g043h7  (fluidigm)

Journal: Science Advances

Article Title: Nanocollision promotes locomotion of dendritic cells for tumor therapy

doi: 10.1126/sciadv.aeb7714

Figure Lengend Snippet: ( A ) Synthesis process of an NCG. (PFH, liquid-gas phase-change core; PDA, stabilizer; FA, solid-liquid phase-change shell) capable of gas-liquid-solid triphasic conversion. ( B ) Schematic of the strategy for NCG-triggered nanocollision for enhanced locomotion of DCs. First, ultrasound induces vaporization of PFH. Then, the gasified PFH generates a critical internal pressure, triggering instantaneous rupture of the FA shell with subsequent outward ejection of fragmented particulates. Subsequently, the fragmented particulates induce nanocollisions with DCs, eliciting localized fluctuation of plasma membrane. IV, Piezo1 detects the fluctuation and mediates Ca 2+ influx through its central pore. Finally, Ca 2+ influx induces F-actin polymerization (enhanced intrinsic locomotion) and high expression of CCR7 (enhanced chemotaxis). ( C ) Locomotion-enhanced DCs potentiate antigen capture and lymph node homing, thereby activating T cells to amplify antitumor immunity.

Article Snippet: Antibodies and reagents used for Western blot are as follows: rabbit anti–IL-12 (bs-0767R), rabbit anti–IFN-γ (bs-0480R), CCR7 rabbit polyclonal antibody (pAb) (bs-1305R), and β-actin mouse monoclonal antibody (bsm-33036 M) from Bioss; cPLA2 pAb (YT1084), cPLA2 (phospho Ser 505 ) pAb (YP0868), PIEZ1 rabbit pAb (YT8073), AP-1 (phospho Tyr 170 ) pAb (YP0018), and AP-1 (Acetyl Lys271) pAb (YK0062) from Immunoway; anti-calreticulin rabbit pAb ( GB112134 ), anti-HMGB1 rabbit pAb (GB11103) from Servicebio.

Techniques: Clinical Proteomics, Membrane, Expressing, Chemotaxis Assay

Journal: Science Advances

Article Title: Nanocollision promotes locomotion of dendritic cells for tumor therapy

doi: 10.1126/sciadv.aeb7714

Figure Lengend Snippet: ( A and B ) Representative flow cytometry dot plots (A) and percentage (B) of CFSE-stained DCs in adjacent lymph nodes. ( n = 5). ( C ) Western blot analysis of CCR7 expression and oligomerization. ( D ) cPLA 2 pathway–related gene alterations, heatmap. (C versus B). ( E and F ) Western blot analysis of expression for proteins in the cPLA 2 pathway, including Piezo1, p-cPLA 2 , CCR7, and their quantitative analysis. ( G ) Representative images of Ca 2+ diffusion from the protrusions to the cell interior. ( H ) Schematic illustration of Ca 2+ influx–induced CCR7 expression in DC. ( I ) Heatmap of collagen and integrin-related genes (A versus B versus C). ( J ) Sankey bubble plot of pathway changes corresponding to genes. (C versus B). ( K ) Density plot of RNA-seq. (A versus B versus C). ( L ) Bubble plot of nanocollision-mediated alterations in DC signaling cascades. (C versus B). ( M and N ) Representative fluorescence images (M) and statistical graphs (N) of deep infiltration of DCs. (A, control; B, magnetic nanospheres; C, magnetic nanospheres with magnetic field, which can achieve collision. n = 6) ( P values: ns, not significant, * P < 0.05, ** P < 0.01.)

Article Snippet: Antibodies and reagents used for Western blot are as follows: rabbit anti–IL-12 (bs-0767R), rabbit anti–IFN-γ (bs-0480R), CCR7 rabbit polyclonal antibody (pAb) (bs-1305R), and β-actin mouse monoclonal antibody (bsm-33036 M) from Bioss; cPLA2 pAb (YT1084), cPLA2 (phospho Ser 505 ) pAb (YP0868), PIEZ1 rabbit pAb (YT8073), AP-1 (phospho Tyr 170 ) pAb (YP0018), and AP-1 (Acetyl Lys271) pAb (YK0062) from Immunoway; anti-calreticulin rabbit pAb ( GB112134 ), anti-HMGB1 rabbit pAb (GB11103) from Servicebio.

Techniques: Flow Cytometry, Staining, Western Blot, Expressing, Diffusion-based Assay, RNA Sequencing, Fluorescence, Control

Journal: Science Advances

Article Title: Nanocollision promotes locomotion of dendritic cells for tumor therapy

doi: 10.1126/sciadv.aeb7714

Figure Lengend Snippet: ( A and B ) Representative fluorescence images of intracellular Ca 2+ distribution in DCs with different treatments. ( C to E ) Locomotion trajectory (C), accumulated distance (in 5 min) (D), and locomotion speed (E) of DCs with different treatments. ( F and G ) Western blot analysis of the nanocollision-induced alterations of monomeric and oligomeric CCR7 (F) and Piezo1 expression (G). ( H and I ), Representative fluorescence images showing the antigen capture (H) and antigen presenting (I) capabilities of DCs with different treatments. ( J ) Western blot analysis of IL-12 and IFN-γ expression. ( K to N ) Transcriptomics analysis of gene expression in DCs. RNA-seq density plot [(K), A versus B versus C], heatmap of immune activation-related genes [(L) A versus B versus C], bubble plot [(M) C versus B], and sankey bubble plot [(N) C versus B]. [(A) Control; (B) R848; (C) R848 + NCG US(40°C) ]. P values: ns, not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001.)

Article Snippet: Antibodies and reagents used for Western blot are as follows: rabbit anti–IL-12 (bs-0767R), rabbit anti–IFN-γ (bs-0480R), CCR7 rabbit polyclonal antibody (pAb) (bs-1305R), and β-actin mouse monoclonal antibody (bsm-33036 M) from Bioss; cPLA2 pAb (YT1084), cPLA2 (phospho Ser 505 ) pAb (YP0868), PIEZ1 rabbit pAb (YT8073), AP-1 (phospho Tyr 170 ) pAb (YP0018), and AP-1 (Acetyl Lys271) pAb (YK0062) from Immunoway; anti-calreticulin rabbit pAb ( GB112134 ), anti-HMGB1 rabbit pAb (GB11103) from Servicebio.

Techniques: Fluorescence, Western Blot, Expressing, Gene Expression, RNA Sequencing, Activation Assay, Control

Journal: EMBO Reports

Article Title: DNA methylation-mediated memory of obesity in CD4 T lymphocytes perpetuates immune dysregulation

doi: 10.1038/s44319-026-00765-w

Figure Lengend Snippet: ( A ) Representative western blots (top) and densitometric quantification (bottom) of Stk26 protein levels within peripheral LNs CD4 T cells isolated from female C57BL/6J mice on different diet groups for 14 weeks (groups 1–3 in Fig. ). Data are presented as mean +/− SD ( n = 3 mice per group). Unpaired nonparametric Mann–Whitney T test; n.s. ( B ) Representative flow cytometry histograms (left) showing LC3II expression in splenocytes from the three 14-week mouse groups (groups 1–3 in Fig. ). Splenocytes were cultured ex vivo in the presence of 100 μM of chloroquine within RPMI for 4 h. Scatter plots (right) show autophagy flux, calculated as the difference in mean fluorescence intensity (MFI) of LC3II between chloroquine-treated and vehicle control groups. Analysis was gated on live (near IR-) CD4 + CD44 + CXCR3+ or CD4 + CD44 + LFA1 + T cell populations. Data are from one experiment ( n = 6 female C57BL/6J mice per group). Results are shown as mean +/− SD. Unpaired nonparametric T test (Mann–Whitney), ** P < 0.01. ( C ) Representative flow cytometry plots (left) of CD4 + KLRG1+ cells gated within the live (near IR-) CD4 T cell population from peripheral LNs of female C57BL/6J mice on a 14-week CD or HFD-RE. Scatter plots (right) show the percentage of CD4 + KLRG1+ cells within the CD4 + CD44+ or CD4 + LFA1+ populations. Data is from one experiment ( n = 5–6 mice per group) and is presented as mean +/− SD. Unpaired nonparametric T test (Mann–Whitney), * P < 0.05. ( D ) Scatter plots showing CD4 and CD8 (CD45RA + CCR7-) human TEMRA from PBMCs of control ( n = 10) and individuals with Alstrom syndrome ( n = 10). Data are presented as mean +/− SD. Paired two-tailed Wilcoxon T test; * P < 0.05. ( E ) Scatter plots showing the percentage of human CD4 TEMRA (CCR7-CD45RO-) cells from PBMCs from individuals ( n = 13) before and after semaglutide treatment. All samples were gated on live (near IR-) populations. Paired nonparametric Wilcoxon T test; n.s. ( F ) Flow cytometry analysis of human CD4 + TEMRA cells (CD3 + CD4 + CD45RA + CD27 − ) in subcutaneous abdominal adipose tissue and lysed whole blood. In adipose, data are expressed as a percentage of CD4 + T cells. Samples are from participants of a 10-week randomized controlled trial of exercise training ( n = 14) compared to control ( n = 13). Pre-intervention = day one of the intervention (before exercise). Post-intervention= 36 h after the 10-week intervention/control period. Repeated measures analyses of variance (ANOVAs); n.s. ( G ) Heatmap displaying transcriptomic data of selected genes with previous implications with autophagy and/or senescence from murine CD44+ memory T cells isolated from spleen of female C57BL/6J mice on different diet groups for 14-week ( n = 3 female C57BL/6J mice per group; groups 1–3 in Fig. ). The analysis was performed using the Nanostring Mouse PanCancer Immune Profiling panel, covering 770 genes including genes related to autophagy and/or senescence. .

Article Snippet: Human CCR7 (clone: REA108) , Miltenyi Biotech , Cat #130-120-463.

Techniques: Western Blot, Isolation, MANN-WHITNEY, Flow Cytometry, Expressing, Cell Culture, Ex Vivo, Fluorescence, Control, Two Tailed Test

Journal: EMBO Reports

Article Title: DNA methylation-mediated memory of obesity in CD4 T lymphocytes perpetuates immune dysregulation

doi: 10.1038/s44319-026-00765-w

Figure Lengend Snippet: ( A ) Scatter box plots showing flow cytometry analysis of the percentage of human CD4 and CD8 Tem (CD45RA-CCR7-) Tem cells from PBMCs isolated from control individuals ( n = 10) or individuals with Alstrom syndrome ( n = 10). Data are presented as mean +/− SD. Unpaired nonparametric Mann–Whitney T test; * P < 0.05. ( B , C ) Flow cytometry analysis of subcutaneous human abdominal adipose tissue ( B ) and lysed whole blood ( C ), including CD8 + T cells, and the CD8 + T cell sub-populations; naive (CD3 + CD8 + CD45RA + CD27 + ), central memory (CD3 + CD8 + CD45RA − CD27 + ), effector memory (CD3 + CD8 + CD45RA − CD27 − ). In adipose, CD8 + T cells are expressed as a percentage of CD3+ events, and CD8 + T cell sub-populations are expressed as a percentage of CD8+ events. Samples are from participants of a 10-week randomized controlled trial of exercise training ( n = 14) compared to a control ( n = 13). Pre-intervention = day one of the intervention (before exercise). Post-intervention = 36 h after the 10-week intervention/control period. Repeated measures analyses of variance (ANOVAs); n.s.

Article Snippet: Human CCR7 (clone: REA108) , Miltenyi Biotech , Cat #130-120-463.

Techniques: Flow Cytometry, Isolation, Control, MANN-WHITNEY

Journal: EMBO Reports

Article Title: DNA methylation-mediated memory of obesity in CD4 T lymphocytes perpetuates immune dysregulation

doi: 10.1038/s44319-026-00765-w

Figure Lengend Snippet: ( A ) Scatter plots showing flow cytometry analysis of the percentage of human CD4 T cells, CD4 Tem (CCR7-CD45RO + ) and inflammatory CD4 T cells (CXCR3 + ) from PBMCs isolated from individuals ( n = 13) before and after semaglutide treatment. All samples were gated on live populations. Paired nonparametric Wilcoxon T test; n.s. ( B , C ) Flow cytometry analysis of human subcutaneous abdominal adipose tissue ( B ) and lysed whole blood ( C ), including CD3 + T cells, CD4 + T cells, and the CD4 + T cell sub-populations; naive (CD3 + CD4 + CD45RA + CD27 + ), central memory (CD3 + CD4 + CD45RA-CD27 + ), effector memory (CD3 + CD4 + CD45RA − CD27 − ). In adipose, CD3 + T cells are expressed as a percentage of CD45+ events, CD4 + T cells are expressed as a percentage of CD3+ events, and CD4 + T cell sub-populations are expressed as a percentage of CD4+ events. Samples are from participants of a 10-week randomized controlled trial of exercise training ( n = 14) compared to a control ( n = 13). Pre-intervention = day one of the intervention (before exercise). Post-intervention = 36 h after the 10-week intervention/control period. Repeated measures analyses of variance (ANOVAs); n.s. .

Article Snippet: Human CCR7 (clone: REA108) , Miltenyi Biotech , Cat #130-120-463.

Techniques: Flow Cytometry, Isolation, Control

Journal: EMBO Reports

Article Title: DNA methylation-mediated memory of obesity in CD4 T lymphocytes perpetuates immune dysregulation

doi: 10.1038/s44319-026-00765-w

Figure Lengend Snippet: ( A ) Representative flow cytometry plot (left) showing human CD4 T cell subsets: naive CD4 T cells (CCR7 + CD45RO-), Tcm (CCR7 + CD45RO + ), Tem (CCR7-CD45RO + ), and TEMRA (CCR7-CD45RO-). Quantification (right) displays the frequencies of Tem (CD45RO + CCR7-) within live CD4 + T cells. Isolated CD4 T cells were treated with 50 μM of oleic acid, palmitate, or stearic acid overnight (right) followed by activation with plate-bound anti-CD3 (2.5 μg/mL) and anti-CD28 (1.5 μg/mL) for 48 h. The untreated control group received the ethanol:BSA (1:4) solution. Each point represents a technical replicate ( n = 4). Kruskal–Wallis with Dunn’s correction; data are presented as mean ± SD, * P < 0.05. ( B ) Representative flow cytometry plot (left) showing the expression of human CD4 Treg (CD4+Foxp3 + ). Quantification (right) shows the frequency of CD4 Treg (CD4+Foxp3 + ) pre-treated overnight with 50 μM oleic acid, palmitate, or stearic acid, then activated for 48 h as described above. The untreated control group received the vehicle control solution. Each point represents a technical replicate from n = 3 donors. Kruskal–Wallis with Dunn’s correction; data are presented as mean ± SD, * P < 0.05. ( C ) Scatter box plots showing relative STK26 mRNA expression in human CD4 T cells isolated from PBMCs from healthy volunteers and pre-treated overnight with 50 μM palmitate without activation, followed by either no activation or 48 h activation with plate-bound anti-CD3/CD28. Untreated controls received the vehicle control solution. Expression was normalized to the housekeeper gene 18S. Each point represents a technical replicate from n = 4 biological replicates. Data are presented as mean +/− SD; Unpaired nonparametric T test (Mann–Whitney), *** P < 0.001. ( D ) Representative western blot images (left) and densitometric quantification (right) of Stk26 and β-actin protein levels in human CD4 T cells pre-treated overnight with 50 μM palmitate or vehicle control, followed by either no activation or activation with plate-bound anti-CD3/CD28 for 48 h. Data are presented as mean +/− SD ( n = 3 donors). Two-tailed Student’s T test with Shapiro–Wilk normality test; * P < 0.05. ( E ) Representative western blot images (left) and densitometric quantification (right) showing LC3II and β-actin protein levels in human CD4 T cells pre-treated overnight with 50 μM palmitate or vehicle control and activated with CD3/CD28 beads for 48 h. To inhibit autophagy, palmitate or vehicle-control-treated CD4 T cells were treated with 25 μM chloroquine (CQ) or vehicle-control overnight. Data are presented as mean +/− SD ( n = 4 donors). Unpaired nonparametric T test (Mann–Whitney); * P < 0.05. ( F ) Scatter box plots of STK26 mRNA expression in human CD4 T cells cultured overnight with adipose-conditioned media from healthy range BMI or BMI > 30 osteoarthritis patients, followed by activation with CD3/CD28 beads for 48 h. Gene expression was normalized to β-actin. Data are presented as mean +/− SD ( n = 6 donors of adipose-conditioned media). Unpaired nonparametric T test (Mann–Whitney); * P < 0.05. ( G ) Scatter box plots showing relative CDKN1C mRNA expression in human CD4 T cells pre-treated overnight with 50 μM palmitate without activation, followed by either no activation or 48 h activation with plate-bound anti-CD3/CD28. Controls received the vehicle control solution. Expression was normalized to the housekeeper 18S. Each point represents a technical replicate ( n = 3–4 donors). Data are presented as mean +/− SD; unpaired nonparametric T test (Mann–Whitney); ** P < 0.01, *** P < 0.001. ( H ) Scatter plots showing the percentages of human CD57+ and IL-4 + CD4 TEMRA cells (CD4 + CD45RO-CCR7-CD57+ and CD4 + CD45RO-CCR7-IL-4 + ) pre-treated overnight with 50 μM oleic acid, palmitate, or stearic acid without activation. Cells were then either left non-activated or activated with plate-bound anti-CD3/CD28 for 48 h. Controls received the vehicle control solution. Each point represents a technical replicate from n = 3 donors. Data are presented as mean +/− SD; Kruskal–Wallis with Dunn’s correction; * P < 0.05, ** P < 0.01, *** P < 0.001. .

Article Snippet: Human CCR7 (clone: REA108) , Miltenyi Biotech , Cat #130-120-463.

Techniques: Flow Cytometry, Isolation, Activation Assay, Control, Expressing, MANN-WHITNEY, Western Blot, Two Tailed Test, Cell Culture, Gene Expression