sting  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: Loss of CD44 re-educates pancreatic cancer-associated fibroblasts modulating their fibrotic and immunosuppressive functions

doi: 10.1038/s41419-026-09155-5

Figure Lengend Snippet: A Conditioned media (CM) from CAFs and CAF ΔCD44 were collected and analyzed via cytokine array. Relevant proteins were highlighted by colored rectangles and subsequently quantified using ImageLab software. The mean intensity of the array spots was evaluated and normalized. The normalized protein expression of IL-6, IL-8, and MCP-1 is depicted as ratios comparing CAFs to CAF ΔCD44 . N = 3. Data are means ± S.E. Statistical significance was determined using the one sample t test. ** p value < 0.01; *** p value < 0.001; **** p value < 0.0001. B RNA of CAFs and CAF ΔCD44 was extracted 48 h after seeding and subjected to qPCR analysis of expression of CCL2 and IL8 . GAPDH was used as a reference gene. N = 3. Data are means ± S.E. Statistical significance was determined using the one sample t test. ** p value < 0.01; **** p value < 0.0001. C DC2.4 were either cultured with 50% CAFs/CAF ΔCD44 conditioned media (CM) or indirectly co-cultured with CAFs/CAF ΔCD44 (as indicated in the illustrations: Treffert, S. (2026) https://BioRender.com/ifl9gho ) for 48 h and treated with LPS. At 24 h after seeding, 1 µg/ml lipopolysaccharide (LPS) was added to the cultures. Il10 and Ido1 expression was assessed via qPCR. Gapdh and β actin were used as reference genes. Data are means ± S.E. Statistical significance was determined using the one sample t test. N = 3. * p value < 0.05; ** p value < 0.01; *** p value < 0.001. D RNA of CAFs and CAF ΔCD44 was extracted 48 h after seeding and subjected to qPCR analysis of TGFβ1 and IL10 expression. GAPDH was used as a reference gene. Data are means ± S.E. Statistical significance was determined using the one sample t test. N = 3. * p value < 0.05; ** p value < 0.01. E Fluorescence analysis of tumor cell confluency (CellTracker TM Green) in different experimental conditions including or not human T cells (CD45-AF647, purple), CD3xPSMA BiTEs as well as control medium, CAF CM or CAF ΔCD44 CM. Imaging was conducted with the Cell Voyager CQ1 Confocal Quantitative Image Cytometer (Yokogawa) for 24 h in intervals of 30 min. Scale bar, 50 µm. F Fitted curves of tumor cell confluency were used to calculate the half-maximal effect (HME) of the two CM conditions. G The maximal killing rates at the HME of both conditions were calculated. Statistical significance was determined using a paired, two-tailed t test. N = 4, n = 2. * p value <0.05; *** p value < 0.001.

Article Snippet: The CD3xPSMA BiTEs (MedChemExpress, Monmouth Junction, NJ, USA; HY-P99802) were prepared in the respective experimental and control medium and added to the co-culture at 10 μg/ml.

Techniques: Software, Expressing, Cell Culture, Fluorescence, Control, Imaging, Cytometry, Two Tailed Test