Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: Immunostaining of the EGFR on Hek 293T cells (left) and the MDA MD 468 cells (right). Images were taken via confocal laser scanning microscopy.

Article Snippet: The MDA-MB-468 (ATCC cat. HTB-132) and Hek 293T (ATCC cat. CRL-3519) cell lines were cultured in Thermo ScientificTM NuncTM Cell Culture Treated Flasks with Filter Caps.

Techniques: Immunostaining, Confocal Laser Scanning Microscopy

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: a) Schematic representation of the experimental findings on NR selectivity: the differential binding profile between the targeted cell line (MDA MD 468) and the non-targeted cell line (Hek 293T) with the targeted NRs (NRs_18Ab and NRs_18Apt). For the MDA MD 468 cells, binding events with the targeted NRs are characterized by very long trajectories, indicating specific binding, while the binding trajectories with the Hek 293T are typically much shorter, indicating non-specific binding. b) Representative image of NRs_18Ab trajectories at 60 minutes after their incubation with MDA MD 468 and Hek 293T cells. Cell contours are indicated by the dotted line. Color bar indicates the diffusion coefficients (ranging from 0 to 4 µm 2 /s). c) Scatter plot of all the binding events for non-functionalized NRs, NR_18Ab and NR_18Apt (i.e. all the trajectories with D ≤ 1), plotted against their respective trajectory length (y-axis). d) Bar plot displaying the differential specific binding percentage between MDA MD 468 and Hek 293T for the different NR designs (NR_18Ab and NR_18Apt) at 3 distinct time points (10 min, 30 min and 60 min). Results are shown as the mean +-standard error of the mean. n = 3 biological replicates (per biological replicate, the trajectories of 5 different movies were combined, i.e. 5 technical replicates) *: significant difference between groups with p ≤ 0.05, ** : significant difference between groups with p ≤ 0.01, *** : significant difference between groups with p ≤ 0.001.

Article Snippet: The MDA-MB-468 (ATCC cat. HTB-132) and Hek 293T (ATCC cat. CRL-3519) cell lines were cultured in Thermo ScientificTM NuncTM Cell Culture Treated Flasks with Filter Caps.

Techniques: Binding Assay, Incubation, Diffusion-based Assay

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: a) Scheme of the binding kinetics between targeted NRs (NR_18Ab and NR_18Apt) and cells (left panel). The corresponding formula of the binding kinetics is displayed in the right panel. b) Comparison of the total number of specific binding events between NRs with 8 or 18 binding ligands (antibody or aptamer) in both MDA MD 468 and Hek 293T cells. The amount of binding events can be directly related to k on . c) Example of the exponential decay fitting for the binding time of NR functionalized with 18 antibodies in MDA MD 468 cells, where dotted line represents the fitted courve and the corresponding equation is displayed. d) τ B values obtained via an exponential decay fitting of all the binding events for each NR design and cell type. This value is inversely proportional to k off . A comparison was made between NRs with 8 or 18 binding ligands (antibody or aptamer) in both MDA MD 468 or Hek 293T. Results are shown as mean fitted value, where error bars represent the standard error of the fitting for each NR design in the different cell lines.

Article Snippet: The MDA-MB-468 (ATCC cat. HTB-132) and Hek 293T (ATCC cat. CRL-3519) cell lines were cultured in Thermo ScientificTM NuncTM Cell Culture Treated Flasks with Filter Caps.

Techniques: Binding Assay, Comparison

Journal: The Journal of Experimental Medicine

Article Title: GSAP regulates lipid homeostasis and mitochondrial function associated with Alzheimer’s disease

doi: 10.1084/jem.20202446

Figure Lengend Snippet: GSAP and its binding proteins are involved in novel biological pathways. (A) Schematic of the experimental design to characterize the GSAP interactome. HA-EV was used as a negative control. (B) GO pathway enrichment analysis for GSAP-binding proteins. Top 20 significantly enriched pathways (P < 0.05) are shown based on P value (dot color) and gene count (dot size). (C) KEGG biological process enrichment analysis for GSAP-binding proteins. Top 20 significantly enriched pathways (P < 0.05) are shown based on P value (dot color) and gene count (dot size). (D) Volcano plot showing differentially enriched proteins (detailed in the methods) in HA-GSAP versus HA-EV co-IP MS experiments in N2a cells. GSAP itself (purple), proteins involved in trafficking (blue), and phosphorylation (red) are highlighted. FC, fold change. (E) Venn diagram showing overlapped protein between different lists. The circle area is not proportional to the sample size. (F) Meta-enrichment analysis of common GO biological pathways shared by two GSAP-binding protein lists. (G) Co-IP validation of GSAP interaction with PP1 and δ-COP (Arcn1) in HEK293T or N2a cells, respectively, via transient transfection. Representative data of three experiments.

Article Snippet: HEK293T cells (ATCC; CRL-11268) and HEK293-APP WT and GKO cells were grown in DMEM containing 10% FBS ( ).

Techniques: Binding Assay, Negative Control, Co-Immunoprecipitation Assay, Phospho-proteomics, Biomarker Discovery, Transfection

Journal: The Journal of Experimental Medicine

Article Title: GSAP regulates lipid homeostasis and mitochondrial function associated with Alzheimer’s disease

doi: 10.1084/jem.20202446

Figure Lengend Snippet: GSAP-binding protein and antibody validation. (A) Heatmap showing GSAP and binding protein levels in bait-expressing (HA-GSAP) versus EV (empty vector expression) samples in N2a co-IP and MS analyses. Proteins enriched in HA-GSAP samples are shown; mitochondrial proteins are highlighted in red. (B) GO biological process association for GSAP from experimental data and computational prediction (humanbase database; http://hb.flatironinstitute.org/gene/54103 ). *, based on previous experimental data. (C) GSAP-binding proteins identified through Y2H were visualized by the STRING App in Cytoscape. (D) Co-IP analysis of GSAP (HA-tagged) interaction with PHB (Flag-tagged) using Flag antibody. Representative data of two experiments. (E) HA-tagged human GSAP plasmid was transfected into HEK293T cells together with control (C) or GSAP siRNA. 48 h after transfection, cell lysates were collected and subjected to SDS-PAGE and immunoblot analysis. GSAP antibody from Thermo Fisher Scientific (Thermo) or R&D Systems (R&D) was used to detect GSAP. Representative data of two experiments.

Article Snippet: HEK293T cells (ATCC; CRL-11268) and HEK293-APP WT and GKO cells were grown in DMEM containing 10% FBS ( ).

Techniques: Binding Assay, Biomarker Discovery, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay, Transfection, Control, SDS Page, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: GSAP regulates lipid homeostasis and mitochondrial function associated with Alzheimer’s disease

doi: 10.1084/jem.20202446

Figure Lengend Snippet: GSAP interacts with Fe65 to regulate APP phosphorylation and trafficking. (A) Immunoblot analysis of protein levels in N2a695 cells transfected with control or GSAP siRNA (left panel). Quantification of APP phosphorylation at Thr668 normalized to total APP level (right panel). Data represent mean ± SEM; unpaired t test, **, P < 0.01. pT668, phospho-Thr668. Representative data of four experiments. (B) Co-IP analysis of full-length GSAP (HA-tagged) interaction with full-length Fe65 (Flag-tagged) using Flag antibody in HEK293T cells. Representative data of two experiments. (C) Co-IP analysis of GSAP C-terminal 16K domain (HA-tagged) coprecipitation with full-length Fe65 (Flag-tagged) using a Flag antibody in HEK293T cells. Representative data of two experiments. (D) Co-IP analysis of endogenous Fe65 interaction with GSAP and PP1 using Fe65 antibody in HEK293T cells. GSAP was detected using an antibody from R&D Systems. Representative data of two experiments. (E) Genomic DNA from CAD WT and Fe65KO cells was isolated, and PCR-amplified fragments flanking the CRISPR-Cas9 cleavage site were generated. PCR fragments were cloned into TOPO vector for Sanger sequencing. A 1-bp insertion (red) and deletion (blue) was identified in Fe65KO CAD cells (upper panel). Immunoblot analysis of proteins from WT and Fe65KO CAD cells (lower panel). (F) Immunoblot analysis of protein levels in CAD cells transiently overexpressing APP (left panel). Quantification of APP phosphorylation at Thr668 normalized to total APP level (right panel). Data represent mean ± SEM; unpaired t test, *, P < 0.05. Representative data from two experiments. (G) Representative confocal microscopy of Fe65 (red) and APP (green) localization in differentiated CAD cells. Arrow denotes the structure of Golgi apparatus. Scale bar, 5 µm. Representative data of ten cells. A.U., arbitrary units. (H) Maximum intensity projection of Airyscan Z-stack of WT (top left) and Fe65KO (top right) CAD cells from 95 slices and 0.173-µm step size and generated in Imaris. Scale bars, 5 µm. The images are representative of four independent experiments. WT (bottom left) and Fe65KO (bottom right) trajectories corresponding to the representative time-lapse image series are shown in the top panel and were reconstructed in MATLAB. Trajectory minimum cutoff time is 10 s. (I) Violin plots showing the velocity (left) and diffusion coefficient (right) distributions of single APP-GFP vesicles in WT and Fe65KO CAD cells. The median value is shown as the horizontal line in the box. The box presents interquartile range. The distributions were compared using the Mann–Whitney U test (**, P < 0.001; WT V median = 1.016 µm/s, KO V median = 1.038 µm/s; WT D median = 0.0187 μm2/s, and KO D median = 0.0290 μm2/s). (J) Co-IP analysis of GSAP (HA-tagged) with APP-C99 (Flag-tagged) in WT and Fe65KO (FKO) CAD cells. Representative data of two experiments. (K) Schematic of protein domain interactions within the APP–Fe65–GSAP complex. AICD, APP intracellular domain.

Article Snippet: HEK293T cells (ATCC; CRL-11268) and HEK293-APP WT and GKO cells were grown in DMEM containing 10% FBS ( ).

Techniques: Phospho-proteomics, Western Blot, Transfection, Control, Co-Immunoprecipitation Assay, Isolation, Amplification, CRISPR, Generated, Clone Assay, Plasmid Preparation, Sequencing, Confocal Microscopy, Diffusion-based Assay, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Rapamycin enhances CAR-T control of HIV replication and reservoir elimination in vivo

doi: 10.1172/JCI185489

Figure Lengend Snippet: ( A ) Humanized NSG-BLT mice were constructed with either unmodified HSCs or HSCs modified with D1D2CAR41BB. After immune reconstitution, mice were infected with HIV-1 NFNSXL9 . Four weeks after infection, mice with CAR-modified HSCs were treated with rapamycin or vehicle for 2 weeks. ( B – E ) Representative flow and average percentage of HLA-DR ( B ), CD38 ( C ), PD-1 ( D ), and Tim-3 ( E ) expression among CAR + CD3 + T cells before and after HIV infection as measured by flow cytometry (quantified by gating of percentage positive ± SEM) ( n = 15–25 each group). ( F ) Plasma HIV viral load from mock or anti-HIV CAR mice at 1 week and 3 weeks of infection ( n = 15–18 each group). ( G ) Average percentage of PD-1, Tim-3, HLA-DR, and CD38 expression among blood CAR + CD3 + T cells before and after rapamycin treatment ( n = 10–15 each group). ( H ) Plasma HIV RNA copies from mock mice or CAR mice after 2 weeks of rapamycin or vehicle treatment (5 weeks after HIV infection) ( n = 9–11 each group). The Mann-Whitney test was used to compare 2 groups, and the Kruskal-Wallis test was used for multiple comparisons ( B – E and H ). Each dot represents an individual mouse; horizontal bars indicate median values. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The lentivirus-based D1D2CAR41BB vectors were produced in Lenti-X 293T cells (Takara Bio) using the Lipofectamine 2000 reagent (Invitrogen).

Techniques: Construct, Modification, Infection, Expressing, Flow Cytometry, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Rapamycin enhances CAR-T control of HIV replication and reservoir elimination in vivo

doi: 10.1172/JCI185489

Figure Lengend Snippet: ( A ) Humanized NSG-BLT mice with D1D2CAR41BB-modified HSCs were treated with rapamycin or vehicle for 2 weeks before necropsy. Afterward, splenocytes were isolated, GFP + CAR cells were sorted, and bulk RNA sequencing was performed ( n = 3 per group). ( B ) Representative flow cytometry analysis showing the gating strategy for sorting of GFP + CAR single cells. ( C and D ) Principal component analysis (PCA) ( C ) and heatmap ( D ) showing the relative expression ( z score) of the top 5,000 genes that were differentially expressed between the 2 populations of CAR-T cells derived from rapamycin-treated versus vehicle-treated CAR mice. Genes were divided into downregulated (green) and upregulated (pink) clusters by k -means clustering based on expression. ( E ) Box plot of log-normalized counts of genes important in T cell survival, activation, and exhaustion. Box plots show the interquartile range, median (line), and minimum and maximum (whiskers). ( F ) KEGG pathway analysis of differentially expressed genes among CAR-T cells between rapamycin-treated and vehicle-treated CAR mice. “GeneRatio” is the percentage of total DEGs in the given Gene Ontology term.

Article Snippet: The lentivirus-based D1D2CAR41BB vectors were produced in Lenti-X 293T cells (Takara Bio) using the Lipofectamine 2000 reagent (Invitrogen).

Techniques: Modification, Isolation, RNA Sequencing, Flow Cytometry, Expressing, Derivative Assay, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: Rapamycin enhances CAR-T control of HIV replication and reservoir elimination in vivo

doi: 10.1172/JCI185489

Figure Lengend Snippet: ( A ) Humanized NSG-BLT mice with either D1D2CAR41BB-modified or nonmodified HSCs were infected with HIV and treated with rapamycin or vehicle for 2 weeks. Afterward, while rapamycin or vehicle treatment was continued, mice were treated with ART for 4 weeks, followed by ART interruption for 2–3 weeks. ( B ) PD-1, Tim-3, HLA-DR, and CD38 expression was measured by flow cytometry (quantified by gating of percentage positive cells) on peripheral blood (top) or spleen (bottom) CD3 + CAR + T cells ( n = 8–10 per group). ( C ) Splenocytes from CAR mice treated with ART and rapamycin or vehicle were isolated and stained with intracellular antibodies against human TOX1. MFIs of the TOX1 on CAR + T cells were measured by flow cytometry ( n = 5 per group). ( D ) Longitudinal HIV viral load in plasma from humanized mice after rapamycin or vehicle treatment was measured by real-time PCR ( n = 4–7 per group). Dotted line indicates limit of detection. ( E ) HIV DNA copies per cell from PBMCs, splenocytes, bone marrow, or thymus implant from different groups of mice as measured by real-time PCR. Human RPP30 gene was used as internal control ( n = 7–9 per group). ( F ) Relative HIV cellular RNA expression from multiple lymphoid tissues from different groups of mice as measured by real-time PCR. Human HPRT1 gene expression was used as internal control ( n = 7–8 per group). The Mann-Whitney test was used to compare 2 groups, and the Kruskal-Wallis test was used for multiple comparisons ( D – F ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The lentivirus-based D1D2CAR41BB vectors were produced in Lenti-X 293T cells (Takara Bio) using the Lipofectamine 2000 reagent (Invitrogen).

Techniques: Modification, Infection, Expressing, Flow Cytometry, Isolation, Staining, Real-time Polymerase Chain Reaction, Control, RNA Expression, Gene Expression, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Rapamycin enhances CAR-T control of HIV replication and reservoir elimination in vivo

doi: 10.1172/JCI185489

Figure Lengend Snippet: ( A ) Humanized NSG-IL-15-BLT mice were constructed with either unmodified HSCs or HSCs modified with D1D2CAR41BB. After immune reconstitution, mice were infected with HIV-1 NFNSXL9 . Four weeks after infection, mock mice were treated with ART only. Mice with CAR-modified HSCs were treated with rapamycin or vehicle along with ART. After successful viral load suppression, ART was interrupted, and rapamycin or vehicle treatment continued for 2 additional weeks before discontinuation ( n = 4–6 per group). ( B ) Longitudinal plasma HIV viral load as measured by real-time PCR. ( C ) Survival analysis of time to detectable viral load among mock mice and CAR mice that were treated with vehicle or rapamycin. P < 0.0001 by log-rank test. ( D ) HIV DNA and relative cellular HIV RNA expression from blood PBMCs, splenocytes, and bone marrow as measured by real-time PCR. Mann-Whitney test (unpaired); * P < 0.05, ** P < 0.01.

Article Snippet: The lentivirus-based D1D2CAR41BB vectors were produced in Lenti-X 293T cells (Takara Bio) using the Lipofectamine 2000 reagent (Invitrogen).

Techniques: Construct, Modification, Infection, Real-time Polymerase Chain Reaction, RNA Expression, MANN-WHITNEY