Fig 2: The effect of microtubule (MT) stabilization in the chondrogenesis of SMSCs. (A) Western blot analysis of Ace-Tubulin, Col I, Col II, SOX9, SMAD3, and phosphorylated SMAD3 expression in synovial mesenchymal stem cells (SMSCs) treated with nocodazole (N) and docetaxel (D) in chondrogenic medium (CM) for 1 week. (B) Quantification of the data of (A). n = 5. (C) Immunofluorescence staining of Ace-Tubulin and F-actin. Scale bar, 50 μm. The docetaxel treatment increased the expression level of Ace-Tubulin. (D) Co-immunofluorescence staining of Col I and Col II. Scale bar, 100 μm. (E) Safranin O staining (SO) and Alcian blue (AB) staining of SMSC micro-mass culture grown in chondrogenic medium (CM) treated with nocodazole (N) and docetaxel (D) for 4 weeks. (F) pellet cultures of SMSCs in chondrogenic medium (CM) treated with nocodazole and docetaxel for 4 weeks. SO staining, toluidine blue (TB) staining, and immunohistochemical staining of Col II and Col I were performed to access the chondrogenesis of SMSC pellets. Scale bar, 100 μm. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Srag interacts with Becn1. (A) Colocalization analysis between Srag and Becn1. HeLa cells were transiently cotransfected with GFP-Srag and mCherry-Becn1, followed by confocal microscopy. Colocalizing structures were indicated in yellow (merge). Arrows indicated the signals overlapped between Srag and Becn1. The nuclei were revealed using Hoechst fluorescence. Scale bar: 5 μm. (B) Co-IP analysis between endogenous Srag and Becn1 in testis of the adult Monopterus. The lysates were immunoprecipitated with anti-Srag, anti-Becn1 antibody, or negative serum, respectively, followed by immunoblotting with the anti-Becn1 or anti-Srag antibody, respectively. Arrows indicated the Co-IP band. (C) Schematic diagram of the Monopterus Becn1 wild-type and various deletions. The conserved domains (BH3, CCD, ECD, and C-terminal) are indicated in boxes. (D) Co-IP between Srag and deletion mutants of Becn1. 293T cells were transiently cotransfected with 3xFLAG-Srag and MYC-Becn1, MYC-Becn1-BH3Δ, MYC-Becn1-CCDΔ, or MYC-Becn1-ECDΔ. After transfection for 48 h, the whole-cell lysates were extracted for Co-IP with anti-FLAG or anti-MYC antibody, and anti-MYC or anti-FLAG antibody was used for western blot, respectively. The cell lysates were examined by western blotting using the anti-MYC or anti-FLAG antibody (input). (E) Schematic diagram of the Monopterus Becn1 truncated mutants. The conserved domains (BH3, CCD, ECD, and C-terminal) are indicated in boxes. (F) Co-IP analysis showed that Srag interacted with the C terminus of Becn1. 3xFLAG-Srag was cotransfected with MYC-Becn1 1–137, MYC-Becn1 138–352, MYC-Becn1 353–447, and MYC-Becn1 138–447 into 293T cells. The cell lysates were examined by western blotting using the anti-MYC or anti-FLAG antibody (input). The lysates were immunoprecipitated with the anti-FLAG or anti-MYC antibody, followed by immunoblotting with anti-MYC and anti-FLAG antibody, respectively. 3xFLAG-Srag can interact with MYC-Becn1 353–447 and MYC-Becn1 138–447. (G) Sequence alignments of the conserved C-terminal (pink) of BECN1 in human, Monopterus, and zebrafish. (H) Sox9–Srag–Becn1 pathway in autophagy regulation. srag promoter activity is activated by its transcription factor Sox9, which is bound to the srag promoter region in vivo. Srag interacts with the C-terminal of Becn1 to promote autophagy via PI3K complex upon starvation induction.

Fig 4: Oxidative stress leads to loss of β-cell maturity markers and a concomitant increase in progenitor marker expression. To evaluate the effect of oxidative stress on β-cell maturity markers and progenitor markers, primary human islets (blue graphs) or EndoC-βH1 cells (red graphs) were treated with hydrogen peroxide (H2O2). (A) Oxidative stress induction in human islets by treatment with 200 μM H2O2 for 90 minutes leads to decreased mRNA expression levels of the β-cell-specific genes MAFA, PDX1, KIR6.2, MAFB, FOXA2, PAX6, NKX2.2, NEUROD1 and GLUT1 as measured by qPCR. (B) The level of β-cell-specific protein MAFA is decreased in human islets upon treatment with 200 μM H2O2 for 90 minutes as measured by Western blot. (C) Oxidative stress induction in EndoC-βH1 cells by treatment with 50 μM H2O2 for 24 hours and 200 μM H2O2 for 90 minutes leads to decreased mRNA expression levels of the β-cell-specific genes MAFA, PDX1, PAX4, NKX6.1, insulin, KIR6.2, MAFB, FOXA2, PAX6, NKX2.2, NEUROD1 and GLUT1 as measured by qPCR. (D) The level of β-cell-specific proteins MAFA and PDX1 is decreased in EndoC-βH1 cells upon treatment with 200 μM H2O2 for 90 minutes as measured by Western blot. (E) Oxidative stress induction in EndoC-βH1 cells by treatment with 50 μM H2O2 for 24 hours and 200 μM H2O2 for 90 minutes leads to increased mRNA expression levels of the progenitor cell-specific genes SOX9 and HES1 as measured by qPCR. (F) The level of progenitor cell-specific protein SOX9 is increased in human islets upon treatment with 200 μM H2O2 for 90 minutes as measured by Western blot. Data are presented as means ± SEM of fold change over untreated control islets (blue graphs) or EndoC-βH1 cells (red graphs). n=1-10 donors/batches; each data point represents one donor/batch. *p < 0.05, **p < 0.01, ***p < 0.0005, ****p < 0.0001 vs. untreated control islets/EndoC-βH1 cells as determined by a paired Student’s t test on the dCT values. Black circles = untreated control islets/EndoC-βH1 cells, upward-pointing triangles = H2O2-treated islets/EndoC-βH1 cells (50 μM 24h), downward-pointing triangles = H2O2-treated islets/EndoC-βH1 cells (200 μM 90min). h = hours, min = minutes, H2O2 = hydrogen peroxide.

Fig 5: Effect of MLT+Ptero on apoptosis-related proteins in colon cancer cells. HCT116, SW480, and HT29 colon cancer cells were exposed to MLT (0.5, 1 mM) and/or Ptero (20, 40 µM) for 24 h and then were subjected to Western blotting for PARP, cleaved caspse-3, Bcl-xL, NEDD9, SOX10, SOX9, and β-actin.