Fig 2: The eIF5A/Tsc2 axis averts DNA damage.Assessment of the effect of eIF5A silencing on DNA damage by a alkaline comet analysis (n = 3) and b TUNEL measurements in U87MG subjected to indicated conditions. Data represent mean ± SEM (n = 5). Asterisk indicates p = 0.001 compared to NS siRNA, two-sided student’s t test. c (Top) Representative images of yH2AX foci in U87MG subjected to indicated conditions. Scale bars: 20 µm. (Bottom) analysis of yH2AX foci (n = 5), asterisk indicates p = 0.00001 and 0.00001 (NN eiF5A siRNA, HA eIF5A siRNA) compared to NS siRNA, two-sided Mann–Whitney U test. The top of the box denotes Q3, the bottom of the box represents Q1; middle line denotes median; X represents mean; bottom whisker denotes minimum: 1st quartile—(1.5*IQR); top whisker denotes maximum: 3rd quartile + (1.5*IQR). d TUNEL analyses of the effects of Tsc2 knockdown and mTORC1 inhibition (by Torin 1 and 2) on DNA damage in cells replete or depleted of eIF5A under hypoxia-acidosis conditions. Data represent mean ± SEM (n = 5). An asterisk indicates p = 0.005, 0.004, 0.002, and 0.027 (eIF5A siRNA + vehicle, Tsc2 siRNA + vehicle, and eIF5A siRNA + torin2) compared to NS siRNA + Vehicle. † indicates p = 0.006 eIF5A siRNA compared to Vehicle and Tsc2 siRNA + Vehicle, two-sided student’s t test. e Top panel: representative images of yH2AX foci in U87MG cells depleted of Tsc2, and in eIF5A-replete or -depleted cells treated with mTORC1 inhibitors (Torin 1 and 2) under hypoxia-acidosis conditions. Bottom panel: analysis of yH2AX (n = 5), an asterix indicates p = 0.0005 and 0.00001 (eIF5A siRNA, tsc2 siRNA) compared to NS siRNA + vehicle, two-sided Mann–Whitney U test. Top of the box denotes Q3, the bottom of the box represents Q1; middle line denotes median; X represents mean; bottom whisker denotes minimum: 1st quartile—(1.5*IQR); top whisker denotes maximum: 3rd quartile + (1.5*IQR).

Fig 3: pH-sensing Sirt1 modulates eIF5A through deacetylation.a Representative images of eIF5A immunocytochemistry showing eIF5A subcellular localization in U87MG subjected to indicated conditions. Data represent mean ± SEM (n = 5). Scale bars: 20 µm. b Representative immunoblots of U87MG subjected to indicated conditions. NN normoxia-neutral pH, HN hypoxia neutral, HA hypoxia acidosis (n = 3). Representative c immunoblots (n = 3) and e immunocytochemistry images (n = 5) of U87MG were treated with the indicated compounds under the indicated conditions. Scale bars: 20 µm. Ex-527: Sirt1 inhibitor; AGK2: Sirt2 inhibitor; DMSO: vehicle. d Representative immunoblots of U87MG depleted of Sirt1 (n = 3). f Representative immunoblots of U87MG ribosome-density fractions treated with the indicated compounds. Mono: light monosome fraction. Poly: heavy-polysome fractions (n = 3). g Effect of Sirt1 inhibition (using ex-527) on Tsc2 mRNA subcellular localization under hypoxia-acidosis conditions. NS nonsilencing. Data represent mean ± SEM (n = 3). An asterisk indicates p < 0.05 compared with DMSO vehicle, two-sided student’s t test. h Representative immunoblots of U87MG treated with the Sirt1 inhibitor ex-527 (n = 3). i Summary model of the Sirt1/eIF5A/Tsc2/mTORC1 pathway that enables metabolic depression and proliferative inhibition during anaerobic acidosis to prevent DNA damage.

Fig 4: ATP13A2 regulates TSC2 through MYCBP2-induced ubiquitination.(a) HeLa cells were fixed and analysed by proximity ligation assay (PLA) using primary antibodies against ATP13A2 and MYCBP2. Cells were imaged by epifluorescence microscopy (scale bar, 10 µM). PLA negative controls are shown in the supplements. (b) Endogenous ATP13A2 was immunoprecipitated. Whole-cell lysate and immunoprecipitates were blotted against ATP13A2, MYCBP2, TSC2 and actin. (c) Control and ATP13A2-knockdown HeLa cells were lysed and blotted against phosphoSer1378-TSC2, phosphoThr1462-TSC2, total-TSC2 and actin. (d) Primary cultures of mouse neurons were transduced with Control or ATP13A2 lentiviral shRNAs for 5 days and used for western blotting against TSC2 and actin. (e) HeLa cells were transfected with empty vector, ATP13A2 WT, ATP13A2 delC mutant or ATP13A2 i16 mutant for 24 h. Cells were collected and cell lysates blotted for TSC2 and actin. (f) HeLa cells were transfected with Control or ATP13A2 siRNAs for 4 days. In the last day of transfection, cells were treated with 50 µg ml-1 cycloheximide for the indicated time points (after a pre-treatment of 2 h). Cell lysates were blotted against TSC2 and actin. Densitometric quantification of the bands was performed and the normalized data (TSC2 levels to actin levels) of three independent experiments is plotted in the graph, which represents mean±s.d. *P<0.05 (two-tailed paired Student's t-test). (g) HeLa cells, transfected with two rounds of either Control or ATP13A2 siRNA (50 nM) alone or in combination with MYCBP2 siRNA (50 nM) for 4 days, were further transfected with FLAG-TSC2 and HA-Ubiquitin (1:3 ratio) for 24 h. MG132 (10 µM) was added for the last 5 h. Lysates were used for TSC2 immunoprecipitation. Inputs and immunoprecipitates were blotted against HA, TSC2 and actin. (h) Lysates of HeLa cells transfected with Control or ATP13A2 siRNA alone or in combination with MYCBP2 siRNA (as described before) were blotted against TSC2, phospho p70S6K, total p70S6K, LC3 and actin. The graph represents the mean±s.e.m. of at least three independent experiments. *P<0.05; #P<0.05, ##P<0.01 (two-tailed paired Student's t-test). (i) Control and ATP13A2-knockdown HeLa cells were immunoprecipitated for TSC2. Inputs and immunoprecipitates were blotted against TSC2, MYCBP2 and actin.

Fig 5: Schematic illustrating the role of hTERP in the regulation of TSC2 phosphorylation at Ser1387 by AMPK.