Journal: RSC Advances

Article Title: Highly efficient green synthesis and photodynamic therapeutic study of hypericin and its derivatives †

doi: 10.1039/c8ra03732a

Figure Lengend Snippet: Cell viability of A431 cells (a); HepG2 cells (b); and MCF-7 cells (c) determined by MTT cell viability assay. The cells were incubated with compound 7a–d, respectively, and irradiated with 595-600 nm light for 30 min.

Article Snippet: The breast cancer cell line (MCF-7), human hepatoma cell line (HepG-2) and the human skin basal cell carcinoma (A431) were obtained from American Type Culture Collection.

Techniques: Viability Assay, Incubation, Irradiation

Journal: iScience

Article Title: Genome editing of FTR42 improves zebrafish survival against virus infection by enhancing IFN immunity.

doi: 10.1016/j.isci.2024.109497

Figure Lengend Snippet: Figure 4. FTR42 inhibits TBK1-triggered IFN response dependent on its E3 ligase activity (A) Schematic diagrams of full-length FTR42, DRING and E3-mut. (B) FTR42 but not DRING or E3-mut possessed the E3 ligase activity. HEK293T cells seeded in 10 cm2 dishes were transfected with His-Ub, together with FTR42- Flag or DRING-Flag or E3-mut-Flag (5 mg each). 30 h later, the cells were lysed and incubated with Ni2-NTA overnight, followed by western blotting to determine the ubiquitination of FTR42 and two mutants. (C) FTR42-DRING or E3-mut failed to inhibit TBK1-triggered IFN41 promoter activation. CAB cells seeded in 48-well plates overnight were transfected with DrIFN41pro-luc, TBK1 or empty vector (100 ng each), together with FTR42 or DRING or E3-mut at increasing amounts. 30 h later, the cells were harvested for luciferase assays. (D) FTR42-DRING or E3-mut failed to attenuate TBK1 protein level in vitro. CAB cells seeded in 3.5 cm2 dishes were co-transfected with the indicated plasmids (1 mg each) for 30 h, followed by western blotting assays with tag-specific Abs. (E and F) FTR42-triggered TBK1 degradation was blocked by MG132 but not by NH4Cl or chloroquine. CO cells seeded in 12-well plates were co-transfected with indicated plasmids for 24 h, followed by treatment with MG132, NH4Cl, or chloroquine for additional 6 h. p values were calculated using Student’s t test. ***p < 0.001; ns, not significant. See also Figure S4.

Article Snippet: Raw and analyzed data This paper Lead Contact, Yibing Zhang (ybzhang@ihb.ac. cn) Experimental models: Cell lines HEK293T ATCC CRL-3216 Epithelioma papulosum cyprini cells (EPC) ATCC CRL-2872 Ovary cells of grass carp (CO) Kept in IHB, CAS N/A Crucian carp (C. auratus L.) blastulae embryonic cells (CAB) Kept in IHB, CAS N/A Experimental models: Organisms/strains Zebrafish (Danio rerio) strain AB China Zebrafish Resource Center N/A Oligonucleotides see Table S1 (Continued on next page) 16 iScience 27, 109497, April 19, 2024

Techniques: Activity Assay, Transfection, Incubation, Western Blot, Ubiquitin Proteomics, Activation Assay, Plasmid Preparation, Luciferase, In Vitro

Journal: Frontiers in Immunology

Article Title: Deciphering the Fine-Tuning of the Retinoic Acid-Inducible Gene-I Pathway in Teleost Fish and Beyond

doi: 10.3389/fimmu.2021.679242

Figure Lengend Snippet: Regulation of RIG-I-mediated signal transduction by a conserved set of cellular proteins in vertebrates. (A) Schematic representation of RIG-I pathway including the main downstream components for signal transduction leading to promoter activation and expression of type-I interferon. A set of cellular regulators that are evolutionarily conserved between fish and mammalian species are placed next to their targets. The effect of each regulator is symbolized by (–) for inhibition (in red), by (+) for activation (in blue), or by (+/-) for ambivalent (in green) reported functions on the RIG-I pathway, based on the literature (mainly from studies with mammalian orthologs; see Table 1 ). Other cytosolic sensors or co-receptors involved in the RIG-I pathway are boxed in orange. *Although TRIM25 promotes the degradation of MAVS, this step is required for IRF3 phosphorylation by TBK1 . (B, C) Fathead minnow orthologs of mammalian regulators were amplified from total RNA extracted from EPC cells, cloned into the eukaryotic expression vectors pcDNA1.1/Amp (Invitrogen) and fully sequenced. Nucleotide sequences of each regulator were deposited in GenBank (see Table 1 for accession numbers). To test their effect on the RIG-I pathway, EPC cells were transfected with the indicated plasmids (1 μg) together with a luciferase reporter construct driven by the promoter of IFN1 derived from EPC cells (1 μg) and the RIG-I Nter-eGFP inducer and internal transfection control construct 0.5 µg in (B) or 1 μg in (C) , as previously described . Twenty-four hours after transfection, the cells were lysed for luciferase assays. Luciferase activity was measured and normalized to eGFP fluorescence. No significant variation in eGFP expression was observed between each condition. The percentage of fold-induction were calculated as the ratio of stimulated (+ RIG-I Nter) versus unstimulated (− RIG-I Nter) conditions and compared to the induction control (RIG-I Nter + empty vector). Means of at least three independent experiments are shown together with the standard errors. The color coding used for the histograms is the same as the one used in panel (A) For statistical analysis, a comparison between groups was performed with a one-way ANOVA and Tukey’s multiple comparison tests using GraphPad Prism (GraphPad, San Diego, CA). Groups that are not significantly different from each other are denoted ns (P > 0.05), whereas those that are significantly different are denoted *(P < 0.05), **(P < 0.01), ***(P < 0.001) or ****(P < 0.0001). (D, E) EPC cells were transfected with the indicated plasmids (2 μg each) or an empty vector (pcDNA1.1/Amp) as a control, as previously described . All transfection mixtures were adjusted with an empty vector to contain an equal amount of plasmid DNA. Twenty-four hours after transfection, cells were infected with a fish novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) at an MOI of 1 and incubated at 15°C. Cell monolayers were stained with crystal violet 3 days postinfection (D) . The culture supernatants from infected cells were collected at different times postinfection and the viral titer was determined by plaque assay (E) . Each time point is represented by two independent experiments, and each virus titration was performed in duplicate. Average values are shown. The standard errors were calculated and the error bars are shown. Asterisks indicate significant difference (*p < 0.05; ****p < 0.0001) as determined by two-way ANOVA and Tukey’s multiple comparison tests. ns, not significant.

Article Snippet: Fathead minnow is a relevant fish species for at least two reasons: 1) EPC cells ( Epithelioma Papulosum Cyprini ; ATCC CRL-2872), the most widely used fish cell line in virology, is derived from this fish species , and 2) fathead minnow belongs to the family Cyprinidae together with the zebrafish ( Danio rerio ), an animal model offering great potential for the study of human and fish viral diseases and the development of antiviral drugs ( – ).

Techniques: Transduction, Activation Assay, Expressing, Inhibition, Phospho-proteomics, Amplification, Clone Assay, Transfection, Luciferase, Construct, Derivative Assay, Control, Activity Assay, Fluorescence, Plasmid Preparation, Comparison, Infection, Virus, Incubation, Staining, Plaque Assay, Titration

Journal: Nature Communications

Article Title: Mechanochemical control of epidermal stem cell divisions by B-plexins

doi: 10.1038/s41467-021-21513-9

Figure Lengend Snippet: a Human primary epidermal keratinocytes (hPEKS) and human primary basal cell carcinoma cells (hPBCCs) were lysed, and proteins were detected using the indicated antibodies. b , c hPBCCs engineered to express GFP (“control”) or myc-tagged Plexin-B2 (“Plexin-B2”) and cultured at dense conditions were immunostained for YAP (red) and myc (green). Blue: DAPI. Representative confocal images are shown in b with dashed boxed areas depicted in the right panel. A quantification of YAP nuclear intensity is shown in c (mean ± s.d.; n = 3 independent experiments; p = 0.0495; two-sided unpaired t -test). d hPBCCs were engineered to express GFP (“control”) or myc-tagged Plexin-B2 (“Plexin-B2”), and cultured at dense conditions. Relative mRNA expression levels of the YAP target genes ctgf and cyr61 were determined by quantitative RT-PCR (mean ± s.d.; n = 5 independent experiments; p = 0.0123 for ctgf , p = 0.0002 for cyr61 ; two-sided unpaired t -test).

Article Snippet: Human primary basal cell carcinoma cells (hPBCCs) were purchased from Celprogen and were grown in human basal cell carcinoma cell line complete media with serum (cat. no. M77015-08S, Celprogen).

Techniques: Cell Culture, Expressing, Quantitative RT-PCR