Journal:

Article Title: Carbohydrate Oxidation Acidifies Endosomes, Regulating Antigen Processing and TLR9 Signaling

doi: 10.4049/jimmunol.0903168

Figure Lengend Snippet: GlyAg oxidation leads to intracellular acidification. All images taken with a 63× oil immersion lens. A, APCs were treated with 40 nM BafA to block normal acidification and 2 μM Lyso to detect vesicular pH and then incubated with 50 μg/ml AlexaFluor594-conjugated GlyAg (GlyAg-594) for 18 h. We found that RAW macrophages and WT primary cells showed intense green fluorescence, indicating acidic pH, and significant colocalization (yellow), indicating that the acidic environment occurred primarily in vesicles loaded with GlyAg. In contrast, iNOS−/− cells showed little colocalization, and the green signal remained unchanged (no increase in fluorescence over background levels), collectively suggesting that NO-mediated oxidation in the presence of GlyAg was required for acidification. Colocalization was determined using Imaris imaging software. Scale bar, 20 μm. B, Representative scatterplots of each fluorescent pixel from confocal images with LysoSensor Green fluorescent intensity along the x-axis and GlyAg fluorescent intensity along the y-axis. The shaded area in the upper right quadrant represents colocalized pixels (i.e., double-positive for red and green) above background with the associated Pearson correlation coefficient indicated for all colocalized pixels, as calculated using Imaris CoLoc software. NO-producing cells showed a direct correlation between GlyAg and acidification, which was lost in the absence of NO-mediated oxidation.

Article Snippet: All values, including the Pearson correlation coefficients ( r values) were determined by the Imaris CoLoc software.

Techniques: Blocking Assay, Incubation, Fluorescence, Imaging, Software

Journal:

Article Title: Carbohydrate Oxidation Acidifies Endosomes, Regulating Antigen Processing and TLR9 Signaling

doi: 10.4049/jimmunol.0903168

Figure Lengend Snippet: GlyAg oxidation-mediated acidification is limited to GlyAg+ vesicles. Using three-dimensional reconstructions of the data sets (Figs. 5, ​,6),6), the individual pixel intensities were also mapped in scatterplots to quantify colocalization of OVA processing and GlyAg. Cells given DQ-OVA only (no GlyAg or BafA) displayed a large number of green pixels, which represent processed OVA (A), whereas BafA blocks proteolysis, as seen with a significantly reduced number of green pixels (B). When RAW macrophages were incubated with BafA and GlyAg (C), all bright green regions (i.e., processed OVA) were also positive for GlyAg (double positive, upper right quadrant). Many cell regions were positive for GlyAg only, but no regions were positive for OVA processing alone. The same pattern was observed in primary WT cells (D, F), in that green pixels were nearly always positive for red, but not vice versa. Conversely, NO-deficient cells show dramatically reduced numbers of green pixels and colocalization (E, G). These data confirm that carbohydrate oxidation by NO had a local pH effect within individual GlyAg+ endosomes. All values, including the Pearson correlation coefficients (r values) were determined by the Imaris CoLoc software.

Article Snippet: All values, including the Pearson correlation coefficients ( r values) were determined by the Imaris CoLoc software.

Techniques: Incubation, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The IL1β-IL1R signaling is involved in the stimulatory effects triggered by hypoxia in breast cancer cells and cancer-associated fibroblasts (CAFs)

doi: 10.1186/s13046-020-01667-y

Figure Lengend Snippet: a Scatter plots depicting the correlation between HIF-1α expression and IL-1β levels in TNBC patients of the TCGA and METABRIC cohorts. The Pearson correlation coefficients (r) and the relative p-values are shown in each panel. b Box plots showing the differential IL-1β expression levels in non-TNBC and TNBC patients, as found in the TCGA and METABRIC datasets. The number of patients and p -values are reported in each panel

Article Snippet: The Pearson correlation coefficients (r-values) between the mRNA levels of HIF-1α and the other genes of the TCGA (n. 20,530) dataset in TNBC cohort of patients were assessed in R Studio (version 3.6.1) using the cor.test() function and setting the method as “Pearson”.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The IL1β-IL1R signaling is involved in the stimulatory effects triggered by hypoxia in breast cancer cells and cancer-associated fibroblasts (CAFs)

doi: 10.1186/s13046-020-01667-y

Figure Lengend Snippet: a Venn diagram of metastatic genes up-regulated in MDA-MB-231 cells in an exclusive and shared manner upon hypoxia (2% O2) and IL-1β. The genes up-regulated by both hypoxia and IL-1β are listed in the box according to the gene expression changes induced by hypoxia (from high to low) (see Additional file ). b mRNA expression of IL-1β and COX2 in MDA-MB-231 cells cultured upon normoxia and hypoxia or vehicle and IL-1β in the presence or absence of the IL1R1 antagonist IL1R1a, as evaluated by real-time PCR. Values are normalized to the actin beta (ACTB) expression and shown as fold changes of the mRNA expression induced by hypoxia respect to normoxic cells. c Scatter plot depicting the correlation between the expression of COX2 and IL-1β in TNBC patients of the METABRIC cohort of patients. The Pearson correlation coefficient (r) and the relative p -value are indicated. d Immunoblots of IL-1β and COX2 in MDA-MB-231 cells cultured upon normoxia or hypoxia in combination with IL1R1a, as indicated. e Immunoblots of IL-1β and COX2 in MDA-MB-231 cells treated with vehicle or IL-1β and in combination with IL1R1a. f Immunoblots of COX2 in MDA-MB-231 cells upon normoxia and hypoxia silencing GPER. Side panels show densitometric analysis of the blots normalized to β-actin. Values represent the mean ± SD of three independent experiments performed in triplicate. (*) p < 0.05 for cells cultured under normoxia versus cells cultured under hypoxia or cells treated with IL-1β versus vehicle-treated cells

Article Snippet: The Pearson correlation coefficients (r-values) between the mRNA levels of HIF-1α and the other genes of the TCGA (n. 20,530) dataset in TNBC cohort of patients were assessed in R Studio (version 3.6.1) using the cor.test() function and setting the method as “Pearson”.

Techniques: Gene Expression, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot