For a – c , one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SD. Each experiment was performed three times, with triplicate samples each time. * p < 0.05, *** p < 0.001, and **** p < 0.0001

Article Snippet: The polyvinylidene difluoride (PVDF) membranes were incubated with specific primary antibodies against ACE (R&D Systems, MAB9291, 1:1,000), GAPDH (Sigma-Aldrich, SAB5600208, 1:2,000), β-actin (Sigma-Aldrich, A3854; 1:1000), phosphorylated NF-kB p65 (Novus, NB100-82086, 1:500), phosphorylated STAT1 (R&D Systems, AF2894, 1 μg/ml), phosphorylated STAT3 (Novus, NBP2-24463, 0.5 μg/ml), or phosphorylated STAT6 (Millipore, 06-937, 1:1000).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Phospho-proteomics, Western Blot, Software

Journal: Journal of Pharmaceutical Analysis

Article Title: CXCL8/SDC1 axis mediates tumor stem cell interactions to drive remote transfer in thyroid cancer

doi: 10.1016/j.jpha.2025.101354

Figure Lengend Snippet: Impact of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway on tumor stem cell self-renewal and migration invasion. (A, B) Western blot analysis of JAK-STAT and nuclear factor kappa B (NF-κB) signaling pathway-related proteins: protein expression levels of phosphorylated-Janus kinase 2 (p-JAK2), JAK2, phosphorylated signal transducer and activator of transcription (p-STAT3), STAT3, NF-κB, and phosphorylated NF-κB (p-NF-κB) in FTC238-S cells co-cultured with different modified myeloid‑derived suppressor cells (MDSCs) (A), and protein levels of p-JAK2, JAK2, p-STAT3, and STAT3 in FTC238-S cells treated with anti-immunoglobulin G (anti-IgG) or anti-C-X-C motif chemokine ligand 8 (anti-CXCL8) antibodies (B). (C) Schematic diagram of monocyte treatment and co-culturing with FTC238-S cells. (D) Western blot analysis of JAK-STAT signaling pathway-related proteins in FTC238-S cells from each group. (E) Protein expression levels of stemness markers neuroepithelial stem cell protein (NESTIN), octamer-binding transcription factor 4 (OCT4), and SRY-box transcription factor 2 (SOX2) in FTC238-S cells from each group examined by Western blot. (F) Cell sphere formation assay assessing the sphere formation capability of FTC238-S cells in co-culture systems of each group. (G) Clonogenic assay evaluating the clonogenic capacity of FTC238-S cells in co-culture systems of each group. (H) Cell Counting Kit-8 (CCK-8) assay measuring the proliferation ability of FTC238-S cells in co-culture systems of each group. (I) Transwell assay determining the migration and invasion capability of FTC238-S cells in co-culture systems of each group, ∗ P < 0.05 compared between the two groups, and all cell experiments were repeated three times. M_oe-NC + S_sh-NC: FTC238-S cells transfected with short hairpin RNA-negative control (sh-NC) and treated with conditioned medium from monocytes transfected with oe-NC; M_oe-CXCL8 + S_sh-NC: FTC238-S cells transfected with sh-NC and treated with conditioned medium from monocytes transfected with oe-CXCL8; M_oe-CXCL8 + S_sh-SDC1: FTC238-S cells transfected with short hairpin RNA-SDC1 (sh-SDC1) and treated with conditioned medium from monocytes transfected with oe-CXCL8; M_oe-CXCL8 + S_DMSO: FTC238-S cells treated with conditioned medium from monocytes transfected with oe-CXCL8 and supplemented with an equal amount of dimethyl sulfoxide (DMSO); M_oe-CXCL8 + S_SD_1008: FTC238-S cells treated with conditioned medium from monocytes transfected with oe-CXCL8 and supplemented with SD-1008; THP-1 cells: Tohoku Hospital Pediatrics-1 cells; OD: optical density.

Article Snippet: Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% skim milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4 °C with primary antibodies against CXCL8 (1:1000, ab235584, Abcam, Cambridge, UK), SDC1 (1:2000, ab128936, Abcam, Cambridge, UK), NESTIN (1:100, ab105389, Abcam, Cambridge, UK), OCT4 (1:10000, ab200834, Abcam, Cambridge, UK), SRY-Box transcription factor 2 (SOX2, 1:1500, ab92494, Abcam, Cambridge, UK), phosphorylated STAT3 (p-STAT3) (1:1000, NB100-82213, Novus Biologicals, Centennial, CO, USA), STAT3 (1:1000, MAB1799, Novus Biologicals, Centennial, CO, USA), phosphorylated-JAK2 (p-JAK2) (1:1500, ab32101, Abcam, Cambridge, UK), JAK2 (1:1000, ab108596, Abcam, Cambridge, UK), nuclear factor kappa B (NF-κB, 1:1000, 8242T, CST, Boston, MA, USA), phosphorylated NF-κB (p-NF-κB) (1:1000, 3033T, CST, Boston, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2500, ab9485, Abcam, Cambridge, UK), and Tubulin (1:5000, ab7291, Abcam, Cambridge, UK).

Techniques: Migration, Western Blot, Expressing, Cell Culture, Modification, Binding Assay, Tube Formation Assay, Co-Culture Assay, Clonogenic Assay, Cell Counting, CCK-8 Assay, Transwell Assay, Transfection, shRNA, Negative Control

Journal: Journal of Pharmaceutical Analysis

Article Title: CXCL8/SDC1 axis mediates tumor stem cell interactions to drive remote transfer in thyroid cancer

doi: 10.1016/j.jpha.2025.101354

Figure Lengend Snippet: Impact of the C-X-C motif chemokine ligand 8/syndecan-1 (CXCL8/SDC1) axis on tumor initiation, growth, and metastasis of thyroid cancer (THCA) stem cells i n Vivo . (A) Diagram of the in vivo animal experiment protocol, with the green syringe representing anti-immunoglobulin G (anti-IgG) and anti-CXCL8 treatments. (B) Western blot analysis of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway-related proteins in subcutaneous tumor tissues of nude mice from each group. (C) Gross anatomy of subcutaneous transplant tumors in nude mice (left) and corresponding weight statistics (right). (D) Images of primary tumors in nude mice from each group (left) and diameter analysis (right). (E) Statistical analysis of axillary lymph node (LN) metastasis in subcutaneous transplant tumors of nude mice from each group. (F) Hematoxylin and eosin (H&E) staining to assess bone metastasis of primary tumors in nude mice from each group. (G) Images of lung metastasis in nude mice from each group (left) and diameter analysis (right). (H) H&E staining to evaluate lung metastasis in nude mice from each group. ∗ P < 0.05 compared between two groups, ∗∗ P < 0.01 with 6 nude mice in each group. p-JAK2: phosphorylated-JAK2; p-STAT3: phosphorylated STAT3; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; S_sh-NC: FTC238-S cells transfected with short hairpin RNA-negative control (sh-NC); S_sh-SDC1: FTC238-S cells transfected with short hairpin RNA-SDC1 (sh-SDC1).

Article Snippet: Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% skim milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4 °C with primary antibodies against CXCL8 (1:1000, ab235584, Abcam, Cambridge, UK), SDC1 (1:2000, ab128936, Abcam, Cambridge, UK), NESTIN (1:100, ab105389, Abcam, Cambridge, UK), OCT4 (1:10000, ab200834, Abcam, Cambridge, UK), SRY-Box transcription factor 2 (SOX2, 1:1500, ab92494, Abcam, Cambridge, UK), phosphorylated STAT3 (p-STAT3) (1:1000, NB100-82213, Novus Biologicals, Centennial, CO, USA), STAT3 (1:1000, MAB1799, Novus Biologicals, Centennial, CO, USA), phosphorylated-JAK2 (p-JAK2) (1:1500, ab32101, Abcam, Cambridge, UK), JAK2 (1:1000, ab108596, Abcam, Cambridge, UK), nuclear factor kappa B (NF-κB, 1:1000, 8242T, CST, Boston, MA, USA), phosphorylated NF-κB (p-NF-κB) (1:1000, 3033T, CST, Boston, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2500, ab9485, Abcam, Cambridge, UK), and Tubulin (1:5000, ab7291, Abcam, Cambridge, UK).

Techniques: In Vivo, Western Blot, Staining, Transfection, shRNA, Negative Control

Journal: Journal of Pharmaceutical Analysis

Article Title: CXCL8/SDC1 axis mediates tumor stem cell interactions to drive remote transfer in thyroid cancer

doi: 10.1016/j.jpha.2025.101354

Figure Lengend Snippet: Schematic diagram of the molecular mechanism by which monocytes promote self-renewal, migration, and invasion of thyroid cancer (THCA) stem cells through the C-X-C motif chemokine ligand 8/syndecan-1 (CXCL8/SDC1) axis. JAK2: Janus kinase 2; STAT3: signal transducer and activator of transcription 3.

Article Snippet: Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% skim milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4 °C with primary antibodies against CXCL8 (1:1000, ab235584, Abcam, Cambridge, UK), SDC1 (1:2000, ab128936, Abcam, Cambridge, UK), NESTIN (1:100, ab105389, Abcam, Cambridge, UK), OCT4 (1:10000, ab200834, Abcam, Cambridge, UK), SRY-Box transcription factor 2 (SOX2, 1:1500, ab92494, Abcam, Cambridge, UK), phosphorylated STAT3 (p-STAT3) (1:1000, NB100-82213, Novus Biologicals, Centennial, CO, USA), STAT3 (1:1000, MAB1799, Novus Biologicals, Centennial, CO, USA), phosphorylated-JAK2 (p-JAK2) (1:1500, ab32101, Abcam, Cambridge, UK), JAK2 (1:1000, ab108596, Abcam, Cambridge, UK), nuclear factor kappa B (NF-κB, 1:1000, 8242T, CST, Boston, MA, USA), phosphorylated NF-κB (p-NF-κB) (1:1000, 3033T, CST, Boston, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2500, ab9485, Abcam, Cambridge, UK), and Tubulin (1:5000, ab7291, Abcam, Cambridge, UK).

Techniques: Migration

Journal: BMC Medicine

Article Title: ISG15-driven immune modulation and tumor progression in breast cancer metastasis: insights from single-cell and spatial transcriptomics

doi: 10.1186/s12916-025-04614-w

Figure Lengend Snippet: Transcriptomic analysis of in situ tumor tissues in mouse mammary gland cancer. Note: A The volcano plot of differentially expressed genes in in situ tumor tissues of mouse mammary gland cancer after ISG15 silencing, with red dots representing upregulated genes and blue dots representing downregulated genes; B heatmap depicting the expression levels of differentially expressed genes in the sh-NC group (6 cases) and sh-ISG15 group (6 cases), with the left dendrogram clustering genes based on expression levels, the color bar on the right representing expression levels (red indicating upregulated genes and blue indicating downregulated genes), and the histogram above indicating blue for non-metastatic group and red for metastatic group; C GO analysis of differentially expressed genes, with the color bar on the right representing enrichment scores for different functional categories; D KEGG analysis of differentially expressed genes, with the color bar on the right representing enrichment scores for different pathways; E expression levels of PD-L1 in in situ tumor tissues of mouse mammary gland cancer after ISG15 silencing, with 6 mice in each group; F binding sites of transcriptional activation factor STAT3

Article Snippet: The membrane was blocked with 5% skim milk at room temperature (RT) for 1 h, then incubated overnight at 4 °C with primary antibodies against ISG15 (1:1000, ab308219), NESTIN (1:1000, ab221660), OCT4 (1:10,000, ab200834), SOX2 (1:1500, ab92494), STAT3 (1:1500, ab68153), p-JAK2 (1:1500, ab32101), JAK2 (1:5000, ab108596), PD-L1 (1:1000, ab213480), Arg (1:1000, ab203490), CD206 (1:1200, ab64693), CD163 (1:1000, ab182422), CD86 (1:1200, ab220188), and GAPDH (1:2500, ab9485) as the internal reference (all from Abcam, Cambridge, UK), as well as p-STAT3 (1:1000, NB100-82213, Novus Biologicals).

Techniques: In Situ, Expressing, Functional Assay, Binding Assay, Activation Assay

Journal: BMC Medicine

Article Title: ISG15-driven immune modulation and tumor progression in breast cancer metastasis: insights from single-cell and spatial transcriptomics

doi: 10.1186/s12916-025-04614-w

Figure Lengend Snippet: Influence of ISG15 in CSCs on T cell activation and its potential molecular mechanisms. Note: A Flow cytometry analysis of Ki-67 to determine the proportion of proliferating CD8 + T cells; B flow cytometry analysis of IFN-γ to determine the proportion of activated CD8 + T cells; C western blot analysis of JAK-STAT signaling pathway-related proteins and PD-L1 expression in co-culture systems of different groups; D dual-luciferase assay to examine the impact of ISG15 on PD-L1 promoter activity; E the performance of ChIP assay to examine the enrichment of p-STAT3 on the PD-L1 promoter in 4T1-S cells (left panel) and CSCs isolated from mouse tumor models (right panel); F the utilization of flow cytometry for the detection of Ki-67 expression in T cells; G The application of flow cytometry for the detection of IFN-γ expression in T cells; H the use of flow cytometry for the detection of Ki-67 expression in T cells; I the use of flow cytometry for the detection of IFN-γ expression in T cells; J the performance of western blot for the analysis of PD-L1 expression in each group co-culture system; K schematic representation of the molecular mechanism of ISG15 in T cell activation in CSCs. * indicates statistical significance ( P < 0.05) between the two groups. All cellular experiments were performed in triplicate

Article Snippet: The membrane was blocked with 5% skim milk at room temperature (RT) for 1 h, then incubated overnight at 4 °C with primary antibodies against ISG15 (1:1000, ab308219), NESTIN (1:1000, ab221660), OCT4 (1:10,000, ab200834), SOX2 (1:1500, ab92494), STAT3 (1:1500, ab68153), p-JAK2 (1:1500, ab32101), JAK2 (1:5000, ab108596), PD-L1 (1:1000, ab213480), Arg (1:1000, ab203490), CD206 (1:1200, ab64693), CD163 (1:1000, ab182422), CD86 (1:1200, ab220188), and GAPDH (1:2500, ab9485) as the internal reference (all from Abcam, Cambridge, UK), as well as p-STAT3 (1:1000, NB100-82213, Novus Biologicals).

Techniques: Activation Assay, Flow Cytometry, Western Blot, Expressing, Co-Culture Assay, Luciferase, Activity Assay, Isolation