Journal: PLoS ONE

Article Title: A Photoprotein in Mouse Embryonic Stem Cells Measures Ca 2+ Mobilization in Cells and in Animals

doi: 10.1371/journal.pone.0008882

Figure Lengend Snippet: A. Clone pool analysis of the 114 neomycin resistant clones. Histamine response kinetics (100 µM) were measured at Lumibox luminometer and recorded as RLU (Relative Luminescence Units) values. The experiments were performed in 96 MTP 24 h after cell seeding. Lumibox luminometer conditions: high sensitivity, for 60 seconds. B. mES/mito c-Photina/29 clone histamine dose response. The histamine response (100 nM–500 µM) was measured in 96 MTP 24 h after seeding 20,000 cells/well. Lumibox luminometer conditions: high sensitivity, reading time 60 seconds .

Article Snippet: 7×10 6 mES cells were electroporated using 30 µg of the mito c-Photina DNA, linearized with BglII (New England Biolabs).

Techniques: Clone Assay

Journal: PLoS ONE

Article Title: A Photoprotein in Mouse Embryonic Stem Cells Measures Ca 2+ Mobilization in Cells and in Animals

doi: 10.1371/journal.pone.0008882

Figure Lengend Snippet: Germline transmission results for clone 29 c-Photina mouse embryonic stem cells.

Article Snippet: 7×10 6 mES cells were electroporated using 30 µg of the mito c-Photina DNA, linearized with BglII (New England Biolabs).

Techniques: Transmission Assay

Journal: PLoS ONE

Article Title: A Photoprotein in Mouse Embryonic Stem Cells Measures Ca 2+ Mobilization in Cells and in Animals

doi: 10.1371/journal.pone.0008882

Figure Lengend Snippet: A. Embryoid bodies in suspension at differentiation day 2 (contrast phase image). Embryoid body in suspension at differentiation day 5 before the plating on gelatin-coated dishes (contrast phase image). Embryoid body in adhesion at differentiation day 7 on gelatin-coated dishes (contrast phase image). Scale bar -200 µm. B. Percentage of embryoid bodies containing pulsing areas obtained after morphological analysis (see also and ). C.-E. Immunofluorescence analysis on in vitro differentiated cardiomyocytes from mES/mito c-Photina/29 clone. The immunofluorescence assay was performed using the following antibodies: C. Anti-GATA-4 primary antibody. Scale bar -100 µm. D.-E. Anti-alpha myosin heavy chain primary antibody. Scale bar −100 µm ( D. ) and −50 µm ( E. ). F. CCD camera-based functional test on in vitro differentiated cardiomyocytes from mES/mito c-Photina/29 clone. Tyrode's buffer, endothelin-1 (50 nM) and norepinephrine (100 µM), and KCl (60 mM) responses were measured in 384 MTP 48 h after 5,000 cells/well seeding. Lumibox luminometer conditions: high sensitivity, reading time 60 seconds .

Article Snippet: 7×10 6 mES cells were electroporated using 30 µg of the mito c-Photina DNA, linearized with BglII (New England Biolabs).

Techniques: Immunofluorescence, In Vitro, Functional Assay

Journal: PLoS ONE

Article Title: A Photoprotein in Mouse Embryonic Stem Cells Measures Ca 2+ Mobilization in Cells and in Animals

doi: 10.1371/journal.pone.0008882

Figure Lengend Snippet: A. c-Photina expression profile in PhotoTopo animals. 5 mice were sacrificed: 2 negatives and 3 positives and 7 different tissues/organs were isolated and pooled and used for TaqMan® quantitative PCR analysis in triplicates. The data were normalized relative to the amount of 18S rRNA levels and expressed as REU (Relative Expression Units). B. c-Photina light activity in PhotoTopo animals. 8 transgenic mice and 6 negative mice from the same litter were sacrificed in 3 different experiments and 11 different tissues/organs were explanted. The cells were incubated for 3 h with 20 µM coelenterazine at room temperature. The photoprotein content was measured injecting a solution of 250 mM CaCl 2 and 1% Triton X-100. Lumibox luminometer conditions: high sensitivity, reading time 60 seconds . C.-D. Coelenterazine systemic injection via tail vein in a PhotoTopo mouse and in a negative control. After 3 h, 12 different tissues/organs were explanted from both mice. Half of the material was directly seeded in a white 96 MTP (C.), the other half was further incubated at room temperature for 3 h with 20 µM coelenterazine ( D. ). All the samples were analysed at CCD camera based luminometer for testing the photoprotein content, injecting a solution of 250 mM CaCl 2 and 1% Triton X-100. Lumibox luminometer conditions: high sensitivity, reading time 60 seconds .

Article Snippet: 7×10 6 mES cells were electroporated using 30 µg of the mito c-Photina DNA, linearized with BglII (New England Biolabs).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Activity Assay, Transgenic Assay, Incubation, Injection, Negative Control

Journal: PLoS ONE

Article Title: A Photoprotein in Mouse Embryonic Stem Cells Measures Ca 2+ Mobilization in Cells and in Animals

doi: 10.1371/journal.pone.0008882

Figure Lengend Snippet: A.-D. PhotoTopo derived aortic endothelial cells. A. Flow cytometry analysis on aortic endothelial cells isolated from seven animals (4 positives and 3 negatives) with antibody anti-von Willebrand Factor (vWF) and with only secondary antibody or without any antibody as controls. B.-C. Immunofluorescence analysis with antibody anti vWF ( B. ), and anti CD31 ( C. ). Scale bar −50 µm. D. Lumibox luminometer functional test performed stimulating the aortic endothelial cells with 100 µM TRAP-6 and −10 peptides (agonists for the proteinase-activated receptors) and 50 nM endothelin-1 (agonist for endothelin receptors). Lumibox luminometer with the following settings: high sensitivity, reading time 60 seconds . E.-H. PhotoTopo derived bone marrow monocyte/macrophage cells. E. Flow cytometry analysis on mature cells after 10 days of differentiation obtained from seven animals (4 positives and 3 negatives) with antibody anti-F4/80 and scavenger receptor type III (CD204), and with only secondary antibody or without any antibody as controls. F.-G. Immunofluorescence analysis with antibody anti F4/80 ( F. ) and scavenger receptor type III ( G. ). Scale bar -50 µm. H. Lumibox luminometer functional test performed using UDP, UTP, and ATP (100 µM) as agonist for purinergic receptors. Lumibox luminometer conditions: high sensitivity, reading time 60 seconds . I.-J. CCD camera-based luminometer functional test performed on pancreatic islets isolated from transgenic mito c-Photina mice. Pancreatic islets were isolated from 1 positive transgenic mito c-Photina and 1 negative mouse. After an overnight culture, 10 islets/well were put in a white 96 MTP. I. Two different wells containing 10 islets each were incubated for 3 h with 10 µM coelenterazine and stimulated respectively with 11 mM glucose or 11 mM mannitol as negative control. Luminoskan luminometer. Integration time 0.5 sec. Reading time 150 seconds . J. The islets were then stimulated with 60 mM KCl. Light emitted was measured at Lumibox. Lumibox luminometer conditions: high sensitivity, reading time 60 seconds .

Article Snippet: 7×10 6 mES cells were electroporated using 30 µg of the mito c-Photina DNA, linearized with BglII (New England Biolabs).

Techniques: Derivative Assay, Flow Cytometry, Isolation, Immunofluorescence, Functional Assay, Transgenic Assay, Incubation, Negative Control

Journal: PLoS ONE

Article Title: A Photoprotein in Mouse Embryonic Stem Cells Measures Ca 2+ Mobilization in Cells and in Animals

doi: 10.1371/journal.pone.0008882

Figure Lengend Snippet: A.-E. A single islet ( A. contrast phase) was stimulated with 60 mM KCl in order to activate voltage-gated Ca 2+ channels. The influx of Ca 2+ induces an activation of the c-Photina and the emission of light. The light emitted was acquired using an intensified CMOS based camera with 512×512 pixel resolution at 60 frames/sec. The images were then elaborated with ImageJ software and integrated in a total bin ( B. ). With ImageJ is possible to retrieve the kinetics of the whole islet ( C. ) or those of the single regions ( D. ). E. The polar injection of the stimulus induces a Ca 2+ mobilization visible in sequential integrated picture frames (see also ). Scale bar −50 µm.

Article Snippet: 7×10 6 mES cells were electroporated using 30 µg of the mito c-Photina DNA, linearized with BglII (New England Biolabs).

Techniques: Activation Assay, Software, Injection

Journal: iScience

Article Title: Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

doi: 10.1016/j.isci.2021.102936

Figure Lengend Snippet: The mitochondrial ROS/PKC pathway is involved in eNOS-Thr 495 phosphorylation by ASPD in primary human cerebral endothelial cells (A and B) Primary human endothelial cells were pretreated for 30 min either with bisindolylmaleimide I (Bim I, 5 μM), Y-27632 (Y27, 10 μM), or compound C (CC, 10 μM) in A or with calphostin C (Cal C, 0.3 μM) in B, and were further treated for 6 hr with ASPD (32 nM in A or 35 nM in B) (see “ ”). The ratio of eNOS-P-Thr 495 to eNOS-total was obtained as in Figure 5 C. The ratio in the non-treated cells is shown as 100 (n = 4 in A and 3 in B). As shown in Western blots in B, calphostin C decreased the eNOS-total without affecting cell survival, most likely due to its non-specific inhibition of phospholipase D activity ( Zheng et al., 2004 ). Nevertheless, because both bisindolylmaleimide I and calphostin C completely inhibited the ASPD-induced increase in eNOS-P-Thr 495 , this does not affect the conclusion that ASPD work through PKC activation. Data are presented as means ± S.E. ∗∗ P < 0.01 (ANOVA with Scheffé’s method). (C) The cells were treated with freshly dissolved Aβ 1-42 solution (1 μM) for 6 hr (see “ ”). The ratio of eNOS-P-Thr 495 to eNOS-total was determined as in A. The ratio of PKC-P-Ser 660 to PKC-total was determined by Western blotting of total extracts with antibodies specific for PKC-P-Ser 660 and PKC-total, respectively (n = 4). The results show that ASPD induce eNOS inactivation through PKC activation, whereas freshly dissolved Aβ 1-42 does not. Our findings also confirmed a possible link between Aβ and inactivation of eNOS, the molecular mechanism of which has been largely unknown, as previously reported ( Chisari et al., 2010 ; Gentile et al., 2004 ; Lamoke et al., 2015 ; Suhara et al., 2003 ). AMP kinase and Rho kinase have been also reported to play a role in the physiological regulation of eNOS by phosphorylating Thr 495 . These kinases might be involved in the case of Aβ 1-42 . Data are presented as means ± S.E. ∗ P < 0.05 (Welch's t test). (D and E) The cells were pretreated with tempol (Tem, 3 mM), BAPTA-AM (BAP, 30 μM), or U-73122 (U-73, 10 μM) for 30 min, and were further treated with ASPD (63 nM) for 6 hr (see “ ”). The ratios of eNOS-P-Thr 495 to eNOS-total and that of PKC-P-Ser 660 to PKC-total were obtained as above (n = 4). Data are presented as means ± S.E. ∗∗ P < 0.01 (ANOVA with Scheffé’s method). (F) The cells were pretreated with YCG-063 (YCG, 50 μM), mito-tempol (M-temp, 100 μM), VAS2870 (VAS, 10 μM), or apocynin (Apo, 20 μM) for 30 min (see “ ”), and were further treated with ASPD (35 nM) for 6 hr. ROS production was estimated by monitoring the fluorescence intensity of a ROS fluorescence indicator, CellROX (see “ ”) (n = 4). Representative fluorescence images, along with expanded images of the areas surrounded by hatched lines in the upper panels are shown. The CellROX fluorescence intensities were determined using a laser scanning cytometer CQ1 (see “ ”). Quantification data in ASPD-treated cells are shown as 100. Scale bars: 100 μm for solid line and 20 μm for hatched line. Data are presented as means ± S.E. ∗∗ P < 0.01 (ANOVA with Scheffé’s method).

Article Snippet: Twenty-four hours later, in the case of inhibitor pretreatment, the cells were pretreated with YCG-063 (50 μM; 557354, Merck-Millipore), mito-tempol (100 μM; 18,796, Cayman Chemical), VAS2870 (10 μM; 492000, Merck-Millipore), or apocynin (20 μM; 178385, Merck-Millipore) for 30 min. All cells were then treated with ASPD (35 nM) for 6 hr, washed twice with HBSS, and were then loaded with a fluorescent ROS indicator CellROX (C10443, Thermo Fisher Scientific), according to the manufacturer’s protocol.

Techniques: Western Blot, Inhibition, Activity Assay, Activation Assay, Fluorescence, Cytometry

Journal: iScience

Article Title: Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

doi: 10.1016/j.isci.2021.102936

Figure Lengend Snippet:

Article Snippet: Twenty-four hours later, in the case of inhibitor pretreatment, the cells were pretreated with YCG-063 (50 μM; 557354, Merck-Millipore), mito-tempol (100 μM; 18,796, Cayman Chemical), VAS2870 (10 μM; 492000, Merck-Millipore), or apocynin (20 μM; 178385, Merck-Millipore) for 30 min. All cells were then treated with ASPD (35 nM) for 6 hr, washed twice with HBSS, and were then loaded with a fluorescent ROS indicator CellROX (C10443, Thermo Fisher Scientific), according to the manufacturer’s protocol.

Techniques: Recombinant, Avidin-Biotin Assay, Synthesized, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Derivative Assay, Software, Light Microscopy, Laser-Scanning Microscopy, Imaging, Cytometry

Journal: Scientific Reports

Article Title: Melanoblast development coincides with the late emerging cells from the dorsal neural tube in turtle Trachemys scripta

doi: 10.1038/s41598-017-12352-0

Figure Lengend Snippet: Potential migratory pathways for late neural crest cells and melanoblasts. ( A ) Anti-HNK1 antibody indicated reappearance of migratory neural crest cells in the dorsal neural tube (*) at G16. Paired round areas of HNK-1 positive cells were visible dorsolateral to the neural tube and adjacent to the surface ectoderm (black arrows). ( B ) At G17, HNK-1 positive cells appeared to follow two migratory pathways: a dorsolateral pathway underneath the surface ectoderm for some of the migratory cells and a pathway where cells travel from the CSA over the dermomyotome laterally (red arrow) or ventrally between the dermomyotome and the sclerotome (orange arrow). ( C ) PNA was not seen in the dorsal surface ectoderm, carapacial mesenchyme or in or around the dermomyotome, thus allowing entry of migratory neural crest cells to both dorsolateral and medial pathways. ( D ) Mitf-positive melanoblasts were visible on top of the neural tube, in the CSA, adjacent to the surface ectoderm and in the dermomyotome (boxed area shown in E) and in at G17. ( E ) The mediodorsal tip of dermomyotome had Mitf-positive cells. ( F ) Mitf-positive cells were detectable between the dermomyotome and the sclerotome. ( G ) At G21, c-Kit-positive melanocytes were observed in both the dorsolateral and medial pathways. Some Mitf- and c-Kit-positive cells are indicated by arrows. Scale bar approx. 1 mm ( A , B ), 100 μm ( C , D ) and 200 μm ( E ). c, vertebral cartilage; d, dermomyotome; drg, dorsal root ganglion; l, lung; nt, neural tube; r, rib.

Article Snippet: 4% PFA-fixed paraffin sections were dewaxed, rehydrated, treated to block endogenous peroxidase activity, boiled in citrate buffer pH 6.0 for antigen retrieval (except for PNA staining), preblocked with 0.3% BSA, and incubated with anti-c-Kit (1:500, Abcam cat# ab 32363), anti-GFP (1:1000, Chemicon cat# MAB3580), anti-HNK1 (1:1000, BD Biosciences cat# 559048), anti-Mitf (1:500, Bioss cat# bs-1990R), PNA (peanut agglutinin) (1:500, Sigma cat# L7759), or anti-Slug (1:500, CST cat# 9585) antibody overnight at 4 °C, washed and incubated with the appropriate secondary HRP-conjugated antibody (1:1000) overnight at 4 °C.

Techniques:

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Confocal images (left) and quantification (right) of classical Mo isolated from JDM and SLE patients. Each dot represents the average count from at least ten different micrscopy fields from a study participant sample that was processed, stained and analyzed independently. Scale bar: 20 µm. b, Percentage of IL-1β − MxA + Mo and IL-1β + MxA + Mo in SLE patients without (blue, n=8) or with (red, n=6) circulating Mito + RBCs. c, Confocal images of classical Mo isolated from SLE patients showing evidence of erythrophagocytosis. GPA: Glycophorin A. Scale bar: 20 µm. d, Two-tailed Pearson’s correlation between SLEDAI and the percentage of IL-1β + ISGs + Mo (n=20). Dashed lines represent 95% confidence intervals. Each dot represents a study participant sample that was processed, stained and analyzed independently.

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Isolation, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Phagocytosis of CFSE-labeled RBCs in the presence or absence of IgG opsonizing antibodies. b, Quantification of phagocytosis of CFSE-labeled Mito - or Mito + RBCs by blood Mo. (n=3). c, Immunoblot analysis of whole cell lysate (WCL) and supernatants (Sup) from blood Mo that were treated as described. One representative of two experiments. d, Levels of IP-10 and IL-1β in the supernatants of bone marrow (BM) Mo cultured with media, Mito - or Mito + opsonized RBCs. (n=3). e, Phenotype of BLaER1 cells before (Day 1) and after (Day 6) differentiation into BLaER1 Mo. f, Quantification of phagocytosis of CFSE-labeled Mito - or Mito + RBCs by BLaER1 Mo. (n=3). g, Immunoblot analysis of WCL and Sup from BLaER1 Mo that were treated as described. One representative of two. h, Relative mtDNA abundance in Mito + RBCs generated with vehicle, EtBr (ρ 0 ) or CAM. (n=3). i, Quantification of phagocytosis of CFSE-labeled Mito + RBCs RBCs generated with vehicle or EtBr (ρ 0 ). (n=3). j, Western blot analysis of Mito + RBCs generated with vehicle, EtBr (ρ 0 ) or CAM. One representative of two.

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Labeling, Western Blot, Cell Culture, Generated

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Heat map of differentially expressed genes in blood Mo that phagocytized either Mito - or Mito + RBCs. Data are normalized to Mito - RBCs sample . b, Levels of IP-10 and IL-1β in the supernatants of blood Mo cultured with media, Mito - or Mito + RBCs. (n=6). c, Heat map of differentially expressed genes in BLaER1 Mo that phagocytized Mito - or Mito + RBCs. Data are normalized to Mito - RBCs sample . d, Levels of IP-10 and IL-1β in the supernatants of BLaER1 Mo cultured with media, Mito - or Mito + RBCs. (n=4). e, Experiment scheme (left) and normalized cytokine levels (right) in the supernatants of BLaER1 Mo activated with Mito + RBCs generated in the presence of vehicle, ethidium bromide (EtBr; ρ 0 ) or chloramphenicol (CAM). (n=4).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Cell Culture, Generated

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Normalized IP-10 levels in the supernatants of Mito + RBCs-activated wild type, cGAS KOor STING1 KO BLaER1 Mo. (n=3). b, Normalized IL-1β levels in the supernatants of Mito + RBCs-activated wild type or Caspase-1 KO BLaER1 Mo. (n=3). c, Experimental scheme (left), dot/Western blot analysis (middle) and quantification (left; n=3) of Mito + RBCs (1 - 3) or BLaER1 Mo (2 − 4) labeled with bromodeoxyuridine (BrdU) and then immunoprecipitated (IP) with cGAS or NLRP3 antibodies. As a loading control, cGAS or NLRP3 immunoblot or double-stranded DNA (dsDNA) dot blot (DB) was performed. A.U.: Arbitrary Units.

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, Labeling, Immunoprecipitation, Control, Dot Blot

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Western blot analysis confirming CRISPR/Cas9-mediated KO of cGAS and STING1. b, Normalized IP-10 levels in the supernatants of Mito + RBC-activated BLaER1 Mo in the presence of the cGAS inhibitor RU.521 or the STING inhibitor H151. (n=3). c, Western blot analysis of phosphorylated TBK1 and IRF3 in BLaER1 Mo cultured with media or Mito + RBCs. d, Western blot analysis confirming CRISPR/Cas9-mediated KO of AIM2. e, Normalized IL-1β levels in the supernatants of Mito + RBCs-activated wild type or AIM2 KO BLaER1 Mo. (n=3). f, Flow cytometry analysis showing the internalization of unlabeled (control) or AlexaFluor 488 (AF488)-labeld IgG upon electroporation. g, Percentages of cell death measured by 7AAD incorporation. h, Western blot analysis confirming Trim-Away knockdown of AIM2 (left). Normalized IL-1β levels in the supernatants of activated BLaER1 Mo upon Trim-Away knockdown of AIM2 (right). (n=3). i, Western blot analysis confirming CRISPR/Cas9-mediated KO of Caspase-1. j, Normalized IL-1β levels in the supernatants of Mito + RBCs-activated BLaER1 Mo in the presence of the NLRP3 inhibitor MCC950 or the caspase-1 inhibitor Ac-YVAD-cmk. (n=4). k, Phagocytosis of CFSE-labeled RBCs in wild type, cGAS KO, STING1 KO or caspase-1 KO BLaER1 Mo. (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, CRISPR, Cell Culture, Flow Cytometry, Control, Electroporation, Knockdown, Labeling

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Normalized cytokine levels in the supernatants of blood Mo activated with Mito + RBCs generated in the presence of vehicle, EtBr (ρ 0 ) or CAM. b, Normalized cytokine levels in the supernatants of Mito + RBC-activated blood Mo in the presence of RU.521, H151, MCC950 or Ac-YVAD-cmk. (n=3). c, Normalized cytokine levels in the supernatants of BM Mo activated with Mito + RBCs generated in the presence of vehicle, EtBr (ρ 0 ) or CAM. d, Normalized cytokine levels in the supernatants of Mito + RBCs-activated BM Mo in the presence of RU.521, H151, MCC950 or Ac-YVAD-cmk. (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Generated

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Western blot analysis of cytosolic fraction obtained from media, Mito + RBCs- or dsDNA/Lyovec-activated BLaER1 Mo. WCL: whole cell lysate. Quantification of lactate dehydrogenase (LDH) release (b) or propidium iodide (PI) internalization (c) in BLaER1 Mo activated Mito + RBCs or dsDNA/Lyovec over time. Data are normalized to media-treated cells. (n=3). d, Western blot analysis of ASC oligomers (pyroptosome) formation in BLaER1 Mo activated Mito + RBCs or dsDNA/Lyovec for 18 h. Triton X-100 soluble (TX100 Sol ) and insoluble (TX100 Ins ) fractions were immunoblotted with ASC antibody. One representative of two experiments. e, Normalized IL-1β levels in the supernatants of Mito + RBC- or dsDNA/Lyovec-activated BLaER1 Mo in the presence of the lysosmal acidification inhibitor Bafilomycin A1 (BAf A1). (n=3). f, Normalized IL-1β levels in the supernatants of Mito + RBC- or dsDNA/Lyovec-activated BLaER1 Mo in the presence of extracellular K + . (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Experiment scheme (left) and normalized cytokine levels (right) in the supernatants of BLaER1 Mo, generated in the presence of vehicle, EtBr (ρ 0 ) or CAM and activated with Mito + RBCs. (n=4). b, Relative levels of mtDNA-encoded genes in cGAS (left) or NLRP3 (right) immunocomplexes isolated from Mito + RBC-activated wild type or MAVS KO BLaER1 Mo. (n=3). c, Experiment scheme (left) and normalized cytokine levels (right) in the supernatants of BLaER1 Mo cultured with Mito + RBCs generated in the presence of Actinomycin D (Act D). (n=4). d, Normalized IL-1β levels in the supernatants of Mito + RBCs- or Poly I:C/Lyovec-activated wild type, MAVS KO or RIG- I/MDA5 KO BLaER1 Mo. (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Generated, Isolation, Cell Culture

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Western blot analysis confirming CRISPR/Cas9-mediated KO of mitochondrial DNA polymerase gamma (POLG; Polγ). b, Normalized IL-1β levels in the supernatants of Mito + RBC-activated wild type or POLG KO BLaER1 Mo. (n=3). c, DB analysis of cGAS or NLRP3 immunocomplexes isolated from Mito + RBCs-activated BLaER1 Mo. As a loading control, dsDNA dot blot (DB) was performed. d, 8-OHdG quantification by ELISA in cGAS or NLRP3 immunocomplexes isolated from Mito + RBC-activated BLaER1 Mo. (n=3). e, Experimental scheme (left) and DB analysis (right) of IP cGAS or NLRP3 upon incubation with biotinylated intact (Int) or fragmented (Fgt) mtDNA. As a loading control, cGAS or NLRP3 immunoblot was performed. f, SDD-AGE of MAVS oligomerization in BLaER1 Mo in response to Mito + RBCs (generated with or without Act D) or to transfection with Poly I:C. One representative of three. g, Western blot analysis confirming CRISPR/Cas9-mediated KO of MAVS. h, Relative caspase-1 activity in the lysate of Mito + RBC-activated wild type, MAVS or RIG-I/MDA5 KOBLaER1 Mo. (n=3). i, Normalized IL-1β levels in the supernatants of Poly I:C/Lyovec-activated BLaER1 Mo in the presence of MCC950 or Ac-YVAD-cmk or that were generated in the presence of EtBr. (n=3). j, Relative levels of mtDNA-encoded genes in NLRP3 immunocomplexes isolated from Poly I:C/Lyovec-activated wild type or MAVS KO BLaER1 Mo. (n=3). k, Relative caspase-1 activity in the lysate of Poly I:C/Lyovec-activated wild type, MAVS or RIG-I/MDA5 KO BLaER1 Mo. (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, CRISPR, Isolation, Control, Dot Blot, Enzyme-linked Immunosorbent Assay, Incubation, Generated, Transfection, Activity Assay

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Western blot analysis confirming CRISPR/Cas9-mediated KO of RIG-I or MDA5. b, Normalized IL-1β levels in the supernatants of Mito + RBCs or Poly I:C/Lyovec-activated wild type, RIG-I KO or MDA5 KO BLaER1 Mo. (n=3). c, Western blot analysis confirming CRISPR/Cas9-mediated KO of DHX33. d, Normalized IL-1β levels in the supernatants of Mito + RBCs or Poly I:C/Lyovec-activated wild type or DHX33 KO BLaER1 Mo. (n=3). e, Western blot analysis confirming CRISPR/Cas9-mediated KO of both RIG-I and MDA5. Relative levels of mtDNA-encoded genes in NLRP3 immunocomplexes isolated from Mito + RBCs (f) or Poly I:C/Lyovec (g) activated wild type or RIG-I/MDA5 KO BLaER1 Mo. (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, CRISPR, Isolation

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Western blot analysis of gasdermin-D N-terminal fragment (GSDMD-NT) in total cell lysate isolated from Mito + RBCs, Poly I:C/Lyovec and Nigericin-activated BLaER1 Mo. b , Quantification of lactate dehydrogenase (LDH) release or propidium iodide (PI) internalization in BLaER1 Mo activated Mito + RBCs, Poly I:C/Lyovec or Nigericin over time. Data are normalized to media-treated cells. (n=3). c, Western blot analysis confirming CRISPR/Cas9-mediated KO of GSDMD (left). Normalized IL-1β levels in the supernatants from Mito + RBCs, Poly I:C/Lyovec and Nigericin-activated wild type or GSDMD KO BLaER1 Mo (right). (n=3). Confocal images of classical Mo isolated from SLE patients and stained for IL-1β and ISG15 (d) or IFIT1 (e). Scale bar: 15 µm.

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, Isolation, CRISPR, Staining

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Experimental scheme (left) and Western blot analysis (right) of the microsomal pellet (100k P) and postmicrosomal supernatant (100k S) isolated from Poly I:C/Lyovec-activated BLaER1 Mo. b , Western blot analysis of different fractions collected after sucrose fractionation of microsomal membranes isolated from Poly I:C/Lyovec-activated BLaER1 Mo. ER: endoplasmic reticulum; ERGIC: ER-Golgi intermediate compartment. c, Western blot analysis of the 100k P and 100k S fractions isolated from Poly I:C/Lyovec-activated wild type or MxA KOBLaER1 Mo. d, Normalized IL-1β levels in the supernatants from Mito + RBCs, Poly I:C/Lyovec and dsDNA/Lyovec-activated wild type or MxA KO BLaER1 Mo. (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, Isolation, Fractionation

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Immunoblot analysis of mIL-1β present within liposomes (pellet), or supernatants (sup) after ultracentrifugation of liposomes that were treated with buffer or recombinant human MxA for 0 (control), 30 and 120 min. Densitometry quantification of Western blot band density for mIL-1β release from liposomes is also shown. (n=3). b, Quantification of LDH enzymatic activity release from Lp loaded with LDH and treated with buffer or MxA for indicated times. (n=3). c, Normalized IL-1β levels in the supernatants from Mito + RBCs-activated BLaER1 Mo with the indicated genotype. (n=3). Immunoblot analysis of the total cell lysate is also shown. d, Western blot analysis confirming Trim-Away knockdown of MxA (left). Normalized IL-1β levels in the supernatants of activated BLaER1 Mo upon Trim-Away knockdown of MxA (right). (n=3). e, Western blot analysis of whole cell lysate (WCL) and supernatants (Sup) isolated from BLaER1 Mo overexpressing mIL-1β that were treated with or without recombinant Type I IFN (IFNα2β). One representative of two.

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, Liposomes, Recombinant, Control, Activity Assay, Knockdown, Isolation

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: (1) In SLE patients with active disease, internalization of Mito + RBCs induces Mo to co-express ISGs and mIL-1β. ISG expression depends on cGAS activation by Mito + RBC-derived mtDNA (2), while mIL-1β production requires NLRP3 activation. Upstream of NLRP3, Mito + RBC-derived mt dsRNA activates the RLRs/MAVS pathway, which induces the release of Mo-derived mtDNA fragments that bind NLRP3 (3). Importantly, mIL-1β triggers the oligomerization of MxA on the surface or the TGN (4), which favors the entry of this cytokine into a TGN-mediated unconventional secretory (vesicular?) pathway (5). (6) Cytosolic dsRNA of microbial origin (i.e. Poly I:C/Lyovec) could also activate these pathways via MAVS/IRF3.

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Expressing, Activation Assay, Derivative Assay

Journal: Antioxidants & Redox Signaling

Article Title: Important Role of Sarcoplasmic Reticulum Ca 2+ Release via Ryanodine Receptor-2 Channel in Hypoxia-Induced Rieske Iron–Sulfur Protein-Mediated Mitochondrial Reactive Oxygen Species Generation in Pulmonary Artery Smooth Muscle Cells

doi: 10.1089/ars.2018.7652

Figure Lengend Snippet: Caffeine, norepinephrine, and hypoxia elevated [Ca2+]m that causes [ROS]m in PASMCs. (A) The correlation curve between mit-2mutAEQ-derived relative luminescence and different free Ca2+ concentration was constructed and then used to quantify the effect of caffeine, norepinephrine, and hypoxia on [Ca2+]m in PASMCs. The mit-2mutAEQ-derived relative luminescence was determined using pcDNA3.1+/mit-2mutAEQ vector as described in the Materials and Methods section. (B) Application of caffeine at various concentrations for 5 min induced a large elevation of [Ca2+]m in PASMCs. [Ca2+]m was by quantified by fitting the data into the correlation curve for mit-2mutAEQ-derived luminescence at various free Ca2+ concentrations. Caffeine-induced increase in [Ca2+]m was concentration-dependent and suppressed by pretreatment with the mitochondria Ca2+ uniporter inhibitor Ru360 (10 μM) for 5 min. (C) Norepinephrine, similar to caffeine, also induced a concentration-dependent and Ru360-inhibited elevation of [Ca2+]m in PASMCs. (D) Hypoxia exposure for 5 min caused a significant elevation of [Ca2+]m in PASMCs compared with normoxic exposure. The hypoxic response was inhibited by treatment with Ru360 (10 μM) for 5 min as well. (E) Exogenous free Ca2+ at concentrations ranged from 1 to 30 μM resulted in a concentration-dependent increase in ROS generation in freshly isolated mitochondria from PASMCs. ROS generation was determined by measuring DCF-derived fluorescence by the mean of H2DCF/DA (DCF). (F) Ca2+ concentration-dependent ROS generation in freshly isolated mitochondria was observed by using the chemiluminescent dye lucigenin. (G) Application of Ca2+ concentration-dependent increased ROS generation in isolated complex III from PASMCs. ROS generation was determined by DCF-derived fluorescence. (H) Ca2+ (200 μM) failed to increase ROS generation (DCF-derived fluorescence) in isolated complex I from PASMCs. (I) Ca2+-dependent ROS generation in isolated complex III from PASMCs was further observed using lucigenin. All data were obtained from three separate experiments. In (B, C), *p < 0.05 compared with control; in (D), *p < 0.05 compared with control in normoxia condition. #p < 0.05 compared with control in hypoxia condition; in (E–H), *p < 0.05 compared with no Ca2+-treated group. [Ca2+]m, intramitochondrial Ca2+ concentration; mit-2mutAEQ, mitochondria-targeted double mutated aequorin; [ROS]m, mitochondrial reactive oxygen species generation.

Article Snippet: Following the procedure provided by Addgene ( 6 ), pcDNA3.1+/mit-2mutAEQ was purified and transfected to primary cultured mouse PASMCs for 48 h. After transfection, PASMCs were incubated for 2 h at room temperature in standard medium (145 m M NaCl, 5 m M KCl, 1 m M MgCl 2 , 1 m M CaCl 2 , 10 m M glucose, and 10 m M HEPES [pH 7.4]) with 2 μ M coelenterazine w (Biotium).

Techniques: Derivative Assay, Concentration Assay, Construct, Plasmid Preparation, Isolation, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: CD44+ and CD133+ Non-Small Cell Lung Cancer Cells Exhibit DNA Damage Response Pathways and Dormant Polyploid Giant Cancer Cell Enrichment Relating to Their p53 Status

doi: 10.3390/ijms23094922

Figure Lengend Snippet: Representative microphotographs of immunofluorescently stained spheroids showing DAPI (blue), FAM3C (GFP, green) and MiTF (Cy5, pink) ( a ). The expression of MITF ( b ) and FAM3C ( c ) in spheroid cultures derived from CD-sorted populations of A549 and H1299 cell lines four days after 5 Gy irradiation. Data are means ± SEM of more than three independent experiments. Where: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Spheroids were incubated with primary rabbit polyclonal antibody to FAM3C/ILEI (dilution 1:100, Cat. # ab72182, Abcam, Cambridge, MA, USA), mouse monoclonal Anti-MiTF antibody [C5] (dilution 1:100, Cat. # ab12039, Abcam, Cambridge, MA, USA) and secondary goat anti-rabbit Alexa Fluor 488 conjugated (dilution 1:500; Cat. # A-11008, Merck Millipore, Burlington, VT, USA) and goat anti-mouse Cy5 conjugated (dilution 1:500; Cat. # AP124C, Merck Millipore, Burlington, VT, USA) antibodies.

Techniques: Staining, Expressing, Derivative Assay, Irradiation