Journal: Nature Communications

Article Title: Search-and-remove genome editing allows selection of cells by DNA sequence

doi: 10.1038/s41467-025-66896-1

Figure Lengend Snippet: a Strategy to apply SNIPE to kill cancer cells based on cancer specific mutations. Wild type cells are colored in gray, cells with cancer mutations in purple and dying cells in red. b Cell growth curves of human 409B2 iPSCs measured by resazurin assay (upper panel). The lower panel shows NGS read frequency for cancer mutation (purple) and wild type cells (gray) in a mixed population over time. c Killing of TP53 knock-out 409B2 cells by SNIPE. Different amounts of TP53 knock-out cells (purple) were mixed with wild type cells (gray). Indels are colored in blue and wild type with indels in light purple. d Cell survival after selection by SNIPE related to ( c ). e K562 cells carry a Philadelphia chromosome, characterized by a reciprocal translocation of chromosome 9 and 22 resulting in the fusion oncogene BCR-ABL1 . f The BCR-ABL fusion generates a new gRNA recognition site in K562 cells. g Cell survival of THP-1 and K562 cells after targeting the BCR-ABL junction. The label “Cas9 cut” indicates CRISPR targeting without SNIPE. h Agarose gel of PCR products of healthy chromosome 22 (297 bp, gray) and Philadelphia chromosome (267 bp, purple). i Quantification of band intensity for K562 cells of ( h ). j Cell survival of heterozygous cancer cell lines RD-ES, HT-29 and HuCC-T1 after targeting the disease allele with SNIPE. k NGS read frequency of cancer locus alleles in RD-ES, HT-29 and HuCC-T1 for mock and SNIPE edit. The cancer mutation allele is colored in purple, wild type in grey, indels in blue and wild type with indels in light purple. Independent biological replicates were performed ( n = 3 for b , c , d , g , h , i , j , k , n = 2 for RDES in j and k) and are depicted as dots for the cancer mutation allele sequence reads and cell survival. Error bars show the s.e.m.

Article Snippet: Human intrahepatic cholangiocarcinoma cells HuCC-T1 (Cytion, catalog no. 300469), the human bone sarcoma cell line RD-ES (Cytion, catalog no. 300410) and the human leukemia cell line THP-1 (Cytion, catalog no. 300356) were cultured in RPMI 1640 (ThermoFisher, catalog no. 11875-093) with 10% FBS.

Techniques: Resazurin Assay, Mutagenesis, Knock-Out, Selection, Translocation Assay, CRISPR, Agarose Gel Electrophoresis, Sequencing

Journal: Cancers

Article Title: Oncogenic and Stemness Signatures of the High-Risk HCMV Strains in Breast Cancer Progression

doi: 10.3390/cancers14174271

Figure Lengend Snippet: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. ( A ) Time-course of the viral titer in the supernatant of MRC5 infected with the strains HCMV-B544 and HCMV-B693, as measured by IE1-qPCR. ( B ) Confocal microscopic images of HCMV-IE1 and pp65 staining in HMECs infected with HCMV-B544 and HCMV-B693 (day 1 post-infection). Uninfected HMECs were used as controls. Nuclei were counterstained with DAPI; magnification ×63, scale bar 10 μm. ( C ) HMECs time-course infection with HCMV-B544 and HCMV-B693 strains (MOI = 1). Magnification ×100, scale bar 100 μm. Uninfected HMECs were used as a control. ( D ) Presence of giant cells with blastomere-like morphology (1 and 6), mesenchymal cells (4 and 7), lipid droplet-packed cells (3, 8, and 9), cells displaying multiple nuclei (2) as well as cell budding (4, 5, and 6), and cells with filopodia protrusions (9) in CTH-B544 and CTH-B693 cells. The inverted light microscope scale bar represents 100 µm; magnification ×200.

Article Snippet: HMECs (A10565, Life Technologies, Carlsbad, CA, USA), CTH cells, MDA-MB231 as well as MCF7 (Institut Hiscia, Arlesheim, Switzerland), and MRC5 (RD-Biotech, Besançon, France) were cultured as previously described [ ].

Techniques: Infection, Staining, Light Microscopy

Journal: Virology Journal

Article Title: Antiviral effects against EV71 of pimprinine and its derivatives isolated from Streptomyces sp

doi: 10.1186/s12985-014-0195-y

Figure Lengend Snippet: Antiviral activities of pimprinine, pimprinethine, WS-30581 A and WS-30581 B against EV71 in a dose-dependent manner in RD cells. (A) The inhibition of virus-induced CPE. RD cells were infected with 100 TCID 50 of EV71 mixed with serial dilutions of tested compounds for 1 h at 37°C, the inocula were aspirated and the cells were incubated with DMEM/tested compounds at 37°C, 5% CO 2 for 48 h, the antiviral effects were observed in terms of cellular morphology. (B) The inhibition of progeny virus yield. RD cells were infected with 100 TCID 50 of EV71 in the absence or presence of tested compounds, the culture media and cell lysates were collected for virus titration at 10 h pi. The viral titers were presented as Log10 TCID 50 /mL. (C) Analysis of the dependence of the EC50s on virus titers. RD cells were infected with various TCID 50 of EV71, the tested compounds were added as described in (A) , cell viability was determined at 48 h pi and the EC50 were calculated. The x-axis is in a base 10 logarithmic scale. Each value is the mean of triplicate assays ± standard deviation (SD).

Article Snippet: Human rhabdomyosarcoma cells (RD), Human laryngeal carcinoma cells (Hep-2), Human cervical carcinoma cells (HeLa) and Madin-Darby canine kidney cells(MDCK)(purchased from China Center for type Culture Collection, CCTCC) were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL of penicillin and streptomycin, and 2 mM L-glutamine.

Techniques: Inhibition, Virus, Infection, Incubation, Titration, Standard Deviation

Journal: Virology Journal

Article Title: Antiviral effects against EV71 of pimprinine and its derivatives isolated from Streptomyces sp

doi: 10.1186/s12985-014-0195-y

Figure Lengend Snippet: Analysis of the modes of action of pimprinethine, WS-30581 A and WS-30581 against EV71. (A-B) Analysis of the effective stage. The RD cells were incubated under serial 2-fold dilutions of tested compounds before, simultaneously or after EV71 (100 TCID 50 ) inoculation. Antiviral effects were detected by measuring cell viability (A) and the progeny virus yields (80 μM) (B) after 48 h of infection. (C) Analysis of the effects on EV71 adsorption. Mock- or 160 μM pimprinethine, 80 μM WS-30581A and 80 μM WS-30581 B-treated EV71 (10 4 TCID50) was inoculated onto RD cells and adsorbed for 2 h, the infected cells were harvested and then subjected to virus titrations using the TCID 50 method. (D) The effects on EV71 release from RD cells. RD cells infected with 100 TCID 50 of EV71 were incubated with tested compounds for 12 h, both of cells and supernatants (intra- and extracellular), or one of which was harvested separately for determination of virus yield. Mock: no infection; VC, virus control. Values represent the means ± SDs of three independent experiments. *P <0.05, compared with virus control group.

Article Snippet: Human rhabdomyosarcoma cells (RD), Human laryngeal carcinoma cells (Hep-2), Human cervical carcinoma cells (HeLa) and Madin-Darby canine kidney cells(MDCK)(purchased from China Center for type Culture Collection, CCTCC) were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL of penicillin and streptomycin, and 2 mM L-glutamine.

Techniques: Incubation, Virus, Infection, Adsorption, Control

Journal: Virology Journal

Article Title: Antiviral effects against EV71 of pimprinine and its derivatives isolated from Streptomyces sp

doi: 10.1186/s12985-014-0195-y

Figure Lengend Snippet: Time-of-addition assay. 80 μM pimprinethine, 40 μM WS-30581 A or 40 μM WS-30581 B were added to RD cells at different time periods after EV71 infection. At 12 h pi, the progeny virus yield was determined (-1–0 h: viral infection period; 0–10 h: the period for virus proliferation in the cells). Values are represented as the means ± SDs. *P <0.05, compared with virus control group.

Article Snippet: Human rhabdomyosarcoma cells (RD), Human laryngeal carcinoma cells (Hep-2), Human cervical carcinoma cells (HeLa) and Madin-Darby canine kidney cells(MDCK)(purchased from China Center for type Culture Collection, CCTCC) were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL of penicillin and streptomycin, and 2 mM L-glutamine.

Techniques: Infection, Virus, Control

Journal: Virology Journal

Article Title: Antiviral effects against EV71 of pimprinine and its derivatives isolated from Streptomyces sp

doi: 10.1186/s12985-014-0195-y

Figure Lengend Snippet: Effects of pimprinethine, WS-30581 A and WS-30581 B on EV71 replication in RD cells. RD cells infected with 100 TCID 50 of EV71 were incubated in the absence (VC) or presence of 80 μM pimprinethine, 40 μM WS-30581 A or 40 μM WS-30581 B and harvested at the indicated times pi. (A) The progeny viral yields were determined. Dashed lines indicate virus titer less than the detectable dose. (B) The total RNA was extracted from cells and culture supernatants and EV71 RNA levels were measured. Cellular actin amplification was used for normalization. The △△CT data were calculated from three independent experiments. *P <0.05, compared with the virus control group. (C) EV71-protein was determined by indirect immunofluorescence using a mouse anti-enterovirus 71 monoclonal antibody and an Alexa Fluor 488-Conjugated Affinipure Goat Anti-Mouse IgG (H + L). The nucleus was stained with DAPI and the green foci indicate the presence of EV71 protein.

Article Snippet: Human rhabdomyosarcoma cells (RD), Human laryngeal carcinoma cells (Hep-2), Human cervical carcinoma cells (HeLa) and Madin-Darby canine kidney cells(MDCK)(purchased from China Center for type Culture Collection, CCTCC) were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL of penicillin and streptomycin, and 2 mM L-glutamine.

Techniques: Infection, Incubation, Virus, Amplification, Control, Immunofluorescence, Staining

Journal: Virology Journal

Article Title: Antiviral effects against EV71 of pimprinine and its derivatives isolated from Streptomyces sp

doi: 10.1186/s12985-014-0195-y

Figure Lengend Snippet: The inhibitory effects of pimprinethine, WS-30581 A and WS-30581 B on EV71-induced apoptosis. RD cells were left untreated (A) or were infected with 100 TCID50 of EV71, after viral adsorption, RD cells were incubated in the absence (B) or presence of 80 μM pimprinethine (C) , 40 μM WS-30581 A (D) and 40 μM WS-30581 B (E) for 36–48 h, the cells were stained with Annexin-V- fluorescein and propidium iodide and measured using flow cytometry. Values are represented as means ± SDs. *P <0.05, compared with virus control group.

Article Snippet: Human rhabdomyosarcoma cells (RD), Human laryngeal carcinoma cells (Hep-2), Human cervical carcinoma cells (HeLa) and Madin-Darby canine kidney cells(MDCK)(purchased from China Center for type Culture Collection, CCTCC) were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL of penicillin and streptomycin, and 2 mM L-glutamine.

Techniques: Infection, Adsorption, Incubation, Staining, Flow Cytometry, Virus, Control