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: EMBO Reports

Article Title: Muskelin is a substrate adaptor of the highly regulated Drosophila embryonic CTLH E3 ligase

doi: 10.1038/s44319-025-00397-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-eIF4E (Boster) was used at 1:10000.

Techniques: SYBR Green Assay, Imaging, Recombinant, Sequencing, Software, Microscopy, Ion-Mobility Spectrometry, Mass Spectrometry

Journal: Drug Design, Development and Therapy

Article Title: Saikosaponin A Mediates the Anti-Acute Myeloid Leukemia Effect via the P-JNK Signaling Pathway Induced by Endoplasmic Reticulum Stress

doi: 10.2147/DDDT.S498458

Figure Lengend Snippet: Primary Antibodies Used in Western Blotting

Article Snippet: PERK , MedChemExpress , HY-p80781.

Techniques: Western Blot