Fig 2: The influence of leucettines as DYRK1A inhibitors on insulin, C-peptide, and glucagon production by MIN6 cells.Flow cytometry analysis of (A) insulin, (B) C-peptide, and (C) glucagon levels in MIN6 cells. Cells were treated with a 5 µM inhibitor for 24 h and 72 h later, cells were stained with appropriate antibodies.

Fig 3: The effect of investigated leucettines on the level of DYRK1A in MIN6 cells.Flow cytometry histograms of the level of DYRK1A in MIN6 cells: (A) Expression level of DYRK1A in control cells. (B-E) DYRK1A expression levels in cells treated with 5 µM harmine, leucettine L41, leucettine L43, or leucettine L92, respectively. (F) RT-qPCR results were obtained for control, harmine and leucettine L41 treated cells. Confocal images for MIN6 cells stained with anti-DYRK1A antibody (green fluorescence) and cell membrane marker (red fluorescence): (G) cells without treatment–control cells and (H) cells after treatment with leucettine L41. The nuclei were counterstained with Hoechst33342 dye (blue fluorescence). Scale bars, 20 µm.

Fig 4: Effects of DYRK1A inhibitors on isolated mouse islets.Representative Hoechst 33342 DNA staining (blue) and insulin immunostaining (red) of isolated islets treated with vehicle (A), leucettine L41 (B), and leucettine L41 + LY364947 (C). Scale bars, 20 µm. (D) Insulin quantification. The islets were incubated with the selected inhibitors (alone or with LY364947) and GSIS was performed. Insulin levels were assayed as described in the Experimental Procedures section. The total insulin content was presented as a fold increase compared to the untreated control. Data are expressed as mean ± SEM. The asterisks denote p-values < *0.05, **0.01, ***0.001, compared to the control.

Fig 5: Effects of leucettines as DYRK1A inhibitors on cell cycle and cyclin D1 level.(A) Cell cycle of MIN6 cells analyzed by flow cytometry–representative histograms registered for vehicle-treated control cells and cells treated for 24 h with leucettine L41 and LY364947 (5 µM each). (B) Quantification of G0/G1, S and G2/M phase distribution based on flow cytometry analysis following the cells treatment with 5 µM leucettines. (C) Representative flow cytometry histograms for the level of cyclin D1 after treatment with investigated compounds. (Da, Db) Quantification of cyclin D1 positive cells after 24 h of treatment with each compound (based on flow cytometry analysis). (E) Western blot analysis of the level of cyclin D1 and pThr286-cyclin D1 in MIN6 cells after treatment with selected compounds. GAPDH was used as a reference protein. Data are expressed as mean ± SEM. The asterisks denote p-values < *0.05, **0.01, ***0.001 compared to the control.