Human neuroblastoma SK-N-SH cell line (ATCC, Manassas, Virginia, Cat#HTB-11) was maintained in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum, 1% penicillin/streptomycin (Gibco, CA, USA). Rapamycin and JNK-inhibitor SP600125 were purchased from (Calbiochem/EMD, CA, and USA).

Protein expression levels were measured using western blot analysis as mentioned previously. Briefly, cells were lysed with 1% protein lysis buffer (150 mM NaCl, 25 mM Tris-HCl pH 7.8, 1% NP-40, 10 mM NaF, 1 mM Na3VO4), and 50 µg of total proteins were separated in 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The blots were transferred to the polyvinylidene difluoride membranes and then blocked with SuperBlock T20 blocking buffer (Thermo Scientific, Rockford, IL). The membranes were incubated with SuperBlock T20 blocking buffer containing primary rabbit monoclonal against p-mTOR (Cell Signaling, Danvers, MA, Cat#5536S), mouse monoclonal p-JNK (Cell Signaling, Danvers, MA, Cat#9255), mouse monoclonal PS1 (Millipore, Bedford, MA, Cat#MAB5232), mouse monoclonal beta-Acting (Sigma, St Louis, MO, Cat#A5441) antibodies overnight at 4⁰C. The blots were developed and visualized using enhanced chemiluminescence detection system (Pierce, Rockford, IL, USA).

Cells were plated and cultured in a 24-well plate containing poly-lysine-coated coverslips (Costar, Cambridge, MA). Following treatment with DMSO or rapamycin or SP600125 and culturing at 37 °C for 24 h, the cells were washed with ice-cold TBS (10 mM Tris pH7.5, 150 mM NaCl) at room temperature for 10 min, fixed in 10% formalin for 15 min, permeabilized in 0.5 % Triton X-100 in TBS (TBST) for 10 min, blocked with 5% Goat Serum in Superblock for 45 minutes at room temperature. Then cells were stained with a primary antibodies such as rabbit monoclonal against p-mTOR (Cell Signaling Technology, Danvers, MA, Cat#5536S), mouse monoclonal p-JNK (Cell Signaling Technology, Danvers, MA, Cat#9255), mouse monoclonal PS1 (Millipore, Bedford, MA, Cat#MAB5232). After staining with primary antibody, cells were stained with a secondary antibody donkey anti-rabbit Alexa Fluor F594 (abcam, Cambridge, MA, Cat#150076), or donkey anti-mouse goat Alexa Fluor 488 (abcam, Cambridge, MA, Cat#150105), all in 1:500 in Superblock. Antibody-stained immunofluorescent samples were mounted by anti-fading aqueous mounting medium containing 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) for nuclei staining and covered by cover slips. Extensive washes with TBST (TBS + 0.05% Tween 20) were performed between each step. Omission of the primary antibody in parallel staining was included as a control to ensure no non-specific staining. Images were photographed using a Nikon EX300 microscope. Magnifications are shown in figures with scale bar.

All data was analyzed using prism software (Graphpad software Inc., La Jolla, CA, USA) and presented as means ± SEM. Comparison was made between groups by one-way ANOVA and Student–Newman–Keuls (SNK) test. A probability was considered to be significant with less than 0.05.

We have previously shown that rapamycin was effective at 50 ng/mL, 100 ng/mL and 200 ng/mL concentrations to inhibit p-mTOR in SK-N-SH cells (13). To determine whether rapamycin controls p-JNK levels, SK-N-SH cells were treated with DMSO two different concentrations of rapamycin for 24 h. Cell extracts were prepared and assayed for p-mTOR and p-JNK protein levels by Western blot analysis. Rapamycin was effective at 100 ng/mL and 200 ng/mL concentrations to inhibit p-mTOR as reported by us previously (13). We have previously reported that there was no significant variation of total mTOR protein levels with 100 ng/mL and 200 ng/mL increasing concentration of rapamycin (13). As shown in Figure 1A and Figure 1B, rapamycin decreased phosphorylation of JNK to p-JNK (p54, p46) in a concentration dependent manner.

Figure 1

Rapamycin inhibits phosphorylation of mTOR and phosphorylation of JNK in SK-N-SH cells. (A) SK-N-SH cells were treated with indicated concentrations of rapamycin for 24 h. Then 50 mg of the total cell lysates were subjected to Western blot analysis with antibodies directed against phosphor- JNK (p-JNK). Blots were developed by chemiluminescence and protein gel bands were quantified using Labworks Image Analysis Software. Positions of beta-actin (42-kDa), and p-JNK (54-kDa and 46-kDa subunits) protein bands are marked. (B) The graphs represent means±SEM of p-JNK (p54+p46) expression levels from 4 independent determinations (n=4) normalized against beta-actin. Statistical significance was denoted by asterisks. **P<0.01. (C) Immunostaining of p-mTOR (red), PS1 (green), and nucleus (blue) in SK-N-SH cells after treatment with DMSO (top panel) or 200 ng/mL rapamycin (bottom panel) for 24 h. Yellow signifies colocalization of p-mTOR and p-JNK in the merge (p-mTOR + p-JNK + DAPI) image. Immunofluorescent staining results show a significant decrease of p-mTOR (red) and p-JNK (green) in rapamycin treated cells. Scale bar =20 μm.

We examined the levels of p-JNK, and p-mTOR in SK-N-SH cells after treatment with rapamycin (200 ng/mL) for 24 h. We performed IFS with p-mTOR, and p-JNK antibodies. As shown in Figure 1C, p-mTOR and p-JNK protein levels were drastically reduced in SK-N-SH cells after treatment with rapamycin. These results suggest that rapamycin inhibits both p-mTOR and p-JNK in SK-N-SH cells under non-stressed condition.

It was previously reported that inhibition of p-JNK abolished the phosphorylation of mTOR (p-mTOR) induced by osmotic stress in cells (14). It was also demonstrated that p-JNK was responsible for phosphorylation of mTOR (p-mTOR) in vitro during osmotic stress (14). We have published previously that p-JNK (p54, p46) levels are reduced progressively in concentration dependent manner with 5 µM, 10 µM, 20 µM, 40 µM, and 60 µM concentrations of JNK-inhibitor SP600125 in SK-N-SH cells (15). We observed that 60 µM concentration of JNK-inhibitor SP600125 drastically reduced p-JNK levels in SK-N-SH cells (15). To confirm whether JNK-inhibitor also inhibits the phosphorylation of mTOR, we treated SK-N-SH cells with 60 µM concentration of SP600125 for 24 h. Cell extracts were prepared and assayed for p-mTOR and p-JNK protein levels by Western blot analysis. As shown in Figure 2A and Figure 2B, p-mTOR levels were drastically reduced by JNK-specific inhibitor SP6000125. As expected, p-JNK (p54, p46) levels were drastically reduced by SP600125 (Figure 2A, Figure 2C). Results presented in Figure 1 and Figure 2 appear to suggest that JNK and mTOR may activate each other by mutual phosphorylation in SK-N-SH cells under non-stressed condition.

Figure 2

JNK-inhibitor SP600125 inhibits phosphorylation of mTOR and phosphorylation of JNK in SK-N-SH cells. SK-NSH cells were treated with DMSO or 60 µM concentration of SP600125 in DMSO at ~80% confluence. (A) The effect of SP600125 on p-mTOR and p-JNK (p54 and p46 subunits) protein expression was tested by Western blotting using beta-actin, p-mTOR and p-JNK antibodies. 50 µg of protein were used for Western blot analysis. The blots were visualized using ECL detection system. Positions of beta-actin (42-kDa), p-JNK (54-kDa and 46-kDa subunits), and p-mTOR (250-kDa) protein bands are marked. (B) The band intensities for p-mTOR protein were quantified using Labworks Image Analysis Software. The levels of p-mTOR protein were normalized against actin protein levels and presented as means ± SEM from 2 different replicates with n=3 for each replicate. All data was analyzed using prism software (Graphpad software Inc., La Jolla, CA, USA). Comparison was made between two groups by Student–Newman–Keuls (SNK) test. Statistical significance was denoted by asterisks. * P<0.05. (C) The band intensities for two subunits p54 (54-kDa) and p46 (46-kDa) of p-JNK protein were quantified using Labworks Image Analysis Software. The levels of (p54+p46) protein were normalized against actin protein levels and presented as means ± SEM from 2 different replicates with n=3 for each replicate. Comparison was made between two groups by Student–Newman–Keuls (SNK) test. Statistical significance was denoted by asterisks. * P<0.05.

We have previously published that SP600125 reduced PS1 mRNA, PS1 protein level, and PS1/gamma-secretes activity drastically (10, 15, 16). We have also published that mTOR inhibitor rapamycin drastically inhibited PS1 mRNA, PS1 protein level, and PS1/gamma-secretes activity (13). We also performed immunofluorescent staining (IFS) of SH-N-SH cells with p-JNK and PS1 antibodies (Figure 3). We used 60 µM concentration SPS00125 for our IFS staining experiments because we have published that p-JNK, PS1mRNA, and PS1 protein were reduced drastically with 60 µM concentration of SPS00125 (15). Therefore, results presented in Figure 1, Figure 2, and Figure 3 appear to suggest that PS1 transcription and PS1 protein expression are regulated by both mTOR and JNK signaling pathways under non-stressed condition.

Figure 3

JNK-inhibitor SP600125 inhibits both p-JNK and PS1 expression in SK-N-SH cells. Immunofluorescent staining results show a significant decrease of p-JNK and PS1 in SP600125-treated SK-N-SH cells. Immunostaining of p-JNK (red), PS1 (green), and nucleus (blue) in SK-N-SH cells after treatment with DSMO (top panel) or 60 µM concentration of SP600125 (bottom panel) for 24 h. Yellow signifies colocalization of p-JNK and PS1 in the merge (PS1 + NICD + DAPI) image. Scale bar =20 μm.