Journal: bioRxiv

Article Title: cGAS-activating lupus autoantibody for cancer immunotherapy

doi: 10.1101/2023.02.11.527649

Figure Lengend Snippet: Human U87 and murine PPQ glioma cells treated with IgG control, DX1, or 4H2 were immunostained to detect antibody penetration and counterstained with DAPI. No significant uptake of IgG control was observed in either cell line. DX1 and 4H2 penetrated and localized into the nuclei and cytoplasm of cells, respectively. Representative merged images are shown. Bar = 10 µm.

Article Snippet: After a final series of washes, cells were treated with Prolong Gold Antifade Reagent with DAPI (Cell Signaling) and imaged using an EVOS fl digital fluorescence microscope (Advanced Microscopy Group, Bothell, WA) under light, DAPI, GFP, or RFP filters.

Techniques: Control

Journal: bioRxiv

Article Title: cGAS-activating lupus autoantibody for cancer immunotherapy

doi: 10.1101/2023.02.11.527649

Figure Lengend Snippet: ( a-c ) The effect of supplemental nucleoside on cellular penetration by DX1 and 4H2 into PPQ cells was determined by ImageJ quantification of fluorescence signal. ADE supplementation increased the efficiency of cellular penetration by DX1 by a factor of 1.70±0.01 (****P<0.0001) ( a ) but did not enhance 4H2 penetration ( b ). In contrast, GUO supplementation increased 4H2 cellular penetration by factor of 3.20±0.30 (****P<0.0001) ( c ), correlating with the known affinity of 4H2 for GUO. ( d ) 4H2 penetrates lung adenocarcinoma cells and localizes into cytoplasm. Representative images of control and 4H2-treated Cal12T cells immunostained for antibody penetration and counterstained with DAPI are shown. ( e ) Lysates of Cal12T cells treated with 0-1 mg/mL 4H2 for 24 hours were analyzed by western blot probed with primary actin antibody for loading control and an anti-mouse secondary antibody to detect the actin primary and 4H2 (both murine). 4H2 HC and LC ran at their expected MWs, showing the antibody is not significantly degraded 24 hours after cellular penetration. ( f ) Visualization of live A549 lung adenocarcinoma cells treated with FITC-labeled 4H2 and MitoTracker Red FM and Hoechst counterstains show 4H2 penetration into cytoplasm. Images of Cal12T cells from this experiment are shown in Supp. Fig. 1b . ( g ) Representative images of Cal12T cells treated with 4H2 immunostained for 4H2 (green) and markers of specific organelles (red), including EEA1 for endosomes, LAMP1 for lysosomes, COX IV for mitochondria, and RCAS1 for Golgi apparatus), and DAPI nuclear counterstained (blue). 4H2 signal did not specifically co-localize with any of the organelles, including endosomes and lysosomes. Error bars = SEM. Scale bars = 10 µm.

Article Snippet: After a final series of washes, cells were treated with Prolong Gold Antifade Reagent with DAPI (Cell Signaling) and imaged using an EVOS fl digital fluorescence microscope (Advanced Microscopy Group, Bothell, WA) under light, DAPI, GFP, or RFP filters.

Techniques: Fluorescence, Control, Western Blot, Labeling

Journal: bioRxiv

Article Title: CNPY4 is a Lipid-Binding Regulator of Sphingolipid Homeostasis

doi: 10.1101/2025.10.13.682155

Figure Lengend Snippet: (A) Schematic of SM synthesis and coupled cholesterol flux. CERT transfers ER ceramide to the Golgi in a PI4P-dependent manner. SMS1 converts ceramide to SM, producing DAG, which activates PKD2, which then phosphorylates OSPB1. This OSPB1 then removes PI4P from the Golgi, and this is coupled to moving cholesterol from the ER to the Golgi, which then goes to the plasma membrane. (B) Confocal microscopy imaging of NIH3T3 cells treated with non-targeting siRNA control (siCtrl) alone, si Cnpy4 , or siCtrl with 10 μM ceramide 6:0 for 30 min, fixed 72 h after siRNA treatment. PI4P signal is shown in red, GM130 in green (Golgi), and DAPI in blue (nucleus). The white box marks the region shown zoomed-in at lower right corner. Scale bar is 10 μm. (C) Quantification of Golgi PI4P from images in (B). PI4P/GM130 fluorescence ratios were normalized to the siCtrl mean, data are mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s Honestly Significant Difference (n = 82 for siCtrl, n = 70 for si Cnpy4 , and n = 81 for siCtrl + ceramide), with ****p<0.0001, n.s. p>0.5, and images were taken across 4 independent experiments. (D) Confocal microscopy of NIH3T3 cells with mScarlet-tagged endogenous ARF1 treated with either siCtrl or si Cnpy4 , fixed and imaged 72 h after siRNA treatment. (E) Quantification of ARF1 fluorescence in cells imaged as described in (F). Fluorescence is normalized to the mean of the siCtrl cells. Error bars represent mean ± SD. Statistical significance determined by a two-sided unpaired Welch’s t-test (n = 117 for siCtrl and n = 137 for si Cnpy4 ), with ****p<0.0001, and images were taken across at least 3 independent experiments. (F) FACS analysis of NIH3T3 cells treated with siCtrl or si Cnpy4 and stained for cholesterol (PFO*-AF657), SM bound to cholesterol (OlyA-AF488), and all SM (OlyA_E69A-AF488) 72 h after siRNA treatment, in the presence of 40 μM myriocin or DMSO control, where indicated. Squares represent individual experiments normalized to the mean of the corresponding siCtrl DMSO condition, with the solid bar representing the mean of all experiments. Statistical significance was determined using a one-sample t-test with a null hypothesis that the mean equals 1 (siCtrl DMSO vs si Cnpy4 DMSO) or a two-sided unpaired Welch’s t-test (siCtrl myriocin vs si Cnpy4 myriocin), with **p<0.01, ***p<0.001, n.s. p>0.05. (n=8 from 4 independent trials, 3,000 – 10,000 cells per point were analyzed). (G) Neutral sphingomyelinase activity in NIH3T3 cell lysates, 72h after transfection with either siCtrl or si Cnpy4, and transfected with either an empty vector control or hCNPY4. Activity was normalized to the mean of the siCtrl with empty vector control. Error bars represent mean ± SD (n = 9 from 3 independent experiments with 3 replicates per experiments). Statistical significance determined by One-way ANOVA and Tukey’s Honestly Significant Difference, with ***p<0.001, ****p<0.0001, n.s. p>0.05.

Article Snippet: Prolong Gold AntiFade with DAPI (Cell Signaling Technologies) was applied to stain the nucleus before sealing with a glass cover slip.

Techniques: Clinical Proteomics, Membrane, Confocal Microscopy, Imaging, Control, Fluorescence, Staining, Activity Assay, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: CNPY4 is a Lipid-Binding Regulator of Sphingolipid Homeostasis

doi: 10.1101/2025.10.13.682155

Figure Lengend Snippet: (A) Confocal imaging of NIH3T3 cells treated with either non-targeting siRNA (siCtrl) control or si Cnpy4 , followed by fixation and staining for ceramide (red) with an anti-ceramide antibody, Golgi (green) with an anti-GM130 antibody, and the nucleus (blue) with DAPI. The white box marks the region shown zoomed-in at lower right corner. Scale bar is 10 μm. (B) Quantification of Golgi ceramide in cells imaged as described in (A). Ratio of ceramide to GM130 fluorescence is shown (mean ± SD). Statistical significance was determined by One-way ANOVA and Tukey’s Honestly Significant Difference (n = 74 for siCtrl 16 h, n = 72 for siCtrl 24 h, n = 63 for siCtrl 48 h, n = 70 for siCtrl 72 h, n = 65 for si Cnpy4 16h, n = 84 for si Cnpy4 24h, n = 77 for si Cnpy4 48h, and n = 62 for si Cnpy4 72h), and shown relative to siCtrl 16 h condition, with *p<0.05, *P<0.001, ****p<0.0001, n.s. p>0.5. Images were taken across 3 independent experiments. (C) Confocal microscopy of NIH3T3 cells treated with either siCtrl or si Cnpy4 , incubated with 1 μg/mL fluorescently tagged NBD-ceramide 6:0 for the indicated time intervals, and imaged 72 h after siRNA treatment. The white arrows point to ceramide accumulated in the Golgi. Scale bar is 10 μm. (D) Quantification of total NBD fluorescence in cells imaged as described in (C). Fluorescence was normalized to the mean of the time 0 in siCtrl-treated cells. Error bars represent mean ± SD. Statistical significance was determined by a two-sided unpaired Welch’s t-test (n=55 siCtrl 0 min, n=55 si Cnpy4 0 min, n=60 siCtrl 30 min, and n=52 si Cnpy4 30 min), with ****p<0.0001, n.s. p>0.05. (E) Quantification of the percentage of total NBD fluorescence localized to the Golgi in the same cells as in (D). Error bars represent mean ± SD. Statistical significance was determined by a two-sided unpaired Welch’s t-test (n = 55 for siCtrl 0 min, n = 55 for si Cnpy4 0 min, n = 60 fro siCtrl 30 min, and n = 52 for si Cnpy4 30 min), with ****p<0.0001, n.s. p>0.05.

Article Snippet: Prolong Gold AntiFade with DAPI (Cell Signaling Technologies) was applied to stain the nucleus before sealing with a glass cover slip.

Techniques: Imaging, Control, Staining, Fluorescence, Confocal Microscopy, Incubation

Journal: Mucosal immunology

Article Title: Deficiency of autophagy protein Map1-LC3b mediates IL-17-dependent lung pathology during respiratory viral infection via ER stress associated IL-1

doi: 10.1038/mi.2015.3

Figure Lengend Snippet: A) Bone marrow-derived DCs (BMDCs) were cultured from naïve WT or LC3b −/− mice, then infected for 4 hours with RSV. Cells were fixed, stained with DAPI and fluorescent antibody to ATG5, and imaged by confocal microscopy. Scale bars = 10μm. B) BMDCs were cultured and infected with RSV, followed by harvest at 4, 8, 12, and 24 hours post-infection. Whole cell lysates were run on SDS-PAGE gels, followed by membrane transfer and immunoblot for p62 or β-actin as indicated. C) Cell supernatants from BMDCs infected with 1:1 MOI RSV for 48 hours, analyzed by Bioplex assay. D) BMDCs were infected as above, harvested at 24 hours post-infection, and gene expression analyzed by qPCR. E) Lung-resident CD11c hi Auto lo CD11b + lamina propria DCs (CD11b + LP DCs) were flow-sorted from enzymatically-digested lungs of naïve WT and LC3b −/− mice. Cells were infected ex vivo with 1:1 MOI RSV, and cell supernatants harvested at 48 hours post-infection by Bioplex assay. F) Lung-resident CD11b + LP DCs were isolated and infected with RSV as previously described, followed by gene expression analysis by qPCR at 24 hours post-infection. G) CD4+ T cells were purified from mediastinal lymph nodes of RSV-infected C57Bl/6 mice, 8 days post-infection. Cells were plated at a 10:1 ratio over WT or LC3b −/− BMDCs, infected 2 hours previously with 1:1 MOI RSV. Culture supernatant IL-17a concentrations were measured at 48 hours by Bioplex assay. WT or LC3b −/− BMDCs (H) or CD11b + LP DCs (I) were pulsed with whole ovalbumin protein in the presence or absence of 1:1 MOI RSV, followed by the addition of 10:1 purified splenic OT-II CD4+ T cells. Culture supernatant IL-17a concentrations were measured at 48 hours by Bioplex assay. All results are representative of at least three independent experiments. *p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: Slides were mounted in ProLong Gold antifade reagent plus DAPI, and imaged using 40× oil immersion objective on a Nikon A1 Confocal Microscope System using NIS Elements software (Nikon Instruments).

Techniques: Derivative Assay, Cell Culture, Infection, Staining, Confocal Microscopy, SDS Page, Membrane, Western Blot, BioPlex Assay, Gene Expression, Ex Vivo, Isolation, Purification

Journal: Mucosal immunology

Article Title: Deficiency of autophagy protein Map1-LC3b mediates IL-17-dependent lung pathology during respiratory viral infection via ER stress associated IL-1

doi: 10.1038/mi.2015.3

Figure Lengend Snippet: A) Lung sections from WT and LC3b −/− mice, 6 days post-infection, were stained with antibodies to mouse IL-1β and E-cadherin. Sections were counterstained with DAPI and imaged on an Olympus BX43 fluorescent microscope. Scale bars = 40μm. (B,C) Airway epithelial cell (AEC) cultures were prepared from lungs of naïve WT or LC3b −/− mice, and infected with 1:1 MOI RSV. B) IL-1α and IL-1β expression was measured at 12 hours post-infection by qPCR, while C) IL-1β and IL-6 secretion was measured in culture supernatants at 24 hours by Bioplex assay. D) Single-cell suspensions prepared from enzymatically-digested lungs were surface-stained, followed by intracellular IL-1β staining, and analyzed by flow cytometry. Lung macrophages were gated Auto hi CD11b hi F4/80 + . Dark histograms represent isotype control antibodies, light histograms anti-IL-1β antibody. E) Quantification of median fluorescence intensity staining of lung macrophages for IL-1β. E) mRNA transcripts of ER stress-associated genes HSP90, CHOP, Edem-1, and spliced XBP-1 were assessed in AECs at 12 hours post-infection by qPCR. (F) AECs were treated with the IRE-1α inhibitor 3,5-dibromosalicylaldehyde (DBSA) at 40μM, or the general ER stress inhibitor 4-phenlbutyrate (4-PBA) at 5mM, 30 minutes prior to 1:1 MOI RSV. Cell culture supernatants were assayed at 24 hours post-infection by Bioplex assay. G) AECs were infected for 12 hours with 1:1 MOI RSV, then fixed and stained with antibodies to the Caspase-1 p10 subunit and DAPI. Cells were imaged on a Nikon A1 confocal microscope. Fluorescence intensity per cell was measured and quantified using maximum intensity projections, using ImageJ software. Scale bars = 30μm. Results are representative of at least two independent experiments. *p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: Slides were mounted in ProLong Gold antifade reagent plus DAPI, and imaged using 40× oil immersion objective on a Nikon A1 Confocal Microscope System using NIS Elements software (Nikon Instruments).

Techniques: Infection, Staining, Microscopy, Expressing, BioPlex Assay, Flow Cytometry, Control, Fluorescence, Cell Culture, Software