Journal: eLife

Article Title: Structure of mouse protocadherin 15 of the stereocilia tip link in complex with LHFPL5

doi: 10.7554/eLife.38770

Figure Lengend Snippet:

Article Snippet: Adherent HEK293 tsa201 cells (ATCC CRL- 11268) were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum at 37°C.

Techniques: Recombinant, Software

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps

doi: 10.1016/j.omtm.2022.01.008

Figure Lengend Snippet: P5-associated rAAV contaminants can be translated and immunogenic after infection (A) Bar graph depicting transcriptional activity of forward and reverse direction of AAV2 P5 promoter and outside of AAV2ITRs after plasmid transfection of 293T cells. GFP cassette positioned relative to P5: upstream (blue), downstream (orange), upstream but reverse orientation (green), no GFP cassette (red). GFP upstream of AAV2 ITR: wild-type (gray), self-complementary (purple). Error bars indicate SEM. (B) Bar chart showing quantification of GFP-expressing 293T cells by FACS 72 h after transduction with AAV8 GFP_P5 F8 (red), AAV8 RevGFP_P5 F8 (orange), or AAV8 Empty_P5 F8 (blue) (n = 3). Error bars indicate SEM. (C) Time course graph depicting detectable GFP-positive cells in post-infection samples (5 × 10 6 MOI) with AAV8 GFP_P5 F8 (red), AAV8 RevGFP_P5 F8 (orange), or AAV8 Empty_P5 F8 (blue). Error bars indicate SEM. (D) Immunohistochemistry (IHC) images of C57BL/6J mouse liver sections stained for GFP protein 1 week after 9.4 × 10 11 vg infection with AAV8 GFP_P5 F8 (top left), AAV8 RevGFP_P5 F8 (top right), AAV8 Empty_P5 F8 (bottom left), or 2 × 10 10 vg AAV8-GFP (bottom right) (n = 5). (E) Representative FACS plots and bar chart depicting GFP reactive tetramer positive splenic CD8 + T cells from BALB/cJ mice 9 days after 1 × 10 12 vg infection with AAV8 GFP_P5 F8 or AAV8 Empty_P5 F8 (nonparametric t test n = 5, p = 0.0079). Error bars indicate SEM.

Article Snippet: Before transfection, adherent human embryonic kidney 293T cells (ATCC CRL-3216) were cultured in DMEM (Lonza Bio Whittaker Catalog #12-733Q) supplemented with 10% fetal bovine serum (Fisher Scientific Catalog #SH3007103) and 2 mM L-glutamine (Corning Catalog #MT25005CI) at 37°C 10% CO 2 .

Techniques: Infection, Activity Assay, Plasmid Preparation, Transfection, Expressing, Transduction, Immunohistochemistry, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of cell-based assay for detecting replication-competent adeno-associated virus by qPCR

doi: 10.1016/j.omtm.2025.101529

Figure Lengend Snippet: Schematic describing the finalized rcAAV assay workflow For the first round of transduction (T1), HEK293 cells are transduced with AAV in the presence of Ad5 at MOI 3 with viral combinations listed in . R2C8 is replication competent and contains rep2-cap8 genes. After 72 ± 2 h, supernatant is harvested from T1. For the second round of transduction (T2), fresh HEK293 cells are seeded and transduced in the presence of fresh Ad5 at MOI 3 with T1 supernatant. After 72 ± 2 h, supernatant is harvested from T2. Similarly, third round of transduction (T3) is set up. After each round, DNA is extracted from the cell pellet, normalized to 5 ng/μL, and then qPCR is set up to measure rep2 . In parallel, the “no transduction control” (T0) and other positive, negative, and spike controls are set up.

Article Snippet: Adherent HEK293 cells (ATCC, P/N: CRL-1573) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, P/N: 10564-011) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, P/N: 10100147), penicillin, and streptomycin (Life Technologies, P/N 15140-122) at 37°C in a 5% CO 2 atmosphere.

Techniques: Transduction, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of cell-based assay for detecting replication-competent adeno-associated virus by qPCR

doi: 10.1016/j.omtm.2025.101529

Figure Lengend Snippet: Schematic of the workflow for defining transduction conditions for AAV HEK293 cells were transduced with R2C8 in the presence of Ad5 at MOI 2 for T1. After 48 and 72 ± 2 h, supernatant and lysate were harvested from T1. A small aliquot of supernatant at each time point was set aside for measuring via qPCR. For the second round of transduction (T2), fresh HEK293 cells were seeded and transduced in the presence of fresh Ad5 at MOI 2 either with T1 supernatant or T1 lysate, from both 48 and 72 h time points. Supernatants were harvested after 48 ± 2 h from T2 for both sources of transduction (48 h supernatant T1 and 48 h lysate T1). Similarly, supernatants were harvested after 72 ± 2 h from T2 for both sources of transduction (72 h supernatant T1 and 72 h lysate T1).

Article Snippet: Adherent HEK293 cells (ATCC, P/N: CRL-1573) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, P/N: 10564-011) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, P/N: 10100147), penicillin, and streptomycin (Life Technologies, P/N 15140-122) at 37°C in a 5% CO 2 atmosphere.

Techniques: Transduction

Journal: Viruses

Article Title: Deubiquitinating Enzyme Inhibitors Block Chikungunya Virus Replication.

doi: 10.3390/v15020481

Figure Lengend Snippet: Figure 1. Cytotoxic effect of deubiquitinating enzyme inhibitors. HEK293, Huh-7, and Vero cells were exposed to PR-619 or WP1130 at the indicated concentrations, DMSO 0.1% was used as a control. After 24 h of treatment, cell viability was analyzed using the resazurin assay method. Data are represented as the mean + SD, and statistically significant differences are indicated as * p < 0.05, ** p < 0.01. n.s no significant differences.

Article Snippet: The adherent cell lines HEK293 (ATCC® CRL-11268G-1TM), Vero clone E6 (ATCC® CRL1586TM), Huh-7 (JCRB0403) and BHK-21 (ATCC® CCL-10TM) (ATCC, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma–Aldrich, St Louis, MI, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO, Carlsbad, CA, USA) at 37 ◦C and 5% CO2.

Techniques: Control, Resazurin Assay