Journal: Cancer Research

Article Title: An Oncolytic Virus Expressing a T-cell Engager Simultaneously Targets Cancer and Immunosuppressive Stromal Cells

doi: 10.1158/0008-5472.can-18-1750

Figure Lengend Snippet: Figure 2. EnAd-expressing FAP-BiTE induces T-cell–dependent cytotoxicity of stromal fibroblasts. A, Genome of the BiTE-armed oncolytic adenovirus, EnAd. ITR, inverted terminal repeat; P, promoter; pA, polyadenylation site. B, Genome replication of parental EnAd- or BiTE-expressing viruses in DLD cells infected with 100 vp/cell. C, Viability of DLD cells infected with EnAd or recombinant virus (MTS; 5 days postinfection). D and E, T-cell activation (D; CD25þ) and target cell cytotoxicity (E; LDH release) in cocultures of T cells, target cells, and infected HEK293A supernatants 24 hpi. F, Viability of NHDF and SKOV3 (4:1) cells monitored by xCELLigence in the absence or presence of CD3-purified PBMCs (5:1 effector:target). Mean (solid line) SD (dotted line) of biological triplicates. G, T-cell activation (CD25þ) in virus-infected cocultures of NHDF and SKOV3 cells. H, Representative images showing coculture of NHDF (red; stained with CellTracker Orange CMTMR), DLD cells (unstained), and T cells (blue; stained with CellTrace Violet), followed by infection with EnAd or BiTE-armed virus. Apoptosis was visualized using CellEvent Caspase 3/7 Detection Reagent (green). White arrow, dead fibroblasts. Scale bar, 100 mm. Data show mean SD of biological triplicates (D-G). Significance assessed using one-way ANOVA with Tukey post hoc analysis compared with "EnAd" (B and C) or "uninfected" (D–G; , P < 0.05; , P < 0.001).

Article Snippet: Recombinant BiTE protein was produced by transfecting HEK293A cells with polyethylenimine [PEI, linear, MW 25000, Polysciences; DNA:PEI ratio of 1:2 (w/w)].

Techniques: Expressing, Infection, Recombinant, Virus, Activation Assay, Staining

Journal: PLoS ONE

Article Title: siRNA Has Greatly Elevated Mismatch Tolerance at 3′-UTR Sites

doi: 10.1371/journal.pone.0049309

Figure Lengend Snippet: (A,B) Northern blot was performed with mRNA transcripts harboring perfectly matched or single-nucleotide mismatched targets of siR-04, located in CDS or 3′-UTR respectively. Reporter plasmids mutated at position 12 or 10 at each location were co-transfected into HEK293A cells with (+) or without (-) the siRNA, and then the amount of reporter mRNA was evaluated using biotin-labeled RNA probe. 18S rRNA was included as loading control. (C,D) Comparison of gene silencing in mRNA and protein levels at position 12 (C) and 10 (D). The intensity of mRNA bands was quantified by ImageJ software, while silencing efficacy of siRNA on protein level was reflected by reporter enzyme activity. The target site location and siRNA:mRNA match pattern were given under the x-axis. The y-axis represented protein yield by the remaining mRNAs, and this repression ratio of siR-04 targeting to mismatched site was normalized to that targeting to perfectly matched site (WT) at either location. Error bars represented SD. All experiments were performed at least twice.

Article Snippet: Fifty-six hours after transfection of 100 nM AGO-specific siRNAs, total proteins were extracted from HEK293A cells by cell lysis buffer (Cell Signaling, Danvers, MA).

Techniques: Northern Blot, Transfection, Labeling, Control, Comparison, Software, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: BMPER Improves Vascular Remodeling and the Contractile Vascular SMC Phenotype

doi: 10.3390/ijms24054950

Figure Lengend Snippet: BMPER physically interacts with insulin-like growth factor-binding protein 4 (IGFBP4). ( A ) Representative photomicrographs of in situ proximity ligation assay (PLA) showing the interaction between BMPER and IGFBP4. Human vSMCs were either incubated in 0.4% FBS/EBM or treated with IGF1 (c = 10 ng/mL) for 20 min before the association of BMPER and IGFBP4 was visualized using an in situ PLA (white). As control, respective rabbit and mouse primary IgGs were used to assess unspecific antibody interactions. Nuclei were stained with DAPI (blue). Scale bars: 20 µm. ( B , C ) HEK293A cells were transfected with BMPER-Myc and IGFBP4-V5 or empty vector for 24 h. ( B ) Immunoprecipitation (IP) of V5-tagged IGFBP4 using a V5-specific antibody was performed. ( C ) BMPER was immunoprecipitated with a BMPER-specific antibody from HEK293A cell lysates 24 h post-transfection. Normal IgG antibody served as IP control (-). Input represents lysate not subjected to IPs. IPs were repeated at least 3 times. ( D , E ) BMPER and IGFBP4 protease activity assay. ( D ) Twenty-four hours after plasmid transfection in HEK293A cells, lysates were subjected to Western blot analysis to verify equal overexpression of IGFBP4 between samples. TUBULIN served as loading control. ( E ) Cell-free supernatants were preincubated with IGF1 (c = 25 ng/mL) as indicated before PAPP-A (c = 150 ng/mL) was added. Afterwards, supernatants were concentrated and subjected to Western blot analysis. Arrow indicates cleaved IGFBP4 at approximately 14 kDa, which is quantified in the diagram below. Ponceau S stain served as loading control. Data are mean values ±SEM; n = 6; One-way ANOVA; * p < 0.001 vs. all other conditions. ( F ) Representative surface plasmon resonance (SPR) sensorgram showing the interaction of immobilized IGFBP-4 with soluble BMPER (KD of 6.04 ± 1.86 nM). The 0 – 320 nM BMPER diluted in HBS-EP buffer was injected for 180 s (injection stop) followed by a dissociation for another 180 s. ( G ), Representative BMPER and IGFBP4 PLA photomicrographs of RCCA and LCCA 14 days after carotid ligation. Association of BMPER and IGFBP4 in white dots and nuclei in blue (DAPI). Scale bars: 20 µm.

Article Snippet: For plasmid transfection in HEK293A cells, FuGENE HD transfection reagent (Promega; Mannheim, Germany) was used.

Techniques: Binding Assay, In Situ, Proximity Ligation Assay, Incubation, Control, Staining, Transfection, Plasmid Preparation, Immunoprecipitation, Activity Assay, Western Blot, Over Expression, SPR Assay, Injection, Ligation

Journal: Journal of Biological Chemistry

Article Title: Constitutive role of the Fanconi anemia D2 gene in the replication stress response

doi: 10.1074/jbc.m117.814780

Figure Lengend Snippet: Figure 5. FANCD2 possesses replication fork protection function in the absence of monoubiquitination (A) Immunoblotting of FANCD2 in HEK293A cell background with indicated genotypes. FANCD2- KR: homozygous knock-in of K561R substitution; FANCD2-/--1 and FANCD2-/--2: two independent FANCD2 knockout clones; FANCD2 L: monoubiquitinated form of FANCD2; FANCD2 S: unmodified FANCD2. Cells were treated with 200 ng/ml MMC for 12 h before harvesting for extract preparation. (B) Immunoblotting of γH2AX in cells with indicated genotypes. Protein extracts were prepared from unperturbed proliferating cells. (C) γH2AX immunostaining in 293A WT, FANCL-/-, FANCD2-/-, and FANCD2-KR cells. KR1 and KR2 are two independent homozygous FANCD2 K561R knock-in clones. (D) Quantification of γH2AX foci in (C). (E) Chromosomal aberrations in 293A WT, FANCL-/-, FANCD2-/-, and FANCD2-KR cells treated with 50 ng/ml MMC for 24 hours. Red line marks the mean value of each mutant. P values are derived by the Student’s t-test.

Article Snippet: Cell Culture and Antibodies - HeLa and HEK293A cells were obtained from ATCC.

Techniques: Western Blot, Knock-In, Knock-Out, Clone Assay, Immunostaining, Mutagenesis, Derivative Assay