Journal: Viruses

Article Title: Small-Molecule RAF265 as an Antiviral Therapy Acts against PEDV Infection

doi: 10.3390/v14102261

Figure Lengend Snippet: RAF265 suppressed the synthesis of viral proteins of PEDV. ( A ) Vero-WT and Vero-eIF4E (S209A) cells were challenged with PEDV for different times; cells lysates were harvested for WB analysis with indicated antibodies. ( B ) Vero-WT and Vero-eIF4E (S209A) cells were infected with PEDV in the presence of increasing doses of RAF265 for 24 h. Two cell lines were harvested for WB analysis with indicated antibodies. ( C ) Schematic representation of dual-targeting inhibitor RAF265 as active antiviral. After binding, PEDV enters into Vero cells by endocytosis, RAF265 mediates arrangement of actin cytoskeleton involved in the internalization of viral infection of PEDV. RAF265 also decreases the level of p-eIF4E to inhibit the synthesis of viral proteins. h.p.i, hours post-infection; MOI, multiplicity of infection.

Article Snippet: Rabbit polyclonal antibody to eIF4E (1:1000) was purchased from Proteintech (Wuhan, China).

Techniques: Infection, Binding Assay

Journal: Advanced Science

Article Title: CDKAL1 Drives the Maintenance of Cancer Stem‐Like Cells by Assembling the eIF4F Translation Initiation Complex

doi: 10.1002/advs.202206542

Figure Lengend Snippet: CDKAL1 promotes the assembly of the eIF4F translation initiation complex. A) Western blotting analysis of m 7 GTP precipitate from RD cell lysate. GAPDH, β ‐actin, vinculin, and α ‐tubulin serve as negative controls. B) Western blotting analysis of m 7 GTP precipitate from RD cell lysate with urea‐based denaturation. Numbers indicate the densitometry of signals (percent of control, Urea 0 m ). C) An in situ proximity ligation assay using anti‐myc tag antibody and antibodies against endogenous eIF4A, eIF4G, and eIF4E in parental RD cells and RD cells expressing myc‐CDKAL1 WT . Scale bars, 20 µm. D) An in situ proximity ligation assay using antibodies against endogenous proteins and m 7 G cap. Note that CD133 high RD cells showed elevated expression levels of CDKAL1 compared to CD133 low RD cells (Figure ). E) Western blotting analysis of m 7 GTP precipitate from Control‐shRNA‐ or CDKAL1 ‐shRNA‐expressing RD cell lysate. F) Western blotting analysis of m 7 GTP precipitate from Control‐shRNA‐, CDKAL1 ‐shRNA‐, EIF4G ‐shRNA‐, EIF4E ‐expressing RD cell lysate. G) A cartoon representing the mutants used in this study. H) Results of the rescue experiment with several truncated mutants of CDKAL1 to maintain the self‐renewal capacity and SALL2 protein levels of RD cells ( n = 4, error bars indicate mean ± SD). I) The m 7 GTP pull‐down experiments with CDKAL1 ‐shRNA‐expressing RD cells displaying that the NH 2 ‐terminus of CDKAL1 is essential for sustaining the eIF4F complex formation.

Article Snippet: The following are the antibodies used in this study, listed as [Protein/Source/Identifier]: [GAPDH/Proteintech/ 60004‐1‐Ig]; [CD133/Proteintech/18470‐1‐AP]; [CD133/Proteintech/66666‐1‐Ig]; [CD133/BioLegend/372808]; [CDKAL1/Proteintech/22988‐1‐AP]; [CDKAL1/Santa Cruz Biotechnology/sc‐393447]; [Myosin Heavy Chain/R&D Systems/MAB4470]; [Myc tag/Medical&Biological Laboratories/M192‐3]; [ALDH1/Novus Biologicals/NBP1‐89152]; [CD44/BioLegend/103001]; [SOX2/Santa Cruz Biotechnology/sc‐17320]; [POU3F2/Cell Signaling Technology/12137S]; [SALL2/Bethyl Laboratories/A303‐208A]; [Desmin/Novus Biologicals/NBP1‐45143]; [Myogenin/Santa Cruz Biotechnology/sc‐12732]; [ β ‐Actin/Cell Signaling Technology/3700S]; [eIF4A1/Cell Signaling Technology/2490S]; [eIF4G1/Proteintech /15704‐1‐AP]; [eIF4E/Cell Signaling Technology/2067S]; [Digoxigenin/Roche/11333062 910]; [7‐methylguanosine (m7G)‐Cap/Medical&Biological Laboratories/RN016M]; [OLIG2/Abcam/ab109186]; [BiP/Cell Signaling Technology/3177S]; [Puromycin/Sigma‐Aldrich/618582]; [Donkey anti‐Rabbit IgG, Alexa Fluor Plus 594/Thermo Fisher Scientific/A32754]; [Donkey anti‐Mouse IgG, Alexa Fluor 488/Thermo Fisher Scientific/A21202]; [Donkey anti‐Rabbit IgG, Alexa Fluor Plus 488/Thermo Fisher Scientific/A21206]; [Donkey anti‐Mouse IgG, Alexa Fluor 594/Thermo Fisher Scientific/A21203]; [Donkey anti‐Rat IgG, Alexa Fluor 647/Abcam/ab150155]; [Donkey anti‐Rat IgG, Alexa Fluor 594/Thermo Fisher Scientific/A21209]; [Anti‐Rabbit IgG, HRP‐linked/Cell Signaling Technology/7074P2]; [Anti‐Mouse IgG, HRP‐linked/Sigma‐Aldrich/A9044]; [Anti‐Goat IgG, HRP‐linked/Sigma‐Aldrich/A4174]; [Anti‐Rat IgG, HRP‐linked/Sigma‐Aldrich/A5795].

Techniques: Western Blot, In Situ, Proximity Ligation Assay, Expressing, shRNA

Journal: Molecules

Article Title: Ginsenoside PPD’s Antitumor Effect via Down-Regulation of mTOR Revealed by Super-Resolution Imaging

doi: 10.3390/molecules22030486

Figure Lengend Snippet: Figure 4. PPD affects the transcription and expression of mTOR and its downstream effectors. (A) Quantitative RT-PCR analyses of the effects of PPD on mTOR, 4EBP1 and eIF4E transcription. The relative levels of their mRNA are displayed in a histogram; (B) Western blot analyses of expression levels of mTOR, 4EBP1 and eIF4E in Hep-2 cells with different doses of PPD treatment; (C) The quantization of the Western blot data after correction for the β-actin loading control. Data in (A,C) are expressed as mean ± S.D., * p < 0.05, ** p < 0.01, compared to control, n = 3.

Article Snippet: Primary antibodies used were as follows: mouse anti-human mTOR, eIF4E, 4EBP1, cleaved caspase-3, Bax, Bcl-2 monoclonal antibody (Santa Cruz, Dallas, Texas, USA), CDK1, CDC25 and CCNB1 antibody (Proteintech, Wuhan, China), and rabbit anti-human β-actin.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Molecules

Article Title: Ginsenoside PPD’s Antitumor Effect via Down-Regulation of mTOR Revealed by Super-Resolution Imaging

doi: 10.3390/molecules22030486

Figure Lengend Snippet: Figure 5. PPD regulates the distribution pattern of mTOR and its downstream effectors. (A) direct Stochastic Optical Reconstruction Microscopy (dSTORM) imaging of mTOR, 4EBP1 and eIF4E in Hep-2 cells treated with different doses of PPD. mTOR, 4EBP1 and eIF4E were labeled with individual primary antibodies and Alexa Fluor 647 secondary antibodies, nuclei were labeled with Hoechst 33342. Scale bars are 10 µm; (B,E,H) Normalized total localizations of reconstructed dSTORM images of mTOR (B), 4EBP1 (E) and eIF4E (H) in Hep-2 cells with different concentrations of PPD; (C,F,I) The cluster number of mTOR (C), 4EBP1 (F) and eIF4E (I) per µm2 in Hep-2 cells with or without PPD treatment; (D,G,J) The average cluster area of mTOR (D), 4EBP1 (G) and eIF4E (J) in Hep-2 cells with or without PPD treatment. Data in (B–J) are obtained from 20 cells in four independent experiments. Data are expressed as mean ± S.D., * p < 0.05, *** p < 0.001, compared to control, n = 10.

Article Snippet: Primary antibodies used were as follows: mouse anti-human mTOR, eIF4E, 4EBP1, cleaved caspase-3, Bax, Bcl-2 monoclonal antibody (Santa Cruz, Dallas, Texas, USA), CDK1, CDC25 and CCNB1 antibody (Proteintech, Wuhan, China), and rabbit anti-human β-actin.

Techniques: Microscopy, Imaging, Labeling, Control