Fig 2: S100A8/9 stimulates the PI3K/Akt/mTOR pathway, Bcl-2 and Rictor in HUVECs. (A) S100A8/9 administration stimulates the PI3K/Akt/mTOR pathway by increasing the expression ratios of p-PI3K/t-PI3K, p-Akt/t-Akt and p-mTOR/t-mTOR. (B) The changes in Rictor and PKCα expression were detected by western blotting. (C) S100A8/9 increased protein and mRNA expression of Bcl-2. Data and error bars represent the mean ± SEM (n=3). *P<0.05, **P<0.01 vs. control group. HUVECs, human umbilical vein endothelial cell; S100A, S100 calcium binding protein A; Rictor, rapamycin-insensitive companion of mTOR; PKCα, protein kinase Cα; p-, phosphorylated; t-, total.

Fig 3: S100A8/9-induced HUVEC activation is RAGE-dependent. HUVECs were pretreated with 10 µg/ml RAGE blocking antibody or 100 nM rapamycin for 1 h prior to treating with S100A8/9 for 48 h. Total protein was harvested and subjected to western blotting. (A) RAGE antibody pretreatment blocked the PI3K/Akt/mTOR pathway, whereas rapamycin only reduced mTOR phosphorylation. (B) RAGE antibody pretreatment blocked Rictor and PKCα, whereas rapamycin made no difference. (C) The change in Rictor mRNA expression was measured by reverse transcription-quantitative PCR. Data and error bars represent the mean ± SEM (n=3). *P<0.05, **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. S100A8/9 group. RAGE, receptor for advanced glycation end products; S100A, S100 calcium binding protein A; Rictor, rapamycin-insensitive companion of mTOR; PKCα, protein kinase Cα; p-, phosphorylated; t-, total.

Fig 4: Growth factor signalling modulates reciprocal binding of mTORC2 and Nup358 to VAPB–PTPIP51 complex.(A) mTORC2 interacts with VAPB and PTPIP51. Left: HEK293T cells expressing FLAG-control (Empty vector) or FLAG-mTOR along with HA-PTPIP51 and myc-VAPB were subjected to immunoprecipitation (IP) using FLAG-specific antibodies. Presence of HA-PTPIP51 and myc-VAPB in the IP samples was examined by western analysis. Arrow indicates HA-PTPIP51 or myc-VAPB as shown; * indicates IgG heavy chain and # indicates IgG light chain cross-reaction. Right: In situ interaction of VAPB with mTOR and Rictor. Interaction between endogenous VAPB with mTOR (top) and the mTORC2-specific subunit Rictor (bottom) was confirmed by in situ PLA in HeLa cells. An increase in the number of PLA dots (yellow) was observed with mTOR or Rictor in combination with VAPB antibodies, as compared to single-antibody controls (mTOR, Rictor or VAPB alone). DNA was stained with Hoechst 33342 (blue). Scale bar, 10 µm. (B) Nup358 interacts with PTPIP51 and VAPB. Left: HEK293T cells expressing HA-PTPIP51 and myc-VAPB were subjected to endogenous Nup358 IP (Nup358 IgG IP) using specific antibodies. Presence of HA-PTPIP51 and myc-VAPB was examined by western analysis of the IP samples. Rabbit IgG (Control IgG IP) was used as control. Arrow indicates HA-PTPIP51 and * indicates IgG heavy chain cross-reaction. Right: Interaction between endogenous Nup358 and VAPB was confirmed by in situ PLA using specific antibodies in HeLa cells. Nup358 or VAPB antibody alone was used as control. DNA was stained with Hoechst 33342 (blue). Scale bar, 10 µm. (C) Reciprocal binding of Nup358 and mTORC2 to VAPB is modulated by growth factor signalling. HeLa cells were serum starved for 3 h (12 h for VAPB–Sin1 PLA experiment). Cells were later treated with (+) or without (−) insulin (1–2 nM) for 20 min. Representative images showing the extent of the interaction between Nup358 and VAPB (top first), mTOR and VAPB (top second), Rictor and VAPB (top third) or Sin1 and VAPB (top fourth) as assessed by PLA (yellow dots). DNA was stained with Hoechst 33342 (blue). Scale bar, 10 µm. Bottom panels: Quantitative data depicting the number of PLA dots per cell under the indicated scenario. Number of Nup358–VAPB PLA dots per cell (n = 98 cells for insulin-untreated and n = 97 for insulin-treated condition from three independent experiments), number of mTOR-VAPB PLA dots per cell (n = 131 cells for insulin-untreated and 125 cells for the insulin-treated condition from three independent experiments), number of Rictor-VAPB PLA dots per cell is shown (n = 67 cells for insulin-untreated and 61 cells for insulin-treated condition from three independent experiments) and Sin1-VAPB PLA dots per cell (n = 90 cells, from three independent experiments) is shown. Data are mean ± SD, Student’s t test. P values are indicated. (D) PI3 kinase activity is not required for insulin-dependent decrease in the interaction between Nup358 and VAPB. As indicated, HeLa cells were serum starved for 12 h, post which they were treated without (−) or with (+) the PI3 kinase inhibitor wortmannin (2.5 µM). The cells were then left untreated (−) or treated with 2 nM insulin for 20 min. Left: Cells were lysed and analysed for different proteins as shown. Middle: Cells were subjected to PLA for monitoring the in situ interaction between Nup358 and VAPB (yellow dots). Scale bar, 10 µm. Right: Quantitation of PLA dots per cell for different conditions is shown (n = 90 cells for each indicated condition from three independent experiments). Data are mean ± SD, Student’s t test. P values are indicated. Source data are available online for this figure.

Fig 5: mTORC2, but not mTORC1, activation is required for enhanced ER–mitochondria contacts in Nup358-downregulated cells.(A) Nup358 depletion leads to elevated mTORC2/Akt activation. HeLa cells were treated with Control (siControl) and Nup358 (siNup358) specific siRNA. Top: The cells were then analysed for the extent of mTORC2/Akt activation by western blotting using indicated antibodies. Vinculin was used as a loading control. Bottom: Quantitative representation of the relative levels of pAkt (S473) as compared to total Akt under the above-mentioned experimental conditions (n = 3 independent experiments). Data are mean ± SEM, unpaired Student’s t test. P value is indicated. (B) Increased activation of mTORC2/Akt signalling occurs in Nup358 knockout (KO) HeLa cells as compared to wild-type (WT) cells. Top: Cells were lysed and subjected to western blotting with indicated antibodies. Bottom: Quantitative data depicting the relative level of pAkt (S473) as compared to total Akt under indicated conditions (n = 3 independent experiments). Data are mean ± SEM, unpaired Student’s t test. P value is indicated. (C) Nup358 restricts mTORC2-mediated phosphorylation of Akt at S473 upon growth factor signalling. HeLa cells, transfected with indicated siRNAs, were serum starved for 3 h. The cells were then treated with (+) or without (−) insulin (1 nM for 20 min) and analysed for mTORC2/Akt activation by western blotting. Top: The extent of depletion of indicated proteins analysed by western blotting, with vinculin used as a loading control. Bottom left: The extent of Akt phosphorylation at S473 under indicated conditions was determined. Vinculin was used as a loading control. Bottom right: Quantitative representation of the relative levels of pAkt (S473) as compared to total Akt (n = 3 independent experiments). Data are mean ± SEM, unpaired Student’s t test. P values are indicated. (D) Nup358 depletion leads to mTORC1 activation, which was rescued by co-depletion of Rictor. HeLa cells, treated with indicated specific siRNAs, were analysed for the extent of protein depletion using western blotting, with mTOR being used as a loading control (left). Right top panels: mTORC2 activation was assessed by examining the phosphorylation of Akt at S473. mTORC1 activation was assessed by monitoring the phosphorylation of Akt at T308 and the mTORC1 target S6 at S235 and S236 using western blotting. Lower panels: Quantitation of the relative levels of phosphorylation of specific proteins normalised to GAPDH (for pAkt-S473) or respective total proteins as indicated (n = 3 or 4 independent experiments). Data are mean ± SEM, unpaired Student’s t test. P values are indicated. (E) Co-depletion of Rictor reverses the increase in ER–mitochondria connectivity in Nup358-depleted cells. HeLa cells were treated with specific siRNAs to deplete indicated proteins and analysed for the extent of ERMCSs present using in situ PLA (yellow) with VAPB and PTPIP51 antibodies (left). DNA was stained with Hoechst 33342 (blue). Scale bar, 10 µm. The extent of protein depletion (right top) and quantitation of PLA dots per cell (right bottom) are shown (n = 72 cells for siControl; 59 cells for siNup358; 58 cells for siNup358 + siRictor from three independent experiments). Data are mean ± SD, Student’s t test. P values are indicated. (F) The experiment was conducted as described in (E), except that instead of Rictor, as indicated, Raptor-specific siRNA was used. Left: Representative microscopic images displaying the PLA dots (yellow). DNA was stained with Hoechst 33342 (blue). Scale bar, 10 µm. The extent of protein depletion (right top) and quantitation of PLA dots per cell (right bottom) are shown (n = 90 cells for siControl; 107 cells for siNup358; 98 cells for siNup358 + siRaptor from three independent experiments). Data are mean ± SD, Student’s t test. P values are indicated. Source data are available online for this figure.