incucyte zoom  (Sartorius AG)

Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Detection of SARS-CoV-2 infected cells using high-content imaging under control conditions. Fluorescent signals: green (anti-SARS-CoV NP mAb), blue (Hoechst). (B) Representative flow cytometry analysis plots showing NP+ cells. (C) Measurement of fluorescent intensity in NP+ cells. (D) Percentage of infected MSCs. (E) The supernatants of infected MSCs were collected and titrated on Vero-E6/TMPRSS2 cells to measure infectious viral particle production using the focus-forming assay. (F) After RNA extraction from infected MSCs, the expression of IDO1 was examined using real-time PCR. The graph displays the expression of IDO1 normalized to GAPDH , with the mock group serving as the control. Data are represented as means ± SEM from three independent biological replicates. For , the Delta condition was available from only two independent donors and was therefore presented descriptively and excluded from the statistical analysis. Statistical comparisons among the remaining groups were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. One-way ANOVA showed a significant overall difference among groups, F(2, 6) = 6.794, P = 0.0287. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: The Operetta high-content imaging system (PerkinElmer) was used to detect and analyze the fluorescent signals.

Techniques: Infection, Imaging, Control, Flow Cytometry, Analysis, Focus Forming Assay, RNA Extraction, Expressing, Real-time Polymerase Chain Reaction

Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Representative western blot depicting the expression of NRP1 in AD-MSCs (No siRNA), cells transfected with non-targeting siRNA (siControl), or siNRP1 for 24 hours. β-actin was used as a loading control. (B) Graph displaying the quantification of NRP1 normalized to β-actin (n = 3). This experiment represents three independent biological replications. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. (C) Transfected cells were visualized using high-content imaging. Fluorescent signals: green for anti-NRP1 mAb, blue for Hoechst. (D) Percentage of NRP1-expressing cells normalized to cells transfected with siControl. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was conducted using a paired t-test. Next, siRNA-transfected cells were infected with different variants of SARS-CoV-2. (E) SARS-CoV-2-infected cells were detected using high-content imaging. Fluorescent signals: green for anti-SARS-CoV NP mAb, blue for Hoechst. (F) The fluorescent intensity of NP+ cells was measured. All data represent three independent experiments. Statistical analysis among each group of variant was performed using an unpaired t-test. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: The Operetta high-content imaging system (PerkinElmer) was used to detect and analyze the fluorescent signals.

Techniques: Western Blot, Expressing, Transfection, Control, Analysis, Imaging, Infection, Variant Assay

Journal: Clinical Cancer Research

Article Title: The Pharmacologic Profile of ART6043, a First-in-Class Clinical DNA Polymerase Theta Polymerase Domain Inhibitor Potentiating PARP Inhibitor Efficacy

doi: 10.1158/1078-0432.CCR-26-0493

Figure Lengend Snippet: ART6043 potentiates PARP inhibition in vitro . A, Representative CTG assay combination matrix for DLD-1 BRCA2 −/− (left) and DLD-1 wild-type (right) cells treated with titrated ART6043 and talazoparib for 14 days. Intensity of blue color indicates the level of synergy. % Control indicates relative viability. B, Representative IncuCyte growth curves for DLD-1 BRCA2 −/− cells treated with ART6043 or talazoparib as monotherapy or in combination at the indicated doses for 14 days. C, As for A but using olaparib instead of talazoparib. D, As for B but using olaparib instead of talazoparib. ART6043 concentrations represent free drug in tissue culture medium.

Article Snippet: Cells were imaged on the IncuCyte Live-Cell Imaging System (Essen Biosciences, RRID:SCR_023147).

Techniques: Inhibition, In Vitro, CTG Assay, Control, Tissue

Journal: Clinical Cancer Research

Article Title: Preclinical Characterization of CLSP-1025, a First-in-Class, Mutation-Specific T-Cell Engager Targeting a Neoantigen Derived from a Common p53 Mutation

doi: 10.1158/1078-0432.CCR-26-1252

Figure Lengend Snippet: CLSP-1025 demonstrates functional activity in various cellular systems including cell cocultures and patient-derived organoids. A, CLSP-1025–mediated cytotoxicity in cocultures of PBMCs and HLA-A*02:01 + –p53 R175H+ tumor cells. Cytotoxicity is reported as the percent tumor-cell death after a 96-hour coculture (mean ± SEM; n = 2). B, HLA-A*02:01 surface expression on p53 R175H+ TYK-nu cells after 72 hours of IFN-γ treatment, measured by geometric mean fluorescence intensity (gMFI) via flow cytometry. Data shown as mean; n = 3 technical replicates. C, CLSP-1025–mediated cytotoxicity in PBMC cocultures with TYK-nu cells with or without IFN-γ pretreatment, shown as percent tumor-cell death (mean ± SEM; n = 3 technical replicates). D–E, CLSP-1025–mediated cytotoxicity and IFN-γ secretion in PBMC cocultures with HLA-A*02:01 + –p53 R175H + adenocarcinoma organoids, measured by percent tumor-cell death and MSD, respectively [mean ± standard deviation (SD); n = 1 PBMC donor and technical duplicates for IFN-γ measurements]. F and G, CLSP-1025–mediated cytotoxicity and IFN-γ secretion in PBMC cocultures with HLA-A*02:01 + –p53 WT adenocarcinoma organoids. H, CLSP-1025–mediated killing of GFP + KLE cells when cocultured with isolated CD4 + , CD8 + , or bulk CD3 + T cells. GFP signal was quantified in real time using Incucyte and plotted as normalized fluorescence units [GFP fluorescence units (GFU) × μm 2 /image]. Data shown as mean ± SD; n = 2 technical replicates.

Article Snippet: The plates were incubated in an Incucyte S3 imager (Sartorius: Incucyte S3 live-cell analysis system, RRID: SCR_023147) for 160 hours and whole-well images captured every 4 hours.

Techniques: Functional Assay, Activity Assay, Patient-Derived Organoids, Expressing, Fluorescence, Flow Cytometry, Organoids, Standard Deviation, Isolation