Journal: bioRxiv

Article Title: Temporal analyses of CRISPR-directed gene editing on NRF2, a clinically relevant human gene involved in chemoresistance

doi: 10.1101/799676

Figure Lengend Snippet: Time course analysis of fluorescently labeled RNP using FACS. A549 cells were transfected with 20 pmol of fluorescently labeled RNP complex and analyzed via FACS to determine fluorescence intensity at 1, 4, 8, 12, 24, and 48 hours post nucleofection. The red area (P1) of the left hand graph contains the population of A549 cells that were further analyzed for fluorescence intensity. Quadrant 1 (Q1) would contain cells with GFP fluorescence. Quandrant 2 (Q2) would contain cells with both GFP and ATTO 550 fluorescence. Quadrant 3 (Q3) would contain cells that do not exceed background fluorescence. Quadrant 4 (Q4) would contain cells with ATTO 550 fluorescence. A549 cells with both fluorescently labeled RNP components – Cas (GFP) and tracrRNA (ATTO550), fall within quadrant 2 (outlined in green). The table under each plot contains raw data for each quadrant.

Article Snippet: Human lung carcinoma A549 cells were purchased from ATCC (#CCL-185) (Manassas, VA, USA).

Techniques: Labeling, Transfection, Fluorescence

Journal: bioRxiv

Article Title: Temporal analyses of CRISPR-directed gene editing on NRF2, a clinically relevant human gene involved in chemoresistance

doi: 10.1101/799676

Figure Lengend Snippet: (A) Time course analysis of the cellular localization of fluorescently labeled RNP. A549 cells were transfected with 20 pmol of fluorescently labeled RNP complex and seeded in 4-well chambers for imaging. Representative images of each time point are shown in the figure depicting localization of the fluorescently labeled RNP complex. The brightness of the images is enhanced to better visualize the localization of the RNP complex. Scale bar represents 20 μm. (B) Graphical representation of individual and dual fluorescence from FACS compared to indel formation. The graph displays the %Total of each quadrant at each time point. Dual Label RNP (Q2) represents each %Total from quadrant 2, which contains cells with both fluorescent components. INDEL represents each percentage for total indel efficiency by TIDE analysis of an unsorted bulk population transfected with the dual labeled RNP. Total Fluor (Q1+Q2+Q4) represents the sum of quadrant 1, 2, and 4 to assess the total population of cells with fluorescence. TracrRNA ATTO 550 (Q4) represents each %Total from quadrant 3, which contains cells with ATTO 550 fluorescence only. Cas9 GFP (Q1) represents each %Total from quadrant 1, which contains cells with GFP fluorescence only. (C) Western blot analysis of Cas9 maintenance in cells. A549 cells were transfected with CRISPR/Cas9 RNP targeting Neh2 in Exon 2 and harvested at the indicated time points for western blot analysis using an antibody directed against spCas9. An antibody directed against GAPDH was used as the loading control.

Article Snippet: Human lung carcinoma A549 cells were purchased from ATCC (#CCL-185) (Manassas, VA, USA).

Techniques: Labeling, Transfection, Imaging, Fluorescence, Western Blot, CRISPR, Control

Journal: bioRxiv

Article Title: Temporal analyses of CRISPR-directed gene editing on NRF2, a clinically relevant human gene involved in chemoresistance

doi: 10.1101/799676

Figure Lengend Snippet: Time course analysis of gene editing activity of fluorescently labeled RNP. The total, unsorted population of A549 cells transfected with 20 pmol of fluorescently labeled RNP complex was collected as each time point. Genomic DNA was Sanger sequenced and analyzed for indel efficiency by TIDE. For comparison, the %Total of cells with both fluorescently labeled RNP components (Quadrant 2) from FACS analysis is listed adjacent to the total indel efficiency.

Article Snippet: Human lung carcinoma A549 cells were purchased from ATCC (#CCL-185) (Manassas, VA, USA).

Techniques: Activity Assay, Labeling, Transfection, Comparison

Journal: PLoS ONE

Article Title: Transient Transfection of a Wild-Type p53 Gene Triggers Resveratrol-Induced Apoptosis in Cancer Cells

doi: 10.1371/journal.pone.0048746

Figure Lengend Snippet: MCF-7 (A), H1299 (B), A549 (C), and H460 cells (D) were treated with various concentrations of trans -resveratrol (10–500 µM) diluted in DMSO for 5 min, 24 h and 48 h. The cell viability was measured by the MTT assay. The final concentration of DMSO in the culture medium was 0.5%. The results (n = 4) are expressed as a % of the control value, and the data presented are mean ± S.E.M.

Article Snippet: Human breast carcinoma cell line MCF-7 (p53 wild-type), human non-small lung carcinoma cell line H1299 (p53 negative), adenocarcinomic human alveolar basal epithelial cell line A549 (p53 wild-type), and human non-small lung carcinoma cell line H460 (p53 wild-type) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: MTT Assay, Concentration Assay, Control

Journal: PLoS ONE

Article Title: Transient Transfection of a Wild-Type p53 Gene Triggers Resveratrol-Induced Apoptosis in Cancer Cells

doi: 10.1371/journal.pone.0048746

Figure Lengend Snippet: Effects of resveratrol on p53 and MDM2 protein levels (A) and on p53 mRNA levels (B) in MCF-7 cells. The cells were exposed to various concentrations of resveratrol or DMSO (0.5%) for 24 h. The protein levels were determined by western blotting analysis as described in . GAPDH expression was used as a control. In panels A and B the data represent 3–5 independent experiments, and the band intensities are graphically represented with each group of images. (C) MCF-7 cell viability in the presence of the p53-specific inhibitor pifithrin-α. The cells were exposed to 30 µM pifithrin for 1 h prior to treatment with various trans -resveratrol concentrations (50–200 µM). The cell viability was measured by the MTT assay. The results (n = 3) are expressed as a % of the control, and the data shown are mean ± S.E.M. * p <0.05 in comparison with the respective control (Student's t -test).

Article Snippet: Human breast carcinoma cell line MCF-7 (p53 wild-type), human non-small lung carcinoma cell line H1299 (p53 negative), adenocarcinomic human alveolar basal epithelial cell line A549 (p53 wild-type), and human non-small lung carcinoma cell line H460 (p53 wild-type) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Western Blot, Expressing, Control, MTT Assay, Comparison

Journal: PLoS ONE

Article Title: Transient Transfection of a Wild-Type p53 Gene Triggers Resveratrol-Induced Apoptosis in Cancer Cells

doi: 10.1371/journal.pone.0048746

Figure Lengend Snippet: (A) p53-transfected and non-transfected H1299 cells were treated with resveratrol (100 µM). The cell viability was measured by the MTT assay. The results (n = 4) are expressed as % of control, and the data shown are mean ± S.E.M. * p <0.05 in comparison with the respective control (Student's t -test). (B) The cells were transiently transfected with the full-length p53-EGFP plasmid and treated with resveratrol (100 µM). The cells were then double-labeled with Hoechst 33342 (Molecular Probes, Eugene, OR, USA) and with Click-iT® TUNEL Alexa Fluor® Imaging Assay (Invitrogen, Carlsbad, CA, USA). Hoeschst fluorescence: EX/EM ∼350 nm/461 nm. TUNEL fluorescence: EX/EM ∼495 nm/519 nm. In the merged images, the arrows indicate cell death of p53-transfected cells. (C) The rate of transfection efficiency for H1299 cells. (D) The quantification of apoptotic and non-apoptotic transfected H1299 cells following exposure to resveratrol.

Article Snippet: Human breast carcinoma cell line MCF-7 (p53 wild-type), human non-small lung carcinoma cell line H1299 (p53 negative), adenocarcinomic human alveolar basal epithelial cell line A549 (p53 wild-type), and human non-small lung carcinoma cell line H460 (p53 wild-type) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Transfection, MTT Assay, Control, Comparison, Plasmid Preparation, Labeling, TUNEL Assay, Imaging, Fluorescence

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: ApoSense : a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis

doi: 10.1007/s00259-005-1905-x

Figure Lengend Snippet: Concomitant confocal imaging of HeLa cell undergoing apoptosis by DDC (green fluorescence) and annexin V (red fluorescence). DDC accumulates within the cytoplasm of the apoptotic cell while annexin V is attached to the external membrane

Article Snippet: Human adult T-cell leukemia Jurkat cells (clone E6-1) and cervical carcinoma HeLa cells (CCL-2.2), were purchased from ATCC (Rockville, MD, USA) and grown in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum at 37°C in 5% CO 2 .

Techniques: Imaging, Fluorescence, Membrane

Journal: Scientific Reports

Article Title: Quinoline carboxylic acid derivatives as potent ectonucleotidase inhibitors

doi: 10.1038/s41598-026-36994-1

Figure Lengend Snippet: ( A ) Fluorescence spectroscopy and microscopy analysis of the antagonist drug molecule 4d. ( A ) Fluorescence spectroscopy profiles of 4d , excited between 410 and 500 nm, with corresponding emissions between 400 and 600 nm. ( B ) The bright-field image of A549 cells; Fluorescence microscopy image (red channel) of A549 cells treated with compound 4d; Merged image combining bright-field and fluorescence channels, highlighting the localization of the compound 4d on the cell membrane.

Article Snippet: Fluorescence imaging experiments were performed using A549 human lung carcinoma cells from American Type Cultures Collection (ATCC, Manassas, VA), while untreated A549 cells were used as controls in fluorescence imaging to account for background autofluorescence and confirm the specificity of compound 4d uptake and signal.

Techniques: Fluorescence, Spectroscopy, Microscopy, Membrane

Journal:

Article Title: UXT Is a Novel Centrosomal Protein Essential for Cell Viability D⃞

doi: 10.1091/mbc.E05-08-0705

Figure Lengend Snippet: UXT is a novel centrosomal component. (A) Colocalization of UXT with the centrosome marker protein γ-tubulin. U2OS cells stably expressing EGFP:UXT (green) were labeled with anti-γ-tubulin (red). Images of cells in the G1, S/G2, prometaphase, metaphase, and anaphase are shown. (B) GFP-UXT is localized to the spindle poles during mitosis. U2OS cells stably expressing EGFP:UXT (green) were stained with an anti-α-tubulin (red) antibody. (C) The FLAG:UXT protein is localized to the centrosome. U2OS cells transfected with FLAG:UXT were immunostained with the mouse anti-FLAG antibody (red) and the rabbit anti-γ-tubulin antibody (green).

Article Snippet: Cell Culture and Treatments The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Marker, Stable Transfection, Expressing, Labeling, Staining, Transfection

Journal:

Article Title: UXT Is a Novel Centrosomal Protein Essential for Cell Viability D⃞

doi: 10.1091/mbc.E05-08-0705

Figure Lengend Snippet: Characterization of the anti-UXT antibodies. (A) The anti-UXT antibody 1B2 can detect both the endogenous UXT and the UXT fusion proteins. Cell lysates from HEK293T cell (lane 1), HEK293T cells transfected with EGFP:UXT (lane 2), and HEK293T cells transfected with FLAG-tagged UXT (lane 3) were separated by 15% SDS-PAGE. The expression of UXT was examined with the anti-UXT antibody 1B2 by Western blot analysis. Note that the anti-UXT antibody 1B2 can detect the endogenous UXT protein (18KD), EGFP:UXT (50KD), and FLAG:UXT (25KD). (B and C) Immunofluorescence detection of the endogenous UXT proteins in U2OS cells using the anti-UXT antibodies. U2OS cells were fixed with methanol and stained with anti-γ-tubulin antibody (green) in combination with either the anti-UXT antibody 15A6 (B, in red) or the anti-UXT antibody 6D3 (C, in red). DNA was stained with DAPI (blue). The arrows indicate the centrosomes.

Article Snippet: Cell Culture and Treatments The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Transfection, SDS Page, Expressing, Western Blot, Immunofluorescence, Staining

Journal:

Article Title: UXT Is a Novel Centrosomal Protein Essential for Cell Viability D⃞

doi: 10.1091/mbc.E05-08-0705

Figure Lengend Snippet: Overexpression of UXT disrupts the centrosome structure. (A) Loss of centrosomal γ-tubulin staining in U2OS cells after overexpression of EGFP:UXT. U2OS cells with transient expression of EGFP:UXT (green) were immunostained with the anti-γ-tubulin antibody (red). DNA was stained with DAPI. Note that the γ-tubulin staining on the centrosome is diminished in cells with over expression of GFP-UXT. (B) Electron microscopic image of disorganized centrosome in U2OS cells with overexpression of EGFP: UXT. U2OS cells, transfected with either EGFP or GFP-UXT, were processed for electron microscope imaging. Top, image of the centrosome in U2OS cells transfected with EGFP (control). Bottom, image of the abnormal centrosome in U2OS cells transfected with EGFP:UXT. The arrow indicates a normal centriole.

Article Snippet: Cell Culture and Treatments The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Over Expression, Staining, Expressing, Transfection, Microscopy, Imaging, Control

Journal:

Article Title: UXT Is a Novel Centrosomal Protein Essential for Cell Viability D⃞

doi: 10.1091/mbc.E05-08-0705

Figure Lengend Snippet: UXT siRNA knockdown causes cell death. Two nonoverlapping UXT siRNAs were tested and produced similar results. The data obtained with one UXT siRNA were shown. The data obtained with another UXT siRNA were shown as supplemental data (Figure S2). (A) Efficacy of siRNA knockdown of the endogenous UXT protein. U2OS cells were treated with transfection reagent (NT), nonspecific siRNA (NS siRNA), or siRNA for UXT (UXT siRNA), respectively. The cell lysates were subject to Western blot using either the anti-UXT antibody 1B2 (top) or the anti-β-tubulin antibody (bottom). The protein levels of UXT were specifically reduced after 72-h treatment with siRNA for UXT. (B) UXT knockdown inhibits cell proliferation. U2OS cells were treated with nonspecific siRNA (left) or siRNA for UXT (right). Pictures were taken 72 h later. (C) UXT knockdown leads to cell death. U2OS cells were treated with nonspecific RNA or the UXT siRNA oligos. Seventy-two hours later, all the cells were collected and used for propidium iodide staining of DNA content by fluorescence-activated cell sorting. (D) p53 is not required for cell death caused by UXT knockdown. The HCT116 (p53+/+) or p53-negative HCT116 (p53-/-) cells were treated with either the control siRNA or the UXT siRNA. Seventy-two hours after transfection, the percentage of cell death was assessed by the trypan blue assay. The diagram shows the representative results of three experiments.

Article Snippet: Cell Culture and Treatments The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Knockdown, Produced, Transfection, Western Blot, Staining, Fluorescence, FACS, Control

Journal: Pharmaceutics

Article Title: NIR Stimulus-Responsive PdPt Bimetallic Nanoparticles for Drug Delivery and Chemo-Photothermal Therapy

doi: 10.3390/pharmaceutics12070675

Figure Lengend Snippet: DOX release behaviors and in vitro cytotoxicity of DOX@PdPt@HA NPs. ( A ) NIR laser irradiation triggers release of DOX from DOX@PdPt@HA NPs. ( B ) Cell viability of 4T1 cells treated with different concentration of PdPt@HA NPs, DOX@PdPt@HA NPs or free DOX with or without NIR laser irradiation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ( C , D ) Representative flow cytometry scatter plots of Annexin V/PI and quantification of apoptotic ratio of 4T1 cells with different treatments. ( E ) Fluorescence images of 4T1 cells exposed to the NPs with and without pre-incubation of hyaluronic acid (HA) for 1 h. Value represent mean ± SE of 3 independent experiments. P values: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mouse mammary carcinoma 4T1 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and human foreskin fibroblast (HFF; ATCC, Manassas, VA) cells were cultured in DMEM complete medium containing 100 U mL −1 penicillin, 100 U mL −1 streptomycin and 10% FBS.

Techniques: In Vitro, Irradiation, Concentration Assay, MTT Assay, Flow Cytometry, Fluorescence, Incubation

Journal: Pharmaceutics

Article Title: NIR Stimulus-Responsive PdPt Bimetallic Nanoparticles for Drug Delivery and Chemo-Photothermal Therapy

doi: 10.3390/pharmaceutics12070675

Figure Lengend Snippet: DOX@PdPt@HA NPs for in vivo combination therapy. ( A ) Thermal images of 4T1 tumor-bearing mice recorded by infrared thermal imaging camera. ( B ) Temperature changes of 4T1 tumors in different groups during laser irradiation. ( C , D ) Body weight of mice and growth of 4T1 tumors were monitored during the in vivo study. ( E ) Representative photographs of mice and excised tumors at the end of study. ( F ) Weight of tumor collected from animals in different groups. ns, no significant difference. Values represent mean ± SE ( n = 6 in each group). P values: ** P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mouse mammary carcinoma 4T1 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and human foreskin fibroblast (HFF; ATCC, Manassas, VA) cells were cultured in DMEM complete medium containing 100 U mL −1 penicillin, 100 U mL −1 streptomycin and 10% FBS.

Techniques: In Vivo, Imaging, Irradiation

Journal: PLoS ONE

Article Title: Cross-linking of T cell to B cell lymphoma by the T cell bispecific antibody CD20-TCB induces IFNγ/CXCL10-dependent peripheral T cell recruitment in humanized murine model

doi: 10.1371/journal.pone.0241091

Figure Lengend Snippet: a) Workflow schematics: skinfold chambers were installed on NSG mice (day -2). 48 hours later (day 0): WSU DLCL2 (blue), unstained CT26 cells, and CD2 + T cells (pink) freshly purified from human PBMCs or from HSC-NSG mice were injected intra-dermally in the skinfold chamber together with labeled CD20-TCB (0.5 mg/kg) or with suitable vehicle. Cells were imaged 2 hours post treatment by MP-IVM. Adapted from https://smart.servier.com/ . b) 3D representative rendering of MP-IVM imaging on skinfold chamber of HSC-NSG-NSG mice showing localization of therapy (white) at the contact site between WSU DLCL2 cells (blue) and T cells (pink), 2 hours post treatment. c) MP-IVM analysis of T cells tracks in the skin fold chamber of PBMC-NSG (top) vs HSC-NSG-NSG mice (bottom), +/- CD20-TCB. T cell tracks are plotted according to their displacement in the X and Y axes. Total number of tracks for each plot is: Top left: Vehicle n = 330. Top right: CD20-TCB n = 759. Bottom left: Vehicle n = 741. Bottom right: CD20-TCB n = 185. d-e) Quantification of (d) Track Speed (μm/min) and (e) Track displacement (μm) of T cells in PBMC-NSG or HSC-NSG-NSG mice, +/- CD20-TCB. Shown in yellow are mean values +/- s.d. Unpaired t-test; ****p<0.0001; n.s.: not significant.

Article Snippet: CT26 cells ( Mus Musculus colon carcinoma) were originally obtained from ATCC and after expansion deposited in the Roche Glycart internal cell bank, and were cultured in RPMI containing 10% FCS and 1% Glutamax.

Techniques: Purification, Injection, Labeling, Imaging

Journal: PLoS ONE

Article Title: Cross-linking of T cell to B cell lymphoma by the T cell bispecific antibody CD20-TCB induces IFNγ/CXCL10-dependent peripheral T cell recruitment in humanized murine model

doi: 10.1371/journal.pone.0241091

Figure Lengend Snippet: a-c) Top: Representative histological staining of WSU DLCL2 tumors 24h post second treatment (0.5 mg/kg CD20-TCB or suitable vehicle i.v.). Bottom: Quantification of total number of cells/mm 2 from histological images of vehicle vs CD20-TCB treatment. Whole slide scans quantification of 4 μm FFPE sections with the software (a) Definiens; (b-c) Halo. Statistical analysis: Unpaired 2-tailed t-test with Welch’s correction. *p<0.05, **p<0.005 (a) Red: CD3 staining, brown: CD31 staining. Quantification: Number of CD3 + cells b) red: Ki67, yellow: CD3, blue: DAPI. Quantification: Number of CD3 + Ki67 + cells c) Red: CXCR3, yellow: CD3, Blue: DAPI. Quantification of CD3 + CXCR3 + T cells. d) Percentage of proliferating CD8 + T cells, as assessed by CFSE dilution, freshly purified from PBMCs. Proliferation has been evaluated at 24h, 48h and 72h post CD20-TCB treatment, at the indicated doses, in the presence of WSU DLCL2 cells as target. n = 3 per group, mean and s.d. are shown. One-way Anova, *p<0.05, **p<0.005, ****p<0.0001. e) Workflow schematics: Skinfold chamber were installed on NSG mice. 48h later, WSU DLCL2 (Blue), unstained CT26 cells, and CD2 + T cells freshly purified from HSC-NSG spleens (pink) were injected intra-dermally in the skinfold chamber, together with 0.25 mg/kg of CD20-TCB or with suitable vehicle. Concomitantly, freshly purified CD2 + T cells from HSC-NSG spleens (orange) were injected i.v. to allow visualization of peripheral blood T cells. Cells were imaged 72h post treatment by MP-IVM. f) Representative MP-IVM imaging of the tumors. Blue: WSU DLCL2 cells; Pink: Resident T cells; Orange: Recruited T cells. Images were acquired 72h post intradermal treatment with 0.25 mg/kg CD20-TCB or suitable vehicle. Adapted from https://smart.servier.com/ g) Quantification of peripheral T cells (number/mm 2 ) 72h post treatment. Mean +/- s.d. are shown. Unpaired 2-tailed t-test with Welch’s correction. **p<0.005. h) In the context of the skinfold chamber model, increasing number of T cells (Resident) were co-injected with the tumor and 0.25 mg/kg of CD20-TCB intradermally, while 2.5*10 6 T cells were injected intravenously (Peripheral). 72h post treatment, peripheral blood T cells were counted for each tumor from 5 representative fields. 4 tumors per group were analyzed. Shown is the count of peripheral T cells/mm 2 , Mean +/- s.d. per group. Statistical analysis: One-way Anova. **** p<0.0001. i) 3 hours in vitro chemotaxis assay of T cells toward preconditioned medium derived from WSU DLCL2 co-culture with CD3/CD28 pre-activated T cells. Pre-activated CD8 T cells have been plated with WSU DLCL2 cells at decreasing T cells: Tumor cells ratios, in the presence of 200 ng/ml of CD20-TCB. 24h later the supernatant has been collected and transferred to the bottom chamber of a 24-Transwell plate. In the top chamber 100.000 pre activated T cells, labeled with CFSE, have been seeded and let to migrate for 3 hours. Migration has been evaluated by counting total amount of CFSE positive migrated cells in the bottom chamber, by flow cytometry at constant volume and acquisition speed. Mean fold change and +/- s.d. are shown. n = 5, from two independent experiments 2-way Anova; **p<0.005.

Article Snippet: CT26 cells ( Mus Musculus colon carcinoma) were originally obtained from ATCC and after expansion deposited in the Roche Glycart internal cell bank, and were cultured in RPMI containing 10% FCS and 1% Glutamax.

Techniques: Staining, Software, Purification, Injection, Imaging, In Vitro, Chemotaxis Assay, Derivative Assay, Co-Culture Assay, Labeling, Migration, Flow Cytometry