Fig 2: Schematic illustration of potential targets of beneficial tianeptine action in LPS-stimulated microglial cells. Abbreviations: TIA—tianeptine; LPS—lipopolysaccharide; TLR4—Toll-like receptor 4; NLRP3—the NOD-like receptor pyrin-containing 3 inflammasome; ASC—apoptosis-associated speck-like protein containing a caspase recruitment domain; ERK 1/2, extracellular signal-regulated kinases; NF-?B—nuclear factor; IL—interleukin; casp—caspase; ROS—reactive oxygen species; TNF—tumor necrosis factor; CCL2—chemokine.

Fig 3: Knockdown of CHL1 reduced the phosphorylation levels of ERK and AKT. Western blot was used to analyze the levels of pAkt and pErk in three cell lines after treatment with vehicle control, negative control and CHL1 siRNA for 48 h. pAkt and pErk protein levels in U251 (A), SHG44 (B) and U-87 MG cells (C) were presented. GAPDH was used as a loading control. The data were expressed as the means ± SEM from 3 independent experiments (*p < 0.05 and **p < 0.01 vs. both vehicle control and control siRNA; independent Students t-test).

Fig 4: Sorafenib inhibits MAPK signaling in MV4-11 but activates it in U937 cells. Immunoblot analysis of phosphorylated C-Raf and Erk using 48 h sorafenib (50, 100, 200 nM) treated or untreated MV4-11 (A) and 72 h sorafenib (200 nM) treated or untreated U937 cells (B). Total C-Raf, Erk1/2 and GAPDH served as loading controls. (C) Flow cytometric analysis of BrdU-Alexa Fluor 488/propidium iodide (PI)-stained cells. U937 cells were treated or untreated with sorafenib for 48 h. The percentage of cells in S-phase as well as G1/G2 is displayed.

Fig 5: Effect of shGnb2l1 icv injection on ERK/CREB pathway.(a) shGnb2l1 icv injection significantly decreased the expression of pERK1/2 protein in the CA2 of HP in acquisition and maintenance. The brown cells represent the pERK1/2 positive cells. The scale bar = 0.5 mm. (a,f) saline + shNC mouse; (b,g) saline + shGnb2l1 mouse; (c,h) morphine + shNC mouse; (d,i) morphine + shGnb2l1 mouse; (e) Enlarged, from (h) shows that pyramidal neurons (Pyr) in the DG of the HP are strongly positive for pERK1/2; (a–d,f–i) The scale bar = 2 mm; (e) The scale bar = 0.5 mm.(b) shGnb2l1 icv injection significantly decreased the expression of pCREB protein in the CA1 of HP in acquisition and maintenance. The brown cells represent the pCREB positive cells in the DAB immunostaining. The scale bar = 0.5 mm. (a,f) saline + shNC mouse; (b,g) saline + shGnb2l1 mouse; (c,h) morphine + shNC mouse; (d,i) morphine + shGnb2l1 mouse; (e) Enlarged, from (c) shows that pyramidal neurons (Pyr) in the CA1of the HP are strongly positive for pCREB; (a–d,f–i) The scale bar = 2 mm; (e) The scale bar = 0.5 mm. (c) Representative immunoblot showing the molecular weights of the proteins used to quantify level of ERK1/2 (44/42 kDa), pERK1/2 (43 kDa), CREB (45 kDa) and pCREB (43 kDa) in HP. Signals were normalized to ß-actin (43 kDa). The level of ERK1/2 and CREB in HP were not changed after morphine treatment in acquisition and maintenance experiment. An increase in pERK1/2 and pCREB in HP could be detected in both experiments. shGnb2l1icv injection significantly decreased the protein expressions of pERK1/2 and pCREB in HP; (d) Comparison of the ratio of the pERK/ERK band between the experimental groups, *p < 0.05 compared with saline + shNC group (n = 5); #p < 0.05 compared with morphine + shNC group (n = 5). (e) Comparison of the ratio of the pCREB/CREB band between the experimental groups, *p < 0.05 compared with saline + shNC group (n = 5); #p < 0.05 compared with morphine + shNC group (n = 5).