Journal: Materials Today Bio

Article Title: In situ administration of STING-activating hyaluronic acid conjugate primes anti-glioblastoma immune response

doi: 10.1016/j.mtbio.2024.101057

Figure Lengend Snippet: HA-MSA2 activates STING signaling and expression of downstream genes in innate immune cells. A. JAWSII cells were treated with HA, MSA2 or HA-MSA2 (20 μM MSA2 equivalent concentration) for 0.5h; untreated cells were used as controls. Cells were lysed and analyzed by Western blot, using anti-phosphoSTING (p-STING), anti-STING, anti-phospho-TBK1 ( p -TBK1), anti-TBK1, anti-phopshoIRF3 (pIRF3) and anti-IRF3 antibodies. Anti-GAPDH or anti-alpha actinin were used as loading controls. Representative blots from three replicates; B. Blots were quantified, and phospho-protein signal (normalized to GAPDH or alpha actinin on the same blot) was normalized to the corresponding total protein (normalized to GAPDH or alpha actinin on the same blot). Relative quantifications are shown. **p < 0.01, ****p < 0.0001 (One-way ANOVA with Tukey's post-hoc). C-E. qPCR gene expression of immune-stimulatory markers (IFNβ, TNFα, IL-6 and CXCL10) after treatment with HA alone, MSA2 or HA-MSA2 of C. JAWSII, D. J774 and E. BV-2 cells for 5 and 24 h. Cells were treated with an equivalent concentration of MSA2 of 20 μM. Untreated cells were used as a reference. Data were normalized to results from untreated cells using RLP19 as a housekeeping gene. Mean, error bar = SD, n = 3, statistical analysis performed via two-way ANOVA followed by Dunnet's multiple comparison test with a single pooled variance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Murine dendritic JAWSII cells (ATCC, Manassas, VA, USA) were maintained in MEM α, nucleosides, with no ascorbic acid, 1 mM sodium pyruvate and 5 ng/ml GM-CSF Recombinant Mouse Protein cultured at 37 °C in a 5 % CO 2 atmosphere.

Techniques: Expressing, Concentration Assay, Western Blot, Gene Expression, Comparison

Journal: Materials Today Bio

Article Title: In situ administration of STING-activating hyaluronic acid conjugate primes anti-glioblastoma immune response

doi: 10.1016/j.mtbio.2024.101057

Figure Lengend Snippet: HA-MSA2 induces cell toxicity in SB28 cells associated with hallmarks of immunogenic cell death and favors the maturation of immune cells. A, B. SB28 cells were treated with MSA2 (blue line) and HA-MSA2 (green line) for 72 h and cell viability was measured; IC 50 values were obtained by non-linear regression curve fitting; Mean, error bar = SD, n = 3, statistical analysis performed via unpaired t -test (**p < 0.01); C. Membrane translocation of calreticulin (CRT, calculated as MFI ratio) and D. ATP release induced by HA, MSA2 and HA-MSA2. Mean, error bar = SD, n = 3, statistical analysis performed via Kruskal-Wallis test followed by Dunn's multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). E. Schematic representation of immune-cell maturation assay. Percentage of CD86 + F. JAWSII, G. J774, and H. BV-2 cells after incubation with SB28 medium pretreated with HA, MSA2, and HA-MSA2. Mean, error bar = SD, n = 3, statistical analysis performed via One-way ANOVA test followed by Tukey's multiple comparisons test, with a single pooled variance. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Murine dendritic JAWSII cells (ATCC, Manassas, VA, USA) were maintained in MEM α, nucleosides, with no ascorbic acid, 1 mM sodium pyruvate and 5 ng/ml GM-CSF Recombinant Mouse Protein cultured at 37 °C in a 5 % CO 2 atmosphere.

Techniques: Membrane, Translocation Assay, Comparison, Incubation

Journal: The Journal of Cell Biology

Article Title: Galectin-9 regulates dendritic cell polarity and uropod contraction by modulating RhoA activity

doi: 10.1083/jcb.202404079

Figure Lengend Snippet: cDC2 cell purity and supplementary phenotype analysis. (A) Gating strategy to determine cDC2 purity after isolation from PBMCs. cDC2s were stained for CD1c, CD11c, CD14, and CD20. A first gate is set based on the physical FCS-A/SSC-A parameters followed by selecting on negative cells for CD14 and CD19. cDC2s are then identified as positive cells for CD11c and CD1c. (B) Surface galectin-9 and CCR7 expression in galectin-9 and CCR7-positive cDC2s. Graph shows average ± SEM gMFI of five individual donors. One-way ANOVA followed by Dunnett’s test for multiple comparisons was performed. *P < 0.05; **P < 0.01; ***P < 0.001. gMFI, geometric mean fluorescence intensity.

Article Snippet: Human cDC2s were isolated from peripheral blood mononuclear cells derived from healthy individuals (Sanquin, Nijmegen, The Netherlands) using the MACS CD1c + isolation kit (130-119-475; Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Isolation, Staining, Expressing, Fluorescence