Journal: Journal of Cellular and Molecular Medicine

Article Title: Exosomal HMGB1 Orchestrates NSCLC Progression and Immunosuppressive Macrophage Polarisation Through the TLR4 / NF ‐ κB / IL ‐6/ STAT3 Signalling Cascade

doi: 10.1111/jcmm.71050

Figure Lengend Snippet: Targeting HMGB1 signalling improves therapeutic outcomes in NSCLC. (A) Correlation analysis between immune infiltration scores and HMGB1 expression in 491 LUAD and 500 LUSC patients from the TCGA database. (B) Correlation between HMGB1 expression and the distribution of various immune cell subsets in LUAD and LUSC patients. (C, D) THP‐1–derived M0 macrophages were treated with PBS, HMGB1 (10 or 100 ng) or exosomes derived from vector or HMGB1 OE cells (cell‐to‐exosome ratio = 1:10). M1 macrophage markers (CD86, CD80, iNOS) and M2 markers (CD206, IL‐10, Arg1) were quantified by PCR. (E) Lewis tumour‐bearing mice were treated with PBS, HMGB1 OE‐derived exosomes (1 × 10 10 exosomes per mouse, twice per week), anti‐PD‐1 antibody (RMP1‐14, 200 μg per mouse, twice per week) or combination therapy ( n = 5 per group). Tumour volumes and apoptosis levels in tumour tissues (day 25) were assessed. (F) PC9 cells were treated with PBS or exosomes from HMGB1 OE cells (cell‐to‐exosome ratio = 1:10), followed by Osimertinib (50 nM, 48 h), and apoptosis was measured. (G) A549 and PC9 cells were similarly treated with PBS or HMGB1 OE‐derived exosomes, followed by Cisplatin (5 μM, 48 h), and apoptosis was analysed. (H) A549 and PC9 cells were similarly treated with paclitaxel (10 μM, 48 h) under the same conditions, and cell apoptosis was determined. (I) A549‐bearing mice were treated with HMGB1 OE‐derived exosomes (1 × 10 10 exosomes per mouse), followed by PBS, paclitaxel (PTX, 10 mg/kg, twice per week), STAT3 inhibitor (5 mg/kg, twice per week) or combination therapy. (J) Schematic diagram illustrating the proposed mechanism: HMGB1 upregulates TLR4, thereby activating the NF‐κB–IL‐6 axis and stimulating JAK2/STAT3 signalling to promote tumour progression. Concurrently, HMGB1 facilitates M2 macrophage polarisation.

Article Snippet: Human NSCLC cell lines (A549, PC9), human embryonic kidney cells (HEK293T) and the human monocytic leukaemia cell line THP‐1 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Derivative Assay, Plasmid Preparation

Journal: Clinical and Translational Medicine

Article Title: Integrative single‐cell analysis uncovers distinct tumour microenvironment ecotypes and immune evasion across skin cancers

doi: 10.1002/ctm2.70611

Figure Lengend Snippet: Ecotypes and cell‒cell communication networks in skin tumour microenvironment. (A) Heatmap showing the five ecotypes of skin tumours, which were inferred based on tumour microenvironment cell compositions. Bar plot showing the distribution of various cell lineages in each sample. (B) Pie charts showing ecotype distribution across tumour types and stages. (C) Pie charts showing tumour‐stage compositions across ecotypes. (D) Bar plots of enriched ligand‒receptor interactions for ecotype 1 and ecotype 4. (E) Cell‒cell communication networks showing MHC‐I signalling interactions in ecotype 1 and ecotype 4 (top) and SPP1 signalling interactions in ecotype 1 and ecotype 4 (bottom). Edge thickness indicates interaction strength, and colours represent different cell lineages. (F) Violin plots showing the expression of HLA‐A (top) and SPP1 (bottom) with associated partner genes (CD8A, CD44) across cell types and ecotypes. (G) qRT‐PCR showing Spp1 expression in RAW264.7 macrophages cultured with conditioned medium from B16 melanoma cells. (H) Western blot showing Spp1 protein levels in tumour‐associated macrophages (TAMs) after B16‐conditioned medium treatment, with corresponding quantification. (I) qRT‐PCR showing M2 polarisation markers expression in RAW264.7 macrophages after B16‐conditioned medium treatment. (J) qRT‐PCR showing SPP1 mRNA expression in THP‐1 macrophages transduced with control short hairpin RNA (shRNA) (negative control shRNA [shNC]) or two independent SPP1 ‐targeting shRNAs (shSPP1‐1# and shSPP1‐2#) (K) Western blot showing SPP1 protein levels in shNC, sh SPP1 ‐1# and sh SPP1 ‐2# THP‐1 cells. (L) M2 polarisation markers ( ARG‐1 , MRC1 and CD163) were measured in shNC or sh SPP1 THP‐1 macrophages cultured with SK‐MEL‐28‐conditioned medium. (M) qRT‐PCR showing SPP1 mRNA expression in THP‐1 macrophages transduced with empty vector (EV) or SPP1 overexpression construct (SPP1‐OE). (N) Western blot showing SPP1 protein levels in EV and SPP1 ‐OE THP‐1 cells. (O) M2 polarisation markers ( ARG‐1 , MRC1 and CD163 ) were measured in EV/ SPP1 ‐OE THP‐1 macrophages cultured with SK‐MEL‐28‐conditioned medium. Data are presented as mean ± SD. n = 3 independent repeats. Unpaired, two‐tailed t ‐test; * p < .05, ** p < .01, *** p < .001, **** p < .0001.

Article Snippet: The cell lines THP‐1, SK‐MEL‐28, RAW264.7 and B16 were obtained from the American Type Culture Collection.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Western Blot, Transduction, Control, shRNA, Negative Control, Plasmid Preparation, Over Expression, Construct, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Tet2 deficiency–induced expansion of monocyte-derived macrophages promotes liver fibrosis

doi: 10.1084/jem.20251114

Figure Lengend Snippet: Tet2 deficiency enhances Ccl2 and Ccl8 mRNA stability by modifying 5hmC-dependent RNA – protein interactions. (A and B) Ccl2 (A) and Ccl8 (B) mRNA decay in Tet2 +/+ and Tet2 −/− MDMs ( n = 6 for each group). (C) Tet2 -mediated oxidation of Ccl2 and Ccl8 mRNA 5mC disrupts its binding with Ybx1, Elavl1, and Zfp36. Pull-down assay was performed by incubating C, 5mC, and 5hmC oligos of Ccl2 and Ccl8 mRNA with cell lysate from THP1-derived pMDMs ( n = 3 for each group). (D) Effect of Tet2 deficiency on the binding enrichment of Ybx1, Elavl1, and Zfp36 at 3′UTR of Ccl2 and Ccl8 mRNA. Tet2-binding sites were mapped in the mRNA of Ccl2 and Ccl8 by qPCR of Ybx1, Elavl1, and Zfp36 RIP product in THP1-derived pMDMs ( n = 3 for each group). (E and F) Effect of enzymatic inactivation of Tet2 via catalytic domain mutation on stabilization of Ccl2 (F) and Ccl8 (G) transcripts ( n = 4 for each group). Data are the accumulative results from at least two independent experiments (A, B, D, E, and F) or are representative of at least two independent experiments with similar results (C and D). All data are shown as mean ± SD and were analyzed by two-tailed, unpaired Student’s t test (A, B, and D–F). ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). Source data are available for this figure: .

Article Snippet: THP1 cell line was purchased from ATCC and was cultured in high-glucose DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin (P/S, 100 U/ml; Hycone).

Techniques: Binding Assay, Pull Down Assay, Derivative Assay, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of the TREM2-CD33 pathway. CD33 inhibition acts on SYK activation, upstream of microglial activation pathways AKT, ERK, p38, and JNK. (B) Protein lysates isolated from THP-1 monocytes 48 hours after transduction were used to quantify percent SYK phosphorylation by MSD. (MOI = 31250) (C) 48 hours post-transduction, THP-1 cells (MOI = 31250) were treated with increasing concentrations of TREM2-targeting antibody agonist (0-10 μg/mL). After 5 minutes, protein lysates were isolated and used to quantify percent SYK phosphorylation by MSD. (D) 48-hours post-transduction of increasing concentrations of AAV6-CD33 (MOI = 0-31250), THP-1 cells were treated with a TREM2-targeting antibody agonist (2.5 ug/mL). After 5 minutes, protein lysates were isolated and used to quantify percent SYK phosphorylation by MSD. (E) Percent AKT and GSK3β phosphorylation was quantified from HMC3 cell lysates 48 hours after transduction of AAV6-CD33, quantified by MSD and ELISA, respectively. (F) Percent ERK and p38-MAPK phosphorylation was quantified from HMC3 cell lysates 48 hours after transduction of AAV6-CD33, quantified by ELISA. (G) Percent JNK phosphorylation was quantified from HMC3 cell lysates 48 hours after transduction of AAV6-CD33, quantified by ELISA. (H) (left to right) Percent expression of secreted IL10, IL1β, TNFα, and IL6 in conditioned media from HMC3 cells 24 hours after LPS challenge and 48 hours after transduction of AAV6-CD33 was quantified by MSD. Data represent mean ± SEM using one-tailed unpaired t-test (A, E-G), and two-way ANOVA using uncorrected Fisher’s LSD with a single pooled variance (H). Data points (n-numbers) are plotted on each bar graph and scatter plot each representing an independent experimental replicate. Line plots are representative of three and two independent experimental replicates (C, D, respectively).

Article Snippet: THP-1 Cells: The human monocytic cell line THP-1 (TIB-202) was obtained from ATCC.

Techniques: Inhibition, Activation Assay, Isolation, Transduction, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Expressing, One-tailed Test