Fig 2: SHMT2 promotes PTC metastasis in vitro and in vivo.A. The mRNA levels of EMT markers were determined in BHP10-3 and IHH4 cells with SHMT2 overexpression or knockdown, respectively. Data presented as mean ± SD, n = 3. *: p < 0.05, **: p < 0.01, ***: p < 0.001. B Protein levels of Vimentin were examined in BHP10-3 and IHH4 cells with SHMT2 overexpression or knockdown, respectively. C, D Migration assays were performed in PTC cell lines with SHMT2 overexpression (C) or knockdown (D). Left panel: representative images, scal bar: 50 μm. Right panel: statistical analysis of migrated cells. Data presented as mean ± SD, n = 6. ***: p < 0.001. E Lung metastasis was performed using BHP10-3 cells with SHMT2 overexpression. Left panel: western blot of SHMT2 overexpression (up) and workflow for lung metastasis (bottom). Middle panel: representative H&E images of lung collecting from each group. Scale bar: 100 μm. Right panel: statistical analysis of metastatic area and number of metastatic nodules. Data presented as mean ± SD, n = 5, Scale bar: 200 μm. **: p < 0.01, ***: p < 0.001.

Fig 3: AMPK is not necessary, nor sufficient, to induce erythroid differentiation.(A and B) Western blot analysis of AMPK signaling in K562 (A) and MEL (B) over 8-day culture in 100 nM FA as determined by phosphorylation of ACC and ULK1. (C and D) Western blot analysis of AMPK signaling and hemoglobin expression in (C) K562 and (D) MEL following 6-day treatment with vehicle, 100 nM FA, SHIN1 (1.25 μM), or AICAR (500 nM). (E and F) Cell surface GlyA or Ter119 expression as measured by flow cytometry in (E) K562 and (F) MEL following treatment with the AMPK agonist, GSK126 (10 μM). (G) RT-qPCR analysis of ALAS2 mRNA expression in AMPKa1/a2 wild-type (WT) and DKO K562 cells cultured in 2000 or 100 nM FA for 6 days. (H to K) Comparison of the fold change of 100 nM/2000 nM FA versus SHIN1/vehicle [(H) and (J)] and AICAR/vehicle versus 100 nM/2000 nM [(I) and (K)], in K562 (I) and MEL [(J) and (K)]. UDP, uridine 5′-diphosphate; GlcNAc, N-acetylglucosamine; ns, not significant. All data shown are means (±SD) of three biological replicates.

Fig 4: PKC activation is necessary for inducing erythroid differentiation.(A) RT-qPCR analysis of ALAS2 mRNA expression and surface GlyA following 6-day treatment with low FA, SHIN1, or AICAR, in combination with the PKC inhibitor, GFX (5 μM) in K562. (B) RT-qPCR analysis of Alas2 mRNA expression and surface Ter119 following 6-day treatment with low FA, SHIN1, or AICAR, in combination with GFX in MEL. (C) Western blot analysis of PKC signaling in K562 cells treated with low FA, SHIN1 (1.25 μM), or AICAR (500 nM) treatment plus PKC inhibition with GFX (5uM) for 4 days. (D and E) Selected metabolite abundance following 6-day culture of (D) K562 or (E) MEL in 2000 or 100 nM FA plus GFX (5 μM). (F) Western blot analysis of hemoglobin as treated in (D) and (E). All data shown are means (±SD) of three biological replicates except [(D) and (E)] n = 4.

Fig 5: Blocking AKT activation eliminates SHMT2-induced metastasis in thyroid cancer.A Protein levels of Vimentin were detected in BHP10-3 or IHH4 cells with SHMT2 overexpression and/or MK-2206 treatment by western blot, respectively. B Protein levels of Vimentin were examined in BHP10-3 or IHH4 cells with shSHMT2 and/or AKT transfection by western blot, respectively. C, D Migration assays were performed in BHP10-3 or IHH4 cells with SHMT2 overexpression and/or MK-2206 treatment, receptively. Left panel: representative micrographs of migrated cells. Scale bar: 50 μm. Right panel: statistical analysis of migrated cells. Data presented as mean ± SD, n = 8, n = 6. ***: p < 0.001, ns: no significant. E, F Migration assays were performed in BHP10-3 or IHH4 cells with shSHMT2 and/or AKT transfection. Left panel: representative micrographs of migrated cells. Scale bar: 50 μm. Right panel: statistical analysis of migrated cells. Data presented as mean ± SD, n = 6. **: p < 0.01, ***: p < 0.001, ns: no significant. G SHMT2 overexpressed and control BHP10-3 cells were injected in node mice via tail vein, followed by treatment with MK-2206 (5 mg/kg) for lung metastasis assays. Right panel: work follow of lung metastasis assays. Middle panel: representative histological micrographs of mice lung. Scale bar: 100 μm. Right panel: statistical analysis of metastasis tumor. Data presented as mean ± SD, n = 3. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ns: no significant.