Journal: Frontiers in Microbiology

Article Title: Enterovirus A71 utilizes host cell lipid β-oxidation to promote its replication

doi: 10.3389/fmicb.2022.961942

Figure Lengend Snippet: The replication of EV-A71 was inhibited after knocked down CPT1 gene. (A,B) SiCPT1 with the highest knockdown efficiency was screened. (A) The expression levels of CPT1 gene was detected by RT-qPCR. (B) The protein expression levels of CPT1 was detected. (C)–(E) 4 h, 8 h, and 12 h after virus infection. (C) The cell viability detected by CCK8. (D,E) The expression levels of CPT1 and EV-A71 gene detected by RT-qPCR. The label of control is blank, EV-A71 is be infected with EV-A71, EV-A71 + SiNC is be treated with SiNC and then be infected with EV-A71, EV-A71 + SiCPT1 is be treated with SiCPT1 and then be infected with EV-A71. (F) The expression levels of VP1, PLIN2 and CPT1 proteins were detected after virus infection for 12 h. the label of control is a blank, EV-A71 is be infected with EV-A71, SiNC is be treated with SiNC and then be infected with EV-A71, and SiCPT1 is be treated with SiCPT1 and then be infected with EV-A71. All the experiments were carried out in triplicate. Error bars represent SEM. * means p < 0.05, ** means p < 0.01, *** means p < 0.001.

Article Snippet: The following antibodies and reagents were used: mouse anti-β-actin and mouse anti-BiP (Proteintech, United States); rabbit anti-EV-A71 VP1(Genetex, United States); rabbit anti-CPT1 and rabbit anti-PLIN2(Abcam, China); Cell Counting Kit-8 (CCK8, Dojindo, China); PKR-IN-C16, Tunicamycin and Etomoxir (MedChemExpress, United States); Oleic acid (OA), and 5-tetradecyloxy-2-furoic acid (TOFA; Sigma- Aldrich, United States); C75 (Selleck, China); nontargeting siRNA (SiNC) and targeting CPT1 (SiCPT1; RiboBio, China); Lipofectamine 3000 reagent (Invitrogen, United States).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Virus, Infection, Control

Journal: Communications Biology

Article Title: The PDE4DIP-AKAP9 axis promotes lung cancer growth through modulation of PKA signalling

doi: 10.1038/s42003-025-07621-y

Figure Lengend Snippet: A shRNA-mediated knockdown of PDE4DIP expression was evaluated via Western blotting. H1299 and A549 cells were infected with lentivirus carrying either control shRNA (shNC) or a PDE4DIP-specific shRNA (shP1 or shP2) to generate PDE4DIP-KD cell lines. GAPDH was used as a control. B MTT assay in control (shNC) and PDE4DIP-KD H1299 and A549 cells. The data are representative of at least three independent experiments performed in triplicate. C Growth curves of PDE4DIP-KD H1299 and A549 cells generated via RTCA. The data were obtained from triplicate samples. D Colony formation assay in PDE4DIP-KD lung cancer cells. Left, representative images of the assay; right, data representative of at least three independent experiments performed in triplicate. Scale bar: 1.0 cm. E Growth curve of xenografts generated from control and PDE4DIP-KD NSCLC cells ( n = 8). F Representative images of half of the tumours in the control and PDE4DIP shRNA-transfected groups are shown at 42 (H1299) and 25 (A549) days after cell injection. G Weights of xenograft tumours derived from control and PD4DIP shRNA-transfected NSCLC cells ( n = 8 mice). *** P < 0.001. The data are presented as the means ± SD, and the P values were determined by Student’s t test. ** P < 0.01; *** P < 0.001 vs. shNC.

Article Snippet: The sequences were as follows: shP1, 5′-AAC CTC CAG TGG CTG AAA GAA-3′; shP2, 5′-AAG CAG AGA GAC AGC TCT ATA-3′; and shNC, 5-TTC TCC GAA CGT GTC ACG T-3′. siRNAs targeting Bim and AKAP9 and a nontargeting negative control siRNA (siNC) were synthesized by GenePharma (Shanghai, China).

Techniques: shRNA, Knockdown, Expressing, Western Blot, Infection, Control, MTT Assay, Generated, Colony Assay, Transfection, Injection, Derivative Assay

Journal: Communications Biology

Article Title: The PDE4DIP-AKAP9 axis promotes lung cancer growth through modulation of PKA signalling

doi: 10.1038/s42003-025-07621-y

Figure Lengend Snippet: A KEGG enrichment analysis of the pathways affected by knockdown of PDE4DIP in A549 cells. B PDE4DIP-KD-induced apoptosis was analysed via flow cytometry. Cells were infected with nontargeting control shRNA (shNC) or PDE4DIP shRNA (shP1 or shP2) for 84 h. Left panel, representative images of the apoptosis assay results; right panel, calculated numbers of apoptotic cells (%, Q4). C Apoptosis was examined by a TUNEL assay in PDE4DIP-KD NSCLC tissues from xenograft. Upper panel, representative images of TUNEL positivity, with the arrow indicating TUNEL-positive cells; lower panel, histograms showing the quantification of TUNEL-positive cells. Scale bar: 100 μm. D Flow cytometric analysis of the cell cycle in PDE4DIP-KD NSCLC cells. Cells were infected with lentivirus containing shP1, shP2 or the nontargeting shNC for 84 h. Upper panel, representative images of the cell cycle analysis results; lower panel, histograms showing the quantification of the cell cycle distribution. E Western blot analysis of apoptosis-related proteins in control and PDE4DIP-KD cells. Cl Casp-3/9, cleaved caspase-3/9. F Silencing Bim abolished PDE4DIP-KD-induced apoptosis. Left panel, evaluation of interference with Bim expression by Western blotting; right panel, histograms showing the quantification of apoptosis. G Western blot analysis of cell cycle-related proteins in control and PDE4DIP-KD cells ( B – D and F ). The data are shown as the means ± SD of a representative experiment performed in triplicate. ** P < 0.01; *** P < 0.001 vs. shNC.

Article Snippet: The sequences were as follows: shP1, 5′-AAC CTC CAG TGG CTG AAA GAA-3′; shP2, 5′-AAG CAG AGA GAC AGC TCT ATA-3′; and shNC, 5-TTC TCC GAA CGT GTC ACG T-3′. siRNAs targeting Bim and AKAP9 and a nontargeting negative control siRNA (siNC) were synthesized by GenePharma (Shanghai, China).

Techniques: Knockdown, Flow Cytometry, Infection, Control, shRNA, Apoptosis Assay, TUNEL Assay, Cell Cycle Assay, Western Blot, Expressing

Journal: Communications Biology

Article Title: The PDE4DIP-AKAP9 axis promotes lung cancer growth through modulation of PKA signalling

doi: 10.1038/s42003-025-07621-y

Figure Lengend Snippet: A Western blot analysis of AKAP9 expression in control and PDE4DIP-KD NSCLC cells. B Western blot analysis of PDE4DIP and PKA RIIα (RIIα) expression in control and AKAP9-silenced NSCLC cells. Cells were transfected with control siNC or siRNA targeting AKAP9 (siA1, siA2) for 48 h. C Coimmunoprecipitation of endogenous AKAP9 with PDE4DIP or PKA RIIα in H1299 and A549 cells. Cell lysates were immunoprecipitated with an anti-AKAP9 antibody and subjected to Western blotting with anti-AKAP9, anti-PDE4DIP, and anti-PKA RIIα antibodies. TCL, total cell lysate. D Immunofluorescence analysis of PDE4DIP (green) and PKA RIIα (red) colocalization at the Golgi apparatus in control (siNC) and AKAP9-silenced (siAKAP9/siA1) NSCLC cells. Scale bars, 10 μm. Representative images (left) and colocalization values (Rcoloc) of PDE4DIP and PKA RIIα are shown (right). The data are presented as the means ± SD of three biological replicates, and the P values were determined by Student’s t test. *** P < 0.001 vs. control. E Western blot analysis of PKA RIIα ubiquitination in control (siNC) and AKAP9-silenced (siA1) NSCLC cells.

Article Snippet: The sequences were as follows: shP1, 5′-AAC CTC CAG TGG CTG AAA GAA-3′; shP2, 5′-AAG CAG AGA GAC AGC TCT ATA-3′; and shNC, 5-TTC TCC GAA CGT GTC ACG T-3′. siRNAs targeting Bim and AKAP9 and a nontargeting negative control siRNA (siNC) were synthesized by GenePharma (Shanghai, China).

Techniques: Western Blot, Expressing, Control, Transfection, Immunoprecipitation, Immunofluorescence, Ubiquitin Proteomics

Journal: Current Issues in Molecular Biology

Article Title: A Feedback Loop between TGF-β1 and ATG5 Mediated by miR-122-5p Regulates Fibrosis and EMT in Human Trabecular Meshwork Cells

doi: 10.3390/cimb45030154

Figure Lengend Snippet: TGF-induced fibrosis and EMT is regulated by ATG5. ( A ) The protein levels of ATG-5, LC3-I, LC3-II, SMA, vimentin, fibronectin, and fibrillin in whole-cell lysates of siRNA-mediated knockdown HTM cells with (10 ng/mL) and without TGF-β1. ( B – G ), relative quantification of the proteins obtained using densitometry analysis of the bands. The error bars represent the standard deviation (*— p < 0.05, **— p < 0.01, ***— p < 0.001, by t -test).

Article Snippet: Briefly, 60 pmol/well of siRNAs against autophagy-essential ATG5 (siATG5) (6345S, Cell signalling technology, Beverly, MA, USA)and nontargeting siRNA (negative control, siNC) (6568S, Cell signalling technology, Beverly, MA, USA) in serum-free medium was mixed with Lipofectamine RNAiMAX reagent (13778, Invitrogen, Beverly, MA, USA) and incubated for 60 min according to the instructions provided by the manufacturers.

Techniques: Knockdown, Quantitative Proteomics, Standard Deviation

Journal: Current Issues in Molecular Biology

Article Title: A Feedback Loop between TGF-β1 and ATG5 Mediated by miR-122-5p Regulates Fibrosis and EMT in Human Trabecular Meshwork Cells

doi: 10.3390/cimb45030154

Figure Lengend Snippet: Apoptosis is regulated by ATG5 in HTM cells. HTM cells were treated with TGF-β1 (10 ng/mL) and siRNA and the total number of apoptotic cells was calculated by flow cytometry. ( A – D ) The total number of apoptotic cells in TGF-β1 treated and siRNA knockdown HTM cells.

Article Snippet: Briefly, 60 pmol/well of siRNAs against autophagy-essential ATG5 (siATG5) (6345S, Cell signalling technology, Beverly, MA, USA)and nontargeting siRNA (negative control, siNC) (6568S, Cell signalling technology, Beverly, MA, USA) in serum-free medium was mixed with Lipofectamine RNAiMAX reagent (13778, Invitrogen, Beverly, MA, USA) and incubated for 60 min according to the instructions provided by the manufacturers.

Techniques: Flow Cytometry, Knockdown

Journal: Current Issues in Molecular Biology

Article Title: A Feedback Loop between TGF-β1 and ATG5 Mediated by miR-122-5p Regulates Fibrosis and EMT in Human Trabecular Meshwork Cells

doi: 10.3390/cimb45030154

Figure Lengend Snippet: ATG5-TGF crosstalk may be mediated by miR-122-5p. Relative miRNA expression levels of, miR-424-5p, miR-124-3p, and miR-122-5p in ( A ) Transforming growth factor, TGF-β1 (10 ng/mL) treated human trabecular meshwork (HTM) cells and ( B ) siRNA mediated knockdown HTM cells with (10 ng/mL) and without TGF-β1 exposure. The error bars represent standard deviation ***— p < 0.001, by one-way ANOVA).

Article Snippet: Briefly, 60 pmol/well of siRNAs against autophagy-essential ATG5 (siATG5) (6345S, Cell signalling technology, Beverly, MA, USA)and nontargeting siRNA (negative control, siNC) (6568S, Cell signalling technology, Beverly, MA, USA) in serum-free medium was mixed with Lipofectamine RNAiMAX reagent (13778, Invitrogen, Beverly, MA, USA) and incubated for 60 min according to the instructions provided by the manufacturers.

Techniques: Expressing, Knockdown, Standard Deviation