Fig 1: Evaluation and visualization of immune cell infiltration. (A) Heatmap of immune cells in keloid and control groups via single-sample gene set enrichment analysis in GSE145725 dataset (n = 19). (B) Correlation analysis of TGM2 expression with immune cell infiltration in the GSE145725 dataset. (C) Ranking of immune cell scores in the GSE145725 dataset. (D) Variation of immune cell scores between keloid and control groups in the GSE145725 dataset. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, not significant.
Fig 2: Acid‐adapted cells use collagen remodeling enzymes to engineer their niche. (a) TGM2 validation by (b) Western blot and (c) Immunocytochemistry (ICC). TGM2 expression in acid‐adapted cancer cells is significantly higher compared to non‐adapted cells. (d) Bicarbonate buffer therapy in animal reduced the expression of TGM2 compared to control group, indicating the effect of acidosis on expression of TGM2. (e, f) Translation of TGM2 expression to clinic and patient samples. LAMP2b is a marker of acidosis that we reported in our previous work. The TMA from same patients was stained for TGM2, and expression level of TGM2 and LAMP2b was compared for each sample. There is a correlation between LAMP2b and TGM2 expression in patients. (g) Acid‐adapted MCF‐7 cells not only have higher amount of TGM2 and LOXL2, but also secrete more of these enzymes to the environment. Both enzymes have been shown to play role in collagen crosslinking and stability. This strengthen our acid‐induced niche engineering phenotype of cancer cells that they build the niche they need it to survive the harsh environment such as acidosis
Fig 3: TGM2 transcriptionally activates FN1 by promoting p65 nuclear translocation in PTC cell lines. (A) Overexpression of TGM2 activates p65 nuclear translocation in PTC cells, as measured using western blot assays. Data are presented as the mean ± S.D., as analyzed using an independent samples t-test. *P < 0.05; **P < 0.01 versus NC. (B) Overexpression of TGM2 activates p65 nuclear translocation in PTC, as measured using immunofluorescence staining. (C) qRT-PCR was applied to detect the protein level of FN1 in PTC cells transfected with p65 or NC. Data are presented as the mean ± S.D., as analyzed using an independent samples t-test. **P < 0.01 versus NC. (D) Western blotting was applied to detect the protein level of FN1 in PTC cells transfected with p65 or NC. Data are presented as the mean ± S.D., as analyzed using an independent samples t-test. **P < 0.01 versus NC. (E) Prediction of the p65 binding site in the FN1 promoter region using JASPAR (http://jaspar.genereg.net/). (F) The effect of overexpression of p65 on luciferase activity of a vector containing the FN1 wild-type promoter or mutated promoter. Data are presented as the mean ± S.D., as analyzed using an independent samples t-test. **P < 0.01 versus NC. (G) The effect of p65 overexpression on the luciferase activity of a vector containing the FN1 wild−type promoter or mutation site 1, mutation site 2, mutation site 3, or mutation site 4. Data are presented as the mean ± S.D., as analyzed using an independent samples t-test. **P < 0.01 versus NC.
Fig 4: Suppression of tTG activity decreases the expression level of IL-33/ST2. a The protein expression levels of IL-33 and ST2 in mice liver homogenates detected by Western blotting are displayed on the left panel, and the result of semi-quantitative analysis is shown on the right panel. GAPDH was used as a loading control. b, c The protein expression levels and location of IL-33 (B) and ST2 (C) in mice liver tissue were determined by IHC assay (200× and 400× left, scale-bar: 50 µm), and the result of semi-quantitative analysis of the protein signal using modified H score is shown on the right panel. Data are presented as the mean ± SD. All experiments were performed twice or thrice. Red arrows indicate the Sj eggs (t-test, **P = 0.0014, ****P < 0.0001)
Fig 5: miR-125a-5p reverses epithelial-mesenchymal transition in MCF7/Adr cells. (A) Transfection efficiency of mimics was determined via reverse transcription-quantitative PCR. miR-125a-5p was overexpressed after transfection of mimics in MCF-7/Adr cells. (B) Cell morphological alterations after miR-125a-5p transfection, as indicated by arrows. Images of cells in medium. The cells of NC groups remained elongated and dispersed with a mesenchymal morphology, and the cells of miR-125a-5p groups were closely packed and rounded with a cobblestone epithelial morphology; magnification, ×200 (scale bar, 20 µm). (C) Western blot analysis of TAFAZZIN, TG2, AKT, E-cadherin, N-cadherin, vimentin and ß-catenin in MCF7/Adr cells. (D and E) Semi-quantification of protein expression results from panel (C). GAPDH served as an internal control. Data are presented as the mean ± SD. Similar results were obtained from three independent experiments. *P<0.05, as obtained via unpaired Student's t-test. TAFAZZIN, tafazzin phospholipid-lysophospholipid transacylases; miR, microRNA; p-, phosphorylated; t-, total; TG2, Transglutaminase 2; Adr, Adriamycin; NC, negative control.
Supplier Page from Abcam for Anti-Transglutaminase 2 antibody [EP2957]