mito rbcs  (Santa Cruz Biotechnology)

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

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 ρ° Mito + RBCs, cells were cultured with MG132 (5 mM; Santa Cruz Biotech) and 500 ng/mL of ethidium bromide (EtBr; Sigma−Aldrich) during phase 3 and checked for mitochondrial and nuclear DNA content by RT-PCR.

Techniques: Expressing, Activation Assay, Derivative Assay