Fig 2: Effects of FGF-receptor inhibition on RUNX2, RUNX3, and MEF2C. MPC pellets were subjected to chondrogenic induction for 6 weeks and treated with 250 nM of the FGFR-Inhibitor PD173074 or with the corresponding amount of DMSO from day 7 onward. Protein levels of pERK1/2 and total ERK1/2 as reference were detected by Western blot (A). Shown is one representative of three experiments. (B) Gene expression of COL2A1 was determined during 42 days of chondrogenesis with PD173074 (black line) or DMSO control (dashed line) (n = 3 donors), (C,D) expression of indicated genes was determined by qPCR at day 21 of chondrogenesis, and levels were referred to reference genes HNRPH1 and CPSF6. ALP activity was quantified in culture supernatants (C). Expression levels of indicated genes and ALP activity are referred to DMSO cultures (dashed lines). Box plots were generated as described in Figure 1. *, p ≤ 0.05, Mann–Whitney U test compared to control. (E,F) RUNX3 and MEF2C protein levels were determined in samples with DMSO or PD173074 at indicated timepoints, β-actin levels were determined as internal reference. One representative of three experiments is shown.

Fig 3: Effects of siRNA-mediated knockdown of MEF2C, RUNX2, and RUNX3 in SAOS-2 osteosarcoma cells. SAOS-2 cells were transfected with siMEF2C, siRUNX2, siRUNX3, or a non-targeting control siRNA (siCtrl) 48 h prior to analysis (n = 6 biological replicates from four independent experiments). (A) Target-gene expression was determined by qPCR. (B) MEF2C, RUNX2, and RUNX3 protein levels were determined by Western blot analysis; β-actin levels were determined as internal reference. One representative of four biological replicates (two to three experiments) is shown. Expression of indicated genes was determined by qPCR (C–E), and levels were referred to reference genes HNRPH1 and CPSF6. Expression levels of indicated genes are referred to siCtrl cultures (dashed lines). Box plots were generated as described in Figure 1; outliers are depicted as ∘. Significance compared to siCtrl was determined by Mann–Whitney U test *, p ≤ 0.05, with Bonferroni correction.

Fig 4: Rarb relays the signal of glycemic variability to Runx3/Col6a1 via histone modification. (A) Sketch view of chromatin immunoprecipitation (ChIP) assay. (B) ChIP analysis of Rarb gene shows percentage input of H3K9me2, H2AK119ub, H3K27me3, H3K4me3, and H3K27ac in hyperglycemic and hypoglycemic PDAC cells. Data are presented as mean ± standard deviation, and data from 3 independent experiments are shown; H, hyperglycemic cells; L, hypoglycemic cells. (C) Quantitative RT-PCR analysis shows expression of Rarb, Runx3, and Col6a1 after 10 µmol/L, 20 µmol/L SAHA treatment for 24 hours. Data are presented as mean ± standard deviation, and data from 3 independent experiments are shown. Unpaired t test is applied, *P < .05. (D) ChIP analysis of Rarb gene shows percentage input of H3K9me2, H3K27me3, and H3K27ac in control and SAHA treated hypoglyemic (L) PDAC cells. Data are presented as mean ± standard deviation, and data of 2 independent experiments are shown.

Fig 5: Activity of the Rarb/Runx3/Col6a1 signal axis is inhibited by metformin. Quantitative RT-PCR results show expression of Rarb, Runx3, and Col6a1 after metformin (20 mmol/L) and 2DG (10 mmol/L) treatment for 48 hours in hyperglycemic PDAC cells. (B) Western blot analysis shows expression of Rarb, Runx3, Col6a1, and phospho-AMPKaThr172 after metformin and 2DG treatment for 48 hours in hyperglycemic PDAC cells. (C) Quantitative RT-PCR analysis shows expression of Runx1, Runx2, Rara, Rarg, and Egr2 after metformin (20 mmol/L) and 2DG (10 mmol/L) treatment for 48 hours in hyperglycemic PDAC cells. (D) Glucose uptake assay shows reduced cellular uptake after metformin treatment for 24 hours. (E) Representative H&E pictures (left panel) and metastatic area quantification (right panel) show metastatic colonization in control and metformin treated hyperglycemic PDAC cells. All data are presented as mean ± standard deviation; data of 3 independent experiments are shown. Unpaired t test is applied, *P < .05. (F) Chromatin immunoprecipitation (ChIP) analysis of Rarb gene shows percentage input of H3K9me2, H3K27me3, and H3K27ac in control and metformin treated hyperglycemic PDAC cells. All data are presented as mean ± standard deviation, and data from 2 independent experiments are shown. H, hyperglycemic cells; Met, metformin.