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. 2024 Oct 30;8:246. doi: 10.1038/s41698-024-00742-3

Fig. 4. Ccl3 activates TGFβ signaling in macrophages.

Fig. 4

A Correlation of Ccl3 expression with tumor purity (left) and with TGFB1 expression (right) in PAAD patients from TIMER2.0 (n = 179). B Correlation between Tgfb1 and Ccl3 mRNA expression in tumors from C57BL6/J orthotopic PDAC models (n = 36). C qPCR of Tgfb1 in RAW264.7 unstimulated or stimulated with rCcl3, and as single cultures or co-cultured with RC416, DT4313 or PAN610 transduced as indicated. Results show mean ± s.d of 3 biological replicates. A schematic representation of the co-culture technique used to culture RAW264.7 macrophages with PDAC cell lines is shown. D Representative western blot of secreted Ccl3 and Tgfβ1 proteins in the conditioned medium of RAW264.7 macrophages as single cultures or cocultured with RC416, DT4313 and PAN610 transduced as indicated. Ponceau served as loading control. E Representative western blot of pSmad2 and Smad2 in RAW264.7 as single cultures or co-cultured as indicated. Hsp90 was used as loading control. Quantification of pSmad2/Smad2 is shown. F qPCR of Tgfb1 in RC416, DT4313 or PAN610 transduced as indicated, as single cultures or co-cultured with RAW264.7. Results show mean ± s.d of 3 biological replicates. A schematic representation of the co-culture technique used to culture PDAC cells with RAW264.7 macrophages is shown. P values in (C) and (F) were calculated by one-way ANOVA and Tukey’s test. G Representative western blot of pSmad2 and Smad2 in RC416, DT4313 or PAN610 transduced as indicated, as single cultures or co-cultured with RAW264.7. Hsp90 was used as loading control. Quantification of pSmad2/Smad2 is shown. *p < 0.05; **p < 0.01; ***p < 0.001.