Met dose selection

In this study, an MTT assay was used to select the maximum Met concentration with the minimum cytotoxicity (Fig. 1). Data indicated that the incubation of MSCs with different concentrations of Met, 1, 5, 15, 25, 50, and 75 µM for 48 h led to enhanced viability compared to the non-treated control (p < 0.01; Fig. 1). No statistically significant differences were obtained in terms of MSC survival between the Met-treated groups (p > 0.05). According to data from current experiments and previous studies, 15 µM Met was used for subsequent analyses. To inhibit autophagy, 3 µM 3-MA was used in different assays according to previous data.

Fig. 1

Monitoring MSC survival rate incubated with different doses of Met, 1, 5, 15, 20, 50, and 75 µM, after 48 h. Data indicated a significant increase in MSC viability in Met-treated groups compared to the control cells (n = 8). In this study, 15 µM Met was selected for other analyses. Data are expressed as mean ± SD. One-way ANOVA with Tukey post hoc. **p < 0.01

Autophagy stimulation promoted TNT formation in MSCs

To investigate the relationship between autophagy response, cell morphology and TNT formation, MSCs were treated with 15 µM Met and 3 µM 3-MA for 48 h and examined using bright-field and SEM images (Fig. 2A). Bright-field images indicated the formation of intercellular TNT connections in MSCs after being exposed to autophagy modulators (red arrowheads; Fig. 2A). Based on the results, Met statistically increased the number and length of TNTs compared to the control and 3-MA treated MSCs (p < 0.01; Fig. 2A). Data confirmed several dense granules, or nodules inside the TNTs (yellow arrows), indicating active transportation between the MSCs. In contrast to the Met-treated cells, the number and length of TNTs were statistically reduced in the 3-MA group with faint cytoskeletal remodeling, leading to almost rounded margins and a reduced number of cellular projections (TNTs, filopodia, and lamellipodia). These findings were also confirmed by SEM ultrastructural images (Fig. 2B). SEM images showed the TNT compartments between 2D cultured MSCs in control and Met-treated groups (yellow arrows). TNT structures were like bridges and strands which signified the active inter-MSC connections after autophagy stimulation (Fig. 2B). In the 3-MA treated MSCs, cells exhibited round-shape morphologies with reduced TNT units. These data indicate that the stimulation of autophagy can promote the MSC-to-MSC juxtacrine interaction via the activation of cytoskeletal remodeling and the formation of TNTs. In contrast, autophagy inhibition reduced TNT structures and cellular projections, leading to the reduction of physical contact between the cells.

Fig. 2

TNT formation and projections in MSCs exposed to 15 µM Met, and 3 µM 3-MA after 48 h. Bright-field images (A), and SEM images (B). Data indicated the formation of different cell projection types including TNTs (red arrows) filopodia, and lamellipodia (yellow arrows) in control MSCs (A). Data indicated that the number and length of TNTs (red arrowheads) were increased in Met-treated MSCs compared to the control and 3-MA groups. In Met-treated MSCs, intercellular transfer of cargo can be detected inside the TNTs (yellow arrows). Data showed the lack of significant differences in terms of TNT length and number in 3-MA treated MSCs compared to the control group. SEM images indicated the existence of TNT links between the control and Met-treated MSCs (B; yellow arrows). In the presence of 3-MA, MSCs lose the ability to produce TNT bridges. Data are expressed as mean ± SD. One-way ANOVA with Tukey post hoc. **p < 0.01; ***p < 0.001

Met can induce autophagy flux inside the MSCs

Autophagic flux was monitored in different groups using LysoTracker, an acidotropic dye (Fig. 3A). This compound is pH-dependent fluorescence and indicates the fusion of autophagosomes with lysosomes. According to our data, Met increased the intensity and number of intracellular LysoTracker green particles compared to the non-treated control MSCs. In the 3-MA treated group, intracellular LysoTracker green particles are barely detectable, indicating the reduction or inhibition of autophagy flux. Even, these features were reduced prominently in 3-MA groups less than that of control MSCs. These data indicate that Met can stimulate the autophagy flux and autophagolysosome formation while 3-MA decreases the fusion of lysosomes with autophagosome.

Fig. 3

Monitoring autophagic process in 15 µM Met, and 3 µM 3-MA treated MSCs using LysoTracker staining after 48 h (A). Immunofluorescence (IF) images indicated the increase of acidic compartments and autophagy flux in the presence of Met compared to the control group. The number and intensity of green LysoTracker particles were at the minimum levels in 3-MA treated MSCs related to control cells. Mitochondrial membrane integrity was studied using Green Mito Tracker staining (B). IF showed higher fluorescence intensity and an increase of fluorescent particles in Met-treated MSCs compared to control and 3-MA groups. These features indicated an appropriate ΔΨ feature and mitochondrial number inside the MSCs after being exposed to Met. Flow cytometry analysis of mitochondria internalization in MSCs after treatment with Met and 3-MA. MSCs from different groups were incubated with Green Mito Tracker-stained mitochondria. Despite the increase of mitochondria uptake in 3-MA treated cells, no statistically significant differences were achieved in terms of fluorescence intensity. Data are expressed as mean ± SD. One-way ANOVA with Tukey post hoc

Met increased mitochondrial functional activity in MSCs

To investigate the relationship between stimulation/inhibition of autophagy and mitochondrial membrane integrity, MitoTracker Green staining was used (Fig. 3B). Data indicated that the intensity of MitoTracker Green+ particles was increased in Met-treated MSCs compared to the control and 3-MA groups. While a relatively similar fluorescence intensity pattern was achieved for the control and 3-MA groups (Fig. 3B). These data demonstrate that higher intensity staining of MSCs with MitoTracker Green is associated with increased mitochondrial content and respiration rate. Based on our data, the inhibition of autophagy did not alter the functional activity of mitochondria compared to the non-treated MSCs.

Autophagy inhibition can alter mitochondrial internalization in MSCs

To investigate whether autophagy inhibition/stimulation is involved in mitochondrial reception and internalization, the isolated mitochondria were pre-labeled with MitoTracker Green, added to the supernatant, and internalization rate was analyzed using flow cytometry after 24 h. Data indicated that the number of internalized mitochondrial particles was relatively similar in the control and Met-treated MSCs in which 33.6 ± 1.1, and 37.5 ± 1.2% of MSCs were MitoTracker Green positive, respectively (Fig. 3C). These features were increased in 3-MA-treated MSCs and reached 43.6 ± 10.5%. Despite the lack of significant changes between the 3-MA-treated cells with control and Met groups, it can be said that probably the number and rate of mitochondrial internalization increased after autophagy inhibition.

Met and 3-MA changed the protein levels related to TNT assembly

In this study, western blotting was used to monitor protein levels of BCLN1, LC3-II/LC3-I ratio, and p62 in MSCs exposed to 3-MA and Met (Fig. 4A and B). According to the data, despite the reduction of BCLN1 in 3-MA treated MSCs, no statistically significant differences were obtained compared to the control and Met groups (p > 0.05). Protein levels of p62 and LC3-II/LC3-I ratio were not statistically significant in Met and 3-MA groups compared to the non-treated MSCs (p > 0.05). Data confirmed the lack of significant changes in protein levels of Miro1 and 2 in treated MSCs related to the control group (p > 0.05; Fig. 4A and B). Of note, protein levels of factors involved in TNT assembly were significantly changed in the presence of 3-MA as compared to the control MSCs (p < 0.05; Fig. 4A and B). Data indicated a significant reduction of GTPase Rab8 and p-FAK in MSCs exposed to 3-MA after 48 h. These data indicated that autophagy inhibitor can reduce protein levels of factors involved in TNT assembly and vesicle transportation.

Fig. 4

Western blotting (A and B). Data revealed the lack of significant differences in protein levels of autophagy machinery (BCLN1, LC3-II/LC3-I ratio, and p62) in the presence of 15 µM Met, and 3 µM 3-MA after 48 h. Protein levels of Miro1 and 2 remained unchanged in the presence of Met, and 3-MA. The levels of Rab8 and p-FAK related to TNT assembly and vesicle transport were reduced significantly in 3-MA-treated MSCs. Data are expressed as mean ± SD. One-way ANOVA and Tukey post hoc test (n = 3). *p < 0.05; **p < 0.01

Met and 3-MA altered the expression of genes related to the autophagy signaling pathway

To monitor the expression of ATGs, the transcription of different genes was monitored using PCR array analysis (Table 1). Data showed that the expression of specific genes was changed in the presence of 15 µM Met and 3 µM 3-MA after 48 h. The expression of genes related to vacuole formation, ATG12 (3.31-fold), ATG16L1 (2.29-fold), ATG4C (2.73-fold), ATG5 (2.86-fold), ATG9B (2.69-fold) were up-regulated in Met-treated MSCs. We noted that the expression of other genes from the same signaling transduction such as ATG4A (2.89-fold), ATG4B (4.11-fold), ATG9A (2.89-fold), GABARAP (2.33-fold), GABARAPL2 (2.87-fold), MAP1LC3B (4.11-fold) was also induced MSCs after being exposed to the 3-MA. Based on the data, ATG4C, ATG4A, and ATG4B with proteolytic activity can foster the autophagy flux via interaction with GABARAP to fuse autophagosomes with lysosomes (Supplementary Table 1). According to the obtained data, 3-MA and Met can differently stimulate the expression of genes related to protein transport signaling transduction pathway in which the expression of ATG4A, ATG4B, ATG9A, GABARAP, GABARAPL2, and RAB24 (5.03-fold), ATG10 (1468.37-fold) was induced after 3-MA treatment whereas Met up-regulated the expression of ATG16L1, ATG4C, and ATG10 (467.23-fold). Data confirmed that the expression of shared genes between the autophagy and apoptosis pathway such as ATG12, ATG5, BAD (11.22-fold), EIF2AK3 (8.16-fold), HDAC1 (10.76-fold), HTT (107.49-fold), PTEN (9.37-fold) and TP53 (16.66-fold) was up-regulated in Met-treated MSCs compared to the non-treated control MSCs. Of note, CXCR4 (6.02-fold), TNF (4.06-fold), MAPK8 (2.64-fold), and ACTB (2.28) genes were up-regulated by 3-MA in MSCs from the same signaling pathway. We noted that both Met and 3-MA triggered the CASP3 (4.68-, and 3.97-fold), INS (14.01, and 9.06-fold), SNCA (7.77, and 6.19-fold), SQSTM1 (18.10, and 9-fold), associated with co-regulators of apoptosis and autophagy signaling transduction pathway. The transcription of genes related to intracellular signals like CTSD (4.16-fold), PIK3R4 (19.13-fold), RPS6KB1 (4.05-fold), and TMEM74 (4.85-fold) was also stimulated by Met. A similar trend was achieved in terms of CTSS (4.56-fold) and PIK3C3 (3.61-fold) expression in the 3-MA group. However, Met and 3-MA can activate common genes the ULK2 (11.54-, and 33.13-fold), and GAA (19.81-, and 3.25-fold), respectively from the same signaling axis. We found that the activation of genes associated with cell cycle such as PTEN, TP53, and RB1 (3.13-fold) after the modulation of autophagy by Met. Interestingly, the activity of genes related to chaperone-mediated autophagy (CMA) namely HSPA8 (26579.01-, and 7.89-fold) was induced in both Met and 3-MA groups. However, these values were more prominent in the Met-treated MSCs compared to the 3-MA group. The expression of EIF2AK3 (8.16-fold) belonging to pathogen-mediated autophagy response signaling was changed in the presence of Met while 3-MA was neutral to change the expression of this gene. These data demonstrate that Met and 3-MA can modulate the expression of various genes related to different signaling transduction pathways for autophagic activity.

Table 1 PCR array analysis of human autophagy signaling pathway in MSCs exposed to Met and 3-MAAutophagy response modulation affected the expression of genes related to the wnt signaling pathway

To assess whether the inhibition/stimulation of autophagy can influence the Wnt signaling pathway, PCR array analysis was done (Table 2). Data indicated that the transcription of different genes related to several signaling transduction axes was altered in MSCs exposed to 15 µM Met and 3 µM 3-MA after 48 h. According to the data, treatment with Met can activate specific genes such as FZD9 (2.08-fold), CTNNB1 (2.02-fold), and SFRP4 (2.02-fold) belonging to the Canonical Wnt signaling pathway (Supplementary Table 2, Supplementary Fig. 1, and supplementary data File 1). Along with these changes, the expression of WNT2 (3.13.-fold), 7 A (3.68-fold), and 8 A (4.92-fold), SFRP1 (2.46-fold), NKD1 (2.41-fold), FZD8 (2.26-fold), DVL1 (3.83-fold), LRP5 (3.86-fold), DKK3 (2.03-fold), DIXDC1 (2.19-fold) from same signaling transduction pathway was increased significantly in 3-MA-treated group compared to the Met and control cells. Met had a potential to increase the expression of DAAM1 (2.29-fold) from the planar cell polarity signaling transduction pathway while 3-MA stimulated the transcription of NKD1, WNT2 (3.13.-fold), DVL1 (3.83-fold), VANGL2 (2.03-fold), WNT7A (3.68-fold), WNT8A (4.92-fold), and RHOU (2.25-fold) from the same molecular cascade. On the other hand, Wnt negative regulators such as KREMEN1 (3.83-fold), DKK3 (2.03-fold), NKD1, and SFRP1 (2.46-fold) were activated in the presence of 3 µM 3-MA. Our data also indicated that Met can activate SFRP4 (2.02-fold), CXXC4 (2.82-fold), and WNT16 (2.39-fold) from the same signaling axis compared to the control MSCs. Both Met and 3-MA triggered the FBXW11 (2.69-, and 4.62-fold), and TLE1 (2.71, and 5.65-fold), respectively from the same pathway. We found that Met can affect the MSC fate via the expression of CTNNB1 (2.02-fold). Interestingly, the transcription of VANGL2 belonging to tissue polarity was also increased in 3-MA-treated cells. Wnt signaling target genes such as DAB2, MYC, JUN, CCN4, and MMP7 were also altered in the presence of Met and 3-MA. Data indicated that the expression of genes belonging to cell growth and proliferation such as MYC (2.34-fold), JUN (2.6-fold), CTNNB1 (2.02-fold), DAB2 (2.21-fold), and MMP-7 (11.76-fold) was up-regulated in Met-treated MSCs compared to the non-treated control MSCs. Likewise, 3-MA induced the expression of CCN4 (2.82), LRP5, MMP7 (2.13-fold), and BCL9 (2.79-fold) in MSCs from the same signaling pathway. LRP5 can also control the cell migration capacity. We noted that Met can alter the expression of several genes related to cell cycle (MYC, JUN, and CTNNB1), homeostasis (MYC, and JUN), and activity signature genes (MYC) compared to the non-treated MSCs. 3-MA induced the expression of FBXW4 (2.23-fold) gene related to the Ubl conjugation pathway.

Table 2 PCR array analysis of human wnt signaling pathway in MSCs exposed to Met and 3-MAProtein array analysis

The protein levels of pro- and anti-apoptotic factors were measured using a human apoptosis antibody array in MSCs after being exposed to Met and 3-MA (Fig. 5). Based on the data, incubation of MSCs with Met increased protein levels of anti-apoptosis factors such as Bcl-2 (3.37-fold), Bcl-w (3.41-fold), IGF-I (2.53-fold), Livin (2.17-fold), p27 (2.04-fold), and XIAP (2.05-fold) compared to the non-treated control MSCs (Supplementary Table 3, supplementary Fig. 2, and Supplementary Data File 2). Compared to non-treated control MSCs, pro-apoptotic factors, including Bad (2.63-fold), Bax (2.27-fold), BID (3.67-fold), BIM (4.85-fold), Caspase-3 (4.77-fold), CD40 (2.15-fold), DR6 (2.07-fold), FasL (2.41-fold), HTRA (2.16-fold), IGFBP-4 (2.14-fold), IGFBP-6 (2.25-fold), IGF1-sR (2.26-fold), sTNF-R1 (2.44-fold), sTNF-R2 (2.5-fold), TNF-α (3.23-fold), TNF-β (3-fold), TRAILR-1 (2.18-fold), TRAILR-2 (2.08-fold), and TRAILR-4 (2.25-fold) were also elevated in the presence of Met. Data showed the lack of changes in these proteins in 3-MA-treated MSCs. These data showed that Met, but not 3-MA, altered protein levels of the apoptosis signaling pathway, either pro-apoptotic or anti-apoptotic factors.

Fig. 5

Measuring protein levels of 43 factors (pro- and anti-apoptotic proteins) by human apoptosis antibody array in MSCs exposed to 15 µM Met, and 3 µM 3-MA after 48 h. Data were obtained from three pooled samples

Fatty acid profile was changed after the modulation of autophagy in MSCs

In the current experiment, the fatty acid profile was studied using GS. The level of PUFA (Linoleate18:20, MUFA (Oleate 18:1), and SFA (Myristate14:0, Palmitate 16:0, Stearate 18:0 and Pentadecanoic acid 15:0) was measured in the presence of Met and 3-MA (Fig. 6). Data showed the increase of PUFA + MUFA/SFA ratio in Met-treated MSCs (161.45) compared to control and 3-MA treated MSCs. The PUFA + MUFA/SFA ratios were 76.12, and 95.51, respectively in the control and 3-MA-treated MSCs. These data indicate that autophagy modulators can alter the MSC fatty acid profile.

Fig. 6

Fatty acid profile analysis using gas chromatography. Data indicated the increase of PUFA + MUFA/SFA ratio in Met-treated MSCs compared to control and 3-MA groups. Data were obtained from three pooled samples