Journal: Nature Communications

Article Title: A translational program that suppresses metabolism to shield the genome

doi: 10.1038/s41467-020-19602-2

Figure Lengend Snippet: a Workflow schematic of MATRIX, an unbiased, high-throughput platform that measures the activity of translational assets. MATRIX analysis of differential translation-factor utilization in human cells (U87MG) exposed to hypoxia acidosis (1% O 2 , pH 6.0, 24 h) compared to b basal (21% O 2 , pH 7.4, 24 h) and c hypoxia-neutral pH (1% O 2 , pH 7.4, 24 h) conditions, using the ratio of heavy polysome to free abundance as the readout. Hypoxia acidosis-activated translation factors (dark-blue bars), basal-activated translation factors (red bars), and hypoxia-neutral pH-activated translation factors (light-blue bars). d Representative immunoblots of U87MG ribosome-density fractions from indicated conditions. Mono: monosome fraction. Poly: polysome fractions. f Representative immunoblots of U87MG were subjected to indicated treatments. e Representative images of eIF5A immunocytochemistry in human (WI-38, U87MG) and mouse (NIH/3T3) cell lines subjected to indicated treatments. Data represent mean ± SEM ( n = 3). Scale bars: 20 μm.

Article Snippet: Antibodies used: KI-67 (SantaCruz, sc-23800), P21 (Proteintech, 10355-1-AP), EIF5A (abcam, ab32443), acetylated EIF5A (Lys47) (Boster Bio, P01727), CYR61 (Proteintech, 26689-1-AP), and PAI1 (Proteintech, 13801-1-AP).

Techniques: High Throughput Screening Assay, Activity Assay, Western Blot, Immunocytochemistry

Journal: Nature Communications

Article Title: A translational program that suppresses metabolism to shield the genome

doi: 10.1038/s41467-020-19602-2

Figure Lengend Snippet: a Relative ATP utilization, b transcriptional intensity, and c translational intensity in U87MG replete or depleted of eIF5A under hypoxia acidosis conditions. a , b NS nonsilencing. Data represent mean ± SEM ( n = 3). An asterisk indicates p = 0.024, 0.028 ( a , b ) compared to NS siRNA, two-sided student’s t test. d Ki-67 and p21 immunocytochemistry, e DNA replication (BrdU staining), and f Congo red staining for A bodies in U87MG replete or depleted of eIF5A under hypoxia acidosis conditions (72 h) ( n = 5). Quantitation in ( d ) and ( e ) represents mean ± SEM ( n = 7, 5). Scale bars: 20 μm. g Effect of eIF5A knockdown on steady-state cell numbers in U87MG subjected to indicated treatments. NS nonsilencing. Data represent mean ± SEM ( n = 3). An asterisk indicates p = 0.035 compared to NS siRNA, two-sided student’s t test. h Effect of eIF5A knockdown on steady-state cell numbers in human and mouse cell lines under hypoxia acidosis conditions. NS nonsilencing. Data represent mean ± SEM ( n = 3). An asterisk indicates p = 0.001, 0.001, 0.047, and 0.025 (U87MG, MCF7, HCT116, and WI-38) compared to the corresponding NS siRNA, two-sided student’s t test. i Representative ultrasound images and j tumor volume measurements of mouse xenograft tumor-formation assays using MCF7 replete or depleted of eIF5A and pretreated with hypoxia acidosis for 72 h. Data represent mean ± SEM ( n = 6). An asterisk indicates p = 0.021, 0.031, 0.004, and 0.014 (days 7, 14, 17, and 21) compared to NS siRNA, two-sided student’s t test.

Article Snippet: Antibodies used: KI-67 (SantaCruz, sc-23800), P21 (Proteintech, 10355-1-AP), EIF5A (abcam, ab32443), acetylated EIF5A (Lys47) (Boster Bio, P01727), CYR61 (Proteintech, 26689-1-AP), and PAI1 (Proteintech, 13801-1-AP).

Techniques: Immunocytochemistry, BrdU Staining, Staining, Quantitation Assay, Knockdown

Journal: Nature Communications

Article Title: A translational program that suppresses metabolism to shield the genome

doi: 10.1038/s41467-020-19602-2

Figure Lengend Snippet: a – c Representative immunoblots of U87MG replete or depleted of eIF5A under hypoxia-acidosis conditions ( n = 3). d Representative immunoblots of U87MG replete or depleted of Tsc2 under hypoxia-acidosis conditions ( n = 3). e Effects of eIF5A and Tsc2 knockdown on cellular ATP levels under hypoxia-acidosis conditions. NS nonsilencing. Data represent mean ± SEM ( n = 3). An asterisk indicates p = 0.024 and 0.012 (eIF5A siRNA and Tsc2 siRNA) compared to NS siRNA, two-sided student’s t test. f Representative images of Ki-67 immunocytochemistry in U87MG (left panel) and MCF7 (right panel) replete or depleted of Tsc2 under hypoxia-acidosis conditions. NS nonsilencing. Data represent mean ± SEM ( n = 5). Scale bars: 20 μm. Effect of Tsc2 knockdown on U87MG g cell number, h transcriptional intensity, and i Congo red staining for A bodies. Scale bars: 20 μm. NS nonsilencing. Data represent mean ± SEM ( n = 3 ( g , h ); n = 5 ( i )). An asterisk indicates p = 0.046 and 0.042, p = 0.007 and 0.002 ( g , h eIF5A siRNA and Tsc2 siRNA) compared to NS siRNA, two-sided student’s t test. j Representative images of Ki-67 immunocytochemistry in U87MG replete or depleted of eIF5A and treated with mTORC1 inhibitors rapamycin and everolimus under hypoxia-acidosis conditions. NS nonsilencing. Data represent mean ± SEM ( n = 5). Scale bars: 20 μm. k Effect of mTORC1 inhibition on eIF5A-replete or -depleted U87MG cell number under hypoxia-acidosis conditions. NS nonsilencing. Data represent mean ± SEM ( n = 6). An asterisk indicates p = 0.026 and 0.022 (Rapamycin + eIF5A siRNA, Everolimus + eIF5A siRNA) compared to Vehicle + eIF5A siRNA, two-sided student’s t test.

Article Snippet: Antibodies used: KI-67 (SantaCruz, sc-23800), P21 (Proteintech, 10355-1-AP), EIF5A (abcam, ab32443), acetylated EIF5A (Lys47) (Boster Bio, P01727), CYR61 (Proteintech, 26689-1-AP), and PAI1 (Proteintech, 13801-1-AP).

Techniques: Western Blot, Knockdown, Immunocytochemistry, Staining, Inhibition

Journal: Nature Communications

Article Title: A translational program that suppresses metabolism to shield the genome

doi: 10.1038/s41467-020-19602-2

Figure Lengend Snippet: 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).

Article Snippet: Antibodies used: KI-67 (SantaCruz, sc-23800), P21 (Proteintech, 10355-1-AP), EIF5A (abcam, ab32443), acetylated EIF5A (Lys47) (Boster Bio, P01727), CYR61 (Proteintech, 26689-1-AP), and PAI1 (Proteintech, 13801-1-AP).

Techniques: TUNEL Assay, MANN-WHITNEY, Whisker Assay, Knockdown, Inhibition

Journal: Nature Communications

Article Title: A translational program that suppresses metabolism to shield the genome

doi: 10.1038/s41467-020-19602-2

Figure Lengend Snippet: a Co-immunoprecipitated mRNA levels of eIF5A-regulated and nonregulated mRNAs relative to IgG isotype control pulldown. Data represent mean ± SEM ( n = 3). An asterisk indicates p = 0.005, 0.031, and 0.001 (Tsc2, c-Jun, and Sdc4) compared to IgG control, two-sided student’s t test. b Effect of eIF5A knockdown on mRNA subcellular localization of indicated mRNAs under hypoxia acidosis conditions. NS nonsilencing. Data represent mean ± SEM ( n = 3). An asterisk indicates p = 0.007, 0.041, and 0.027 (Tsc2, c-Jun, and Sdc4) eIF5A siRNA compared to NS siRNA, two-sided student’s t test. c mRNA fluorescent in situ hybridization. NS nonsilencing. One-third exposure level was used for Tsc2 mRNA FISH under eIF5A siRNA conditions relative to all other conditions to avoid signal saturation ( n = 5). Scale bars: 20 μm. d Effect of eIF5A knockdown on translation efficiencies of indicated mRNAs under hypoxia-acidosis conditions. NS nonsilencing. Data represent mean ± SEM ( n = 3). An asterisk indicates p = 0.0.004, 0.002, and 0.006 (Tsc2, c-Jun, and Sdc4) eIF5A siRNA compared to NS siRNA, two-sided student’s t test. e Effect of leptomycin B treatment on eIF5A protein subcellular localization in U87MG under indicated conditions. Vehicle: DMSO ( n = 5). Scale bars: 20 μm. f Effect of leptomycin B treatment on Tsc2 mRNA subcellular localization under hypoxia-acidosis conditions. Vehicle (Veh): DMSO. Data represent mean ± SEM ( n = 5). An asterisk indicates p = 0.015 compared to DMSO vehicle, two-sided student’s t test. g Representative immunoblot of Tsc2 protein levels in U87MG treated with leptomycin B ( n = 3).

Article Snippet: Antibodies used: KI-67 (SantaCruz, sc-23800), P21 (Proteintech, 10355-1-AP), EIF5A (abcam, ab32443), acetylated EIF5A (Lys47) (Boster Bio, P01727), CYR61 (Proteintech, 26689-1-AP), and PAI1 (Proteintech, 13801-1-AP).

Techniques: Immunoprecipitation, Control, Knockdown, In Situ Hybridization, Western Blot

Journal: Nature Communications

Article Title: A translational program that suppresses metabolism to shield the genome

doi: 10.1038/s41467-020-19602-2

Figure Lengend Snippet: 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.

Article Snippet: Antibodies used: KI-67 (SantaCruz, sc-23800), P21 (Proteintech, 10355-1-AP), EIF5A (abcam, ab32443), acetylated EIF5A (Lys47) (Boster Bio, P01727), CYR61 (Proteintech, 26689-1-AP), and PAI1 (Proteintech, 13801-1-AP).

Techniques: Immunocytochemistry, Western Blot, Inhibition

Journal: Ecotoxicology and environmental safety

Article Title: PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

doi: 10.1016/j.ecoenv.2023.114772

Figure Lengend Snippet: Fig. 5. NaF exposure induces elevated phos phorylation of PRKAA1 expression in vivo and in vitro. (A) The hierarchical cluster heatmap of phosphorylated proteins in rat hippocampus. (B) Statistics of different phosphorylation sites and proteins in rat hippocampus. (C) Volcano plot of differentially phosphorylated proteins in rat hippocampus. The criteria for significance were set at logarithmic fold change (log2 FC) of > 1.2 or < 0.8 (P < 0.05). (D) Phosphorylation of PRKAA1 protein in rat hippocampus. (E-F) Representative western blot and relative quan tifications of PRKAA1 and p-PRKAA1 in rat hippocampus. (G-H) Representative western blot and relative quantifications of PRKAA1 and p-PRKAA1 in SH-SY5Y cells. All experiments were performed independently and repeated 3 times. The data were presented as the means ± SD. *P < 0.05 compare with the control group.

Article Snippet: PRKAA1, p-PRKAA1 (Thr172), Parkin, and Cyt C antibodies were obtained from Boster Biotech (China).

Techniques: Expressing, In Vivo, In Vitro, Phospho-proteomics, Western Blot, Control

Journal: Ecotoxicology and environmental safety

Article Title: PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

doi: 10.1016/j.ecoenv.2023.114772

Figure Lengend Snippet: Fig. 6. DM inhibits aberrant mitophagy by pre venting the phosphorylation of PRKAA1 in vitro. (A-B) Representative western blot and relative quantifications of PRKAA1and p-PRKAA1 in SH- SY5Y cells. (C-D) Representative western blot and relative quantifications of PINK1 and Parkin in SH-SY5Y cells. (E-F) Representative images and relative quantifications of immunofluores cence staining of PINK1 and Mito-tracker, after DM intervention. The scale bar represents 50 µm. (G-H) Representative western blot and relative quantifications of TOMM-20 in SH-SY5Y cells. All experiments were performed independently and repeated 3 times. The data were presented as the means ± SD. *P < 0.05 compare with the control group, @P < 0.05 compare with the NaF group.

Article Snippet: PRKAA1, p-PRKAA1 (Thr172), Parkin, and Cyt C antibodies were obtained from Boster Biotech (China).

Techniques: Phospho-proteomics, In Vitro, Western Blot, Staining, Control