Journal:

Article Title: Analysis of Cell Type-Specific Responses Mediated by the Type IV Secretion System of Helicobacter pylori

doi: 10.1128/IAI.73.8.4643-4652.2005

Figure Lengend Snippet: Cell lines used for this study

Article Snippet: Cells were grown in the indicated media and subcultured every 2 to 3 days. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Cell line Reference or source a (no.) Cell type Growth medium b 293T ATCC (CRL 11268) Human embryonal kidney DMEM (Gibco BRL), 10% fetal bovine serum (FBS; Gibco BRL), 2 mM l -glutamine, 1 mM sodium pyruvate AGS ATCC (CRL 1739) Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine CHO K1 ATCC (CCL 61) Chinese hamster ovary MEM alpha (Gibco BRL), 10% FBS Cos1 ATCC (CRL 1650) Green African monkey kidney RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine GLC4 42 Human lung carcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Hec1.b ATCC (HTP 113) Human endometrial adenocarcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine Hela ATCC (CCL 2) Human cervix epithelial carcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HepG2 ATCC (HB 8065) Human hepatocellular carcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HL ECACC (96121720) Human lung, fibroblast-like RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HT29 ATCC (HTB 38) Human colon adenocarcinoma Mc Coy's 5A (Gibco BRL), 10% FBS J774.A DSMZ (ACC 170) Mouse monocytic macrophages RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Kato3 ATCC (HTB 103) Human gastic carcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine L929 ATCC (CCL 1) Mouse fibroblasts RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine MDCK ATCC (CCL 34) Canine kidney RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine MKN-28 27 Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine MKN-45 DSMZ (ACC 409) Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine SR4987 DSMZ (ACC 323) Mouse stromal fibroblast-like Mc Coy's 5A (Gibco BRL), 10% FBS SYF+src ATCC (CRL 2498) Mouse fibroblasts overexpressing Src DMEM (Gibco BRL), 10% FBS, 2 mM l -glutamine, 1 mM sodium pyruvate THP1 ATCC (TIB 202) Human monocytes from acute monocytic leukemia RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Open in a separate window a ATCC, American Type Culture Collection ( www.atcc.org ); DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ( www.dsmz.de ); ECACC, European Collection of Cell Cultures ( www.ecacc.org.uk ). b DMEM, Dulbecco's modified Eagle's medium; MEM, minimum essential medium.

Techniques:

Journal:

Article Title: Analysis of Cell Type-Specific Responses Mediated by the Type IV Secretion System of Helicobacter pylori

doi: 10.1128/IAI.73.8.4643-4652.2005

Figure Lengend Snippet: Responses of 19 mammalian cell lines to infection with H. pylori strain P1

Article Snippet: Cells were grown in the indicated media and subcultured every 2 to 3 days. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Cell line Reference or source a (no.) Cell type Growth medium b 293T ATCC (CRL 11268) Human embryonal kidney DMEM (Gibco BRL), 10% fetal bovine serum (FBS; Gibco BRL), 2 mM l -glutamine, 1 mM sodium pyruvate AGS ATCC (CRL 1739) Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine CHO K1 ATCC (CCL 61) Chinese hamster ovary MEM alpha (Gibco BRL), 10% FBS Cos1 ATCC (CRL 1650) Green African monkey kidney RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine GLC4 42 Human lung carcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Hec1.b ATCC (HTP 113) Human endometrial adenocarcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine Hela ATCC (CCL 2) Human cervix epithelial carcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HepG2 ATCC (HB 8065) Human hepatocellular carcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HL ECACC (96121720) Human lung, fibroblast-like RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HT29 ATCC (HTB 38) Human colon adenocarcinoma Mc Coy's 5A (Gibco BRL), 10% FBS J774.A DSMZ (ACC 170) Mouse monocytic macrophages RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Kato3 ATCC (HTB 103) Human gastic carcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine L929 ATCC (CCL 1) Mouse fibroblasts RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine MDCK ATCC (CCL 34) Canine kidney RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine MKN-28 27 Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine MKN-45 DSMZ (ACC 409) Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine SR4987 DSMZ (ACC 323) Mouse stromal fibroblast-like Mc Coy's 5A (Gibco BRL), 10% FBS SYF+src ATCC (CRL 2498) Mouse fibroblasts overexpressing Src DMEM (Gibco BRL), 10% FBS, 2 mM l -glutamine, 1 mM sodium pyruvate THP1 ATCC (TIB 202) Human monocytes from acute monocytic leukemia RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Open in a separate window a ATCC, American Type Culture Collection ( www.atcc.org ); DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ( www.dsmz.de ); ECACC, European Collection of Cell Cultures ( www.ecacc.org.uk ). b DMEM, Dulbecco's modified Eagle's medium; MEM, minimum essential medium.

Techniques: Infection

Journal:

Article Title: Analysis of Cell Type-Specific Responses Mediated by the Type IV Secretion System of Helicobacter pylori

doi: 10.1128/IAI.73.8.4643-4652.2005

Figure Lengend Snippet: Chemokine secretion from H. pylori-infected human and murine cell lines. Cells were infected for 20 h with the H. pylori wild-type strain (P1wt) or the type IV secretion-defective knockout mutant P1ΔvirB11. IL-8 or Mip-2 secreted into the medium was determined by ELISA. The amounts of chemokine secretion are presented as percentages relative to the amount during wild-type infection, except for GLC4, L929, SR4987, and SYF+src cells, from which no secretion was detected at all. For most cell lines, chemokine secretion was dependent on the function of the TFSS. In addition, HeLa and THP1 cells showed some IL-8 secretion upon infection withP1ΔvirB11. The Mann-Whitney test indicated that this low level of IL-8 secretion was significantly higher than that in noninfected cells (P < 0.05, asterisks). Infection of the mouse cell line J774.A with P1ΔvirB11 induced a slightly higher Mip-2 secretion level than infection with the wild-type strain. Error bars indicate standard deviations.

Article Snippet: Cells were grown in the indicated media and subcultured every 2 to 3 days. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Cell line Reference or source a (no.) Cell type Growth medium b 293T ATCC (CRL 11268) Human embryonal kidney DMEM (Gibco BRL), 10% fetal bovine serum (FBS; Gibco BRL), 2 mM l -glutamine, 1 mM sodium pyruvate AGS ATCC (CRL 1739) Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine CHO K1 ATCC (CCL 61) Chinese hamster ovary MEM alpha (Gibco BRL), 10% FBS Cos1 ATCC (CRL 1650) Green African monkey kidney RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine GLC4 42 Human lung carcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Hec1.b ATCC (HTP 113) Human endometrial adenocarcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine Hela ATCC (CCL 2) Human cervix epithelial carcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HepG2 ATCC (HB 8065) Human hepatocellular carcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HL ECACC (96121720) Human lung, fibroblast-like RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HT29 ATCC (HTB 38) Human colon adenocarcinoma Mc Coy's 5A (Gibco BRL), 10% FBS J774.A DSMZ (ACC 170) Mouse monocytic macrophages RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Kato3 ATCC (HTB 103) Human gastic carcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine L929 ATCC (CCL 1) Mouse fibroblasts RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine MDCK ATCC (CCL 34) Canine kidney RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine MKN-28 27 Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine MKN-45 DSMZ (ACC 409) Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine SR4987 DSMZ (ACC 323) Mouse stromal fibroblast-like Mc Coy's 5A (Gibco BRL), 10% FBS SYF+src ATCC (CRL 2498) Mouse fibroblasts overexpressing Src DMEM (Gibco BRL), 10% FBS, 2 mM l -glutamine, 1 mM sodium pyruvate THP1 ATCC (TIB 202) Human monocytes from acute monocytic leukemia RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Open in a separate window a ATCC, American Type Culture Collection ( www.atcc.org ); DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ( www.dsmz.de ); ECACC, European Collection of Cell Cultures ( www.ecacc.org.uk ). b DMEM, Dulbecco's modified Eagle's medium; MEM, minimum essential medium.

Techniques: Infection, Knock-Out, Mutagenesis, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal:

Article Title: Analysis of Cell Type-Specific Responses Mediated by the Type IV Secretion System of Helicobacter pylori

doi: 10.1128/IAI.73.8.4643-4652.2005

Figure Lengend Snippet: Phenotypes of H. pylori-infected host cells. Infected cells were analyzed by phase-contrast microscopy. Only AGS cells showed the clearly defined hummingbird phenotype (upper panels). The hummingbird phenotype requires CagA, as it was not observed after infection with P1ΔcagA (not shown). THP1 cells formed aggregates after infection with the wild-type strain (middle panels). Aggregation is independent of CagA but requires the TFSS, as it was induced upon infection with the cagA but not the virB11 knockout mutant (not shown). Kato3 cells and all other cell lines tested did not reveal any obvious change in phenotype upon infection (lower panels). Bar, 100 μm.

Article Snippet: Cells were grown in the indicated media and subcultured every 2 to 3 days. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Cell line Reference or source a (no.) Cell type Growth medium b 293T ATCC (CRL 11268) Human embryonal kidney DMEM (Gibco BRL), 10% fetal bovine serum (FBS; Gibco BRL), 2 mM l -glutamine, 1 mM sodium pyruvate AGS ATCC (CRL 1739) Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine CHO K1 ATCC (CCL 61) Chinese hamster ovary MEM alpha (Gibco BRL), 10% FBS Cos1 ATCC (CRL 1650) Green African monkey kidney RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine GLC4 42 Human lung carcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Hec1.b ATCC (HTP 113) Human endometrial adenocarcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine Hela ATCC (CCL 2) Human cervix epithelial carcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HepG2 ATCC (HB 8065) Human hepatocellular carcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HL ECACC (96121720) Human lung, fibroblast-like RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HT29 ATCC (HTB 38) Human colon adenocarcinoma Mc Coy's 5A (Gibco BRL), 10% FBS J774.A DSMZ (ACC 170) Mouse monocytic macrophages RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Kato3 ATCC (HTB 103) Human gastic carcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine L929 ATCC (CCL 1) Mouse fibroblasts RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine MDCK ATCC (CCL 34) Canine kidney RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine MKN-28 27 Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine MKN-45 DSMZ (ACC 409) Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine SR4987 DSMZ (ACC 323) Mouse stromal fibroblast-like Mc Coy's 5A (Gibco BRL), 10% FBS SYF+src ATCC (CRL 2498) Mouse fibroblasts overexpressing Src DMEM (Gibco BRL), 10% FBS, 2 mM l -glutamine, 1 mM sodium pyruvate THP1 ATCC (TIB 202) Human monocytes from acute monocytic leukemia RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Open in a separate window a ATCC, American Type Culture Collection ( www.atcc.org ); DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ( www.dsmz.de ); ECACC, European Collection of Cell Cultures ( www.ecacc.org.uk ). b DMEM, Dulbecco's modified Eagle's medium; MEM, minimum essential medium.

Techniques: Infection, Microscopy, Knock-Out, Mutagenesis

Journal:

Article Title: Analysis of Cell Type-Specific Responses Mediated by the Type IV Secretion System of Helicobacter pylori

doi: 10.1128/IAI.73.8.4643-4652.2005

Figure Lengend Snippet: Model of the roles of host cell factors required for the cellular responses mediated by the H. pylori TFSS. H. pylori translocates CagA, which is phosphorylated by Src family kinases (SFK). Tyrosine-phosphorylated CagA has been shown to interact with Csk and SHP-2, to inactivate Src family kinases, and to induce tyrosine dephosphorylation of cortactin and ezrin and rearrangements of the actin cytoskeleton. The TFSS also translocates peptidoglycan (PG), which activates the intracellular Nod1 receptor, activates the transcription factor NF-κB, and stimulates the expression and secretion of IL-8 and the upregulation of ICAM-1 on the cell surface. (A) Host cell factors appear to be required for CagA and peptidoglycan translocation. These factors could be inducers or cellular receptors of the TFSS. (B) Processing of CagA only occurs in some cell lines, indicating that specific cellular proteases are involved. (C) The hummingbird phenotype is only observed with AGS cells. Thus, some unknown host cell factors are apparently required for its induction. (D) HeLa and THP1 cells secrete some IL-8 in a TFSS-independent manner, indicating that another receptor can detect H. pylori in these cells.

Article Snippet: Cells were grown in the indicated media and subcultured every 2 to 3 days. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Cell line Reference or source a (no.) Cell type Growth medium b 293T ATCC (CRL 11268) Human embryonal kidney DMEM (Gibco BRL), 10% fetal bovine serum (FBS; Gibco BRL), 2 mM l -glutamine, 1 mM sodium pyruvate AGS ATCC (CRL 1739) Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine CHO K1 ATCC (CCL 61) Chinese hamster ovary MEM alpha (Gibco BRL), 10% FBS Cos1 ATCC (CRL 1650) Green African monkey kidney RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine GLC4 42 Human lung carcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Hec1.b ATCC (HTP 113) Human endometrial adenocarcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine Hela ATCC (CCL 2) Human cervix epithelial carcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HepG2 ATCC (HB 8065) Human hepatocellular carcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HL ECACC (96121720) Human lung, fibroblast-like RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HT29 ATCC (HTB 38) Human colon adenocarcinoma Mc Coy's 5A (Gibco BRL), 10% FBS J774.A DSMZ (ACC 170) Mouse monocytic macrophages RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Kato3 ATCC (HTB 103) Human gastic carcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine L929 ATCC (CCL 1) Mouse fibroblasts RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine MDCK ATCC (CCL 34) Canine kidney RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine MKN-28 27 Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine MKN-45 DSMZ (ACC 409) Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine SR4987 DSMZ (ACC 323) Mouse stromal fibroblast-like Mc Coy's 5A (Gibco BRL), 10% FBS SYF+src ATCC (CRL 2498) Mouse fibroblasts overexpressing Src DMEM (Gibco BRL), 10% FBS, 2 mM l -glutamine, 1 mM sodium pyruvate THP1 ATCC (TIB 202) Human monocytes from acute monocytic leukemia RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Open in a separate window a ATCC, American Type Culture Collection ( www.atcc.org ); DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ( www.dsmz.de ); ECACC, European Collection of Cell Cultures ( www.ecacc.org.uk ). b DMEM, Dulbecco's modified Eagle's medium; MEM, minimum essential medium.

Techniques: De-Phosphorylation Assay, Expressing, Translocation Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Factor H Is Bound by Outer Membrane-Displayed Carbohydrate Metabolism Enzymes of Extraintestinal Pathogenic Escherichia coli and Contributes to Opsonophagocytosis Resistance in Bacteria

doi: 10.3389/fcimb.2020.592906

Figure Lengend Snippet: FH recruitment contributed to the anti-opsonophagocytic ability of ExPEC. ExPEC strain RS218 pre-incubated with purified human FH and incubated with 10% FH-depleted human serum. Then the opsonized bacteria were infected to THP-1 cells or performed plate counting. The phagocytic rates were determined as ratios of the number of bacteria recovered from THP-1 to those recovered from human serum. The data are expressed as the mean ± SEM of three independent experiments. The statistical significance of differences between each pair was determined using the unpaired t -test (*p < 0.05; **p < 0.01; ns, no significance.).

Article Snippet: THP-1 cells (human macrophage cell line, CLS Cat# 300356/p804_THP-1, RRID: CVCL_0006) were cultured in RPMI 1640 media (Gibco, Cat# 72400047) with 10% fetal bovine serum (FBS, Gibco, Cat# 10100139C) at 37°C and 5% CO 2 atmosphere.

Techniques: Incubation, Purification, Bacteria, Infection

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 1. CCR2 is required for efficient SFTSV infection in cells. (A to C) RT-qPCR (A), flow cytometry (B), and Western blot (C) analysis of SFTSV infection at 24 hours after infection in CCR2- or ATF6-knockdown THP-1 cells. n = 6. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) RT-qPCR analysis of virus RNA in CCR2-KO THP-1 cells inoculated with four phylogenetically distinct SFTSV strains, including HBMC16, HNXY2017-50, HNXY2017-66, and WCH, for 24 hours. n = 4. (E) Multistep growth curves of four SFTSV strains in CCR2-KO THP-1 cells. n = 4. (F) Surface expression of CCR2 on BMDMs from CCR2−/−and WT C57BL/6J mice. A representative of three replicates is shown. (G and H) Statistical results (G) and scanned images (H) of the immunological focus assay of SFTSV titers at 24 hours after infection in BMDMs with deletions in CCR2. n = 6. (I) Microscopy of BMDMs immunostained for F4/80, SFTSV NP, and DAPI at 24 hours after infection. (J) SFTSV infection rates at 24 hours after infection determined by flow cytometry analysis in Huh7, HeLa, and Jurkat cells overexpressing CCR2A or CCR2B. n = 6. (K) Representative flow plot of SFTSV infection in Huh7 cells. (L) Supernatant viral titers measured by immunological focus assay in Huh7, HeLa, and Jurkat cells overexpressing CCR2A or CCR2B. n = 6. Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), and (G)]. One-way ANOVA followed by Tukey’s multiple comparisons test was per- formed for comparison of variables among three groups [(J) and (K)].

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Quantitative RT-PCR, Flow Cytometry, Western Blot, Knockdown, Virus, Expressing, Microscopy, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 2. Effect of CCR2 inhibitor and antibody on SFTSV infection in cells. (A to F) Effects of CCR2 antagonist RS102895 and CCR2 antagonist 1 on SFTSV infection in THP-1 (A) to (C) and Huh7 cells (D) to (F). At 24 hours after infection, relative vRNA levels were measured via RT-qPCR [(A), (B), (D), and (E), n = 3], and relative intracellular SFTSV NP levels were measured by Western blotting (C) and (F). Cell viability was measured using CCK-8 [(D) and (E), n = 3]. (G to I) Effects of CCR2 antibody on SFTSV infection in THP-1 cells. The dose-dependent inhibitory effects of the CCR2 antibody were analyzed by detecting virus loads in THP-1 cells at 24 hours after infection [(G), n = 6]. SFTSV infection rates were measured at 24 hours after infection with MOIs of 1 and 5 [(H), n = 6]. Representative flow plot of SFTSV infection in THP-1 cells treated with anti-human CCR2 or isotype control antibody (I). Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), (E), (G), and (H)]. R2 [(A), (B), (D), and (E)] was estimated by a nonlinear regression model (curve fit).

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Virus, Control, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 3. CCR2 mediates SFTSV binding and internalization. (A and B) Effects of CCR2 deletions on the internalization (A) and binding (B) of SFTSV in BMDMs. Relative vRNA levels were measured via RT-qPCR. n = 6. (C) Fluorescence microscopy analysis of the effects of CCR2 deletions on the binding of SFTSV. BMDMs were immunos- tained with F4/80 (green), SFTSV NP (red), and DAPI. A representative of three replicates is shown. (D and E) SFTSV binding assay in CCR2-KO (KO) THP-1 cells by using RT- qPCR (D) and immunofluorescence staining (E). n = 4. Cellular membranes were labeled with WGA. (F) Flow cytometry analysis of SFTSV infection at 2 hours after infection in Huh7, HeLa, and Jurkat cells overexpressing CCR2A or CCR2B. n = 6. (G to I) Effects of the CCR2 antagonist RS102895 (G), CCR2 antagonist 1 (H), and favipiravir (I) on the binding and internalization of SFTSV in THP-1 cells. n = 3. (J) Binding assay of HRTV, RVFV, and AMRV for control and CCR2-KO THP-1 cells. n = 3. (K and L) Effects of CCR2 antagonist RS102895 (K) and CCR2 antagonist 1 (L) on the binding of HRTV, RVFV, and AMRV in THP-1 cells. n = 3. Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), and (J)]. One-way ANOVA followed by Tukey’s multiple comparisons test was performed for comparison of variables among three groups [(F) to (I), (K), and (L)]. ns, no significance; p.i., post-infection.

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Binding Assay, Quantitative RT-PCR, Fluorescence, Microscopy, Immunofluorescence, Staining, Labeling, Flow Cytometry, Infection, Control, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 4. The CCR2 N-terminal extracellular domain mediates SFTSV binding to cells. (A) Coimmunoprecipitation of co-overex- pressed Gn-strep protein and CCR2-flag proteins in HEK293T cells using strep-tag binding beads or anti-Flag antibody beads. Vector-flag, GFP-flag, and SCARB1-flag pro- teins were used as negative controls. (B to D) Effect of CCR2 overexpression on the in- fectivity of SFTSV in Huh7 (B), HeLa (C), and Jurkat (D) cells. SFTSV infection rates were measured at 24 hours after infection by flow cytometry analysis in control-, CCR2A-, CCR2B-, CCR2A-ΔN–, and CCR2B-ΔN–over- expressing cells. N = 6. (E) Surface expres- sion of CCR2 and representative flow plot of SFTSV infection in Jurkat cells. (F and G) Effect of the CCR2 N-terminal extracellular domain on the infectivity of SFTSV in HeLa (F) and Jurkat (G) cells. SFTSV infection rates were measured at 24 hours after infection by flow cytometry in control-, CCR2A-, CCR2A-N14Q–, CCR2A-Y26F–, CCR2B-, CCR2B-N14Q–, and CCR2B-Y26F–overex- pressing cells. n = 6. (H) Binding of CCR2 N- terminal–derived Y26 sulfated peptide (p2), peptide without tyrosine sulfation at Y26 (p1), or the scrambled peptide to SFTSV virions determined by PRM assay. Statistical analysis is shown in the left panel, and PRM transitions are shown in the right panel. (I and J) Effect of CCR2 N-terminal–derived peptides on the infectivity of SFTSV in THP-1 cells. Relative intracellular vRNA levels and supernatant viral titers were measured at 24 hours after infection via RT-qPCR. n = 4. (K) Effect of CCR2 N-terminal–derived peptides on the binding of SFTSV in THP-1 cells. Rel- ative levels of bound virions were measured via RT-qPCR. n = 4. One-way ANOVA fol- lowed by Tukey’s multiple comparisons test was performed for comparison of variables among three groups [(B) to (D) and (F) to (K)].

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Binding Assay, Strep-tag, Plasmid Preparation, Over Expression, Infection, Flow Cytometry, Control, Expressing, Derivative Assay, Quantitative RT-PCR, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 5. CCR2 contributes to SFTSV pathogenesis in mouse models. (A) Serum and spleen viral titers in SFTSV-infected CCR2−/−and WT C57BL/6J mice (four for each group) tested by immunological focus assay at 3 and 5 dpi. (B and C) Survival probability (B) and relative body weight (C) in anti-IFNAR1 antibody–pretreated CCR2−/−(n = 11) and WT (n = 11) C57BL/6J mice after intraperitoneal infection with SFTSV. (D) Viral titers in serum, spleen, liver, and lung samples from anti-IFNAR1 antibody–pre- treated CCR2−/−and WT C57BL/6J mice (four for each group) tested by immunological focus assay at 3 and 5 dpi. (E and F) Representative images of spleen, liver, and lung sections collected at 5 dpi from control and SFTSV-challenged CCR2−/−and WT C57BL/6J mice stained with a rabbit polyclonal antibody against SFTSV NP (E) or with hematoxylin and eosin (F). (G) Viral titers in serum from anti-IFNAR1 antibody–pretreated C57BL/6J mice tested by immunological focus assay at 3 (n = 13 for each group) and 5 (n = 10 for nontreated group and n = 13 for treated group) dpi. (H) Survival probability in anti-IFNAR1 antibody–pretreated C57BL/6J mice with SFTSV infection in the absence (n = 13) or presence (n = 13) of CCR2 antagonist RS102895 and without SFTSV infection (n = 5). Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), and (G)]. The Kaplan-Meier method was used to analyze time-to-event data [(C) and (H)].

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Control, Staining, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 6. CCR2 contributes to SFTSV infectivity in primary human monocytes. (A and B) Comparisons of surface CCR2 expression levels on primary human mono- cytes (left) and the ability of SFTS binding (right) between donors aged <60 (n = 10) and ≥60 (n = 10) years old (A), as well as between healthy donors (n = 8) and donors with DM (n = 8) (B). MFI, mean fluorescent intensity. (C) Association of surface CCR2 expression level on primary human monocytes with virus binding ability or with the age of donors (n = 20). Ra 2 indicates the correlation between the CCR2 expression level and individual age, and Rb 2 indicates the correlation between the CCR2 expression level and the binding ability of SFTSV. (D) Associa- tion of surface CCR2 expression level on primary human monocytes with the peak viral load in serum that was consecutively collected from SFTS patients (n = 45) during the clinical course. Two-tailed Student’s t test was performed for compar- ison of variables between two groups [(A) and (B)]. R2 [(C) and (D)] was estimated by a linear regression model. HC, healthy control; DM, diabetes mellitus; CT, cycle threshold.

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Expressing, Binding Assay, Virus, Two Tailed Test, Control

Journal: Cell Death & Disease

Article Title: Leishmania donovani parasite requires Atg8 protein for infectivity and survival under stress

doi: 10.1038/s41419-019-2038-7

Figure Lengend Snippet: a THP1 monocyte-derived macrophages infected with L. donovani show the highest infection rates with Atg8 -OE as compared with ∆ Atg8 and VT. Arrows indicate the amastigotes within the infected macrophages. Scale bars represent 10 μm. b The upper panel shows the number of amastigotes/macrophage. Data represent mean ± SEM ( n = 3, from counts of 500 macrophages), * P < 0.05. The lower panel shows changes in the percentage of infected macrophages with respect to the duration of infection. Data represent mean ± SEM ( n = 3), * P < 0.05. Note that infection by ∆ Atg8 parasites is low. c Data from spleens ( n = 10) of uninfected mice and those infected with ∆ Atg8 parasites showed a significant difference in spleen size between WT-infected and ∆ Atg8- infected animals. Photomicrographs of splenic imprints show parasite load in mice infected with WT and ∆ Atg8 parasites. d , e Size and weight of mice spleen isolated from Leishmania- infected samples. Increase in weight and dimensions of spleens are evident in mice infected with WT parasites compared with those infected with ∆ Atg8 parasites in which infection was negligible, n = 10. f Parasite burden in terms of Donovan units was calculated from splenic smears. LDU, Leishman–Donovan units, n = 10

Article Snippet: The human monocytic cell line THP1 (ATCC TIB-67) was maintained at 37 °C and 5% CO 2 in Roswell Park Memorial Institute Medium (RPMI; Sigma-Aldrich, St. Louis, MO) containing sodium bicarbonate (350 mg·ml −1 ), glucose (4.5 g·ml −1 ), and 10% FCS.

Techniques: Derivative Assay, Infection, Isolation

Journal: Science Immunology

Article Title: ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection

doi: 10.1126/sciimmunol.abo6294

Figure Lengend Snippet: ( A ) Heatmap depicting the altered pathways in whole blood cell transcriptomes of critically ill (CI) patients compared with non-critically ill (NCI) patients with COVID-19 ( 34 ). NES, normalized enrichment scores. ( B ) IFN-β release in the supernatant of wild type (WT) bone marrow-derived macrophages (BMDMs) infected with mouse hepatitis virus (MHV) or influenza A virus (IAV) (left) or THP-1 cells infected with SARS-CoV-2 or IAV (right) for the indicated time. ( C ) Immunoblot analysis of phosphorylated STAT1 (pSTAT1), total STAT1 (tSTAT1) and ISG15 in WT BMDMs infected with MHV or IAV (left) or THP-1 cells infected with SARS-CoV-2 or IAV (right) for the indicated time. Molecular weight marker sizes in kDa are indicated in small font on the left of each blot. ( D ) Kinetics of IFN-α and IFN-γ responses in SARS-CoV-2– or IAV-infected Calu-3 cells ( 41 , 104 ). Data are representative of at least three independent experiments ( B, C ). **** P < 0.0001. Analysis was performed using the two-way ANOVA ( B ). Data are shown as mean ± SEM.

Article Snippet: Human THP-1 macrophages were stimulated with 50 ng/mL of IFN-β (PBL Assay, 11410-2) for the indicated time.

Techniques: Derivative Assay, Infection, Virus, Western Blot, Molecular Weight, Marker

Journal: Science Immunology

Article Title: ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection

doi: 10.1126/sciimmunol.abo6294

Figure Lengend Snippet: ( A – C ) Immunoblot analysis of ( A ) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); ( B ) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and ( C ) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK3 (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from mock- or IFN-β–treated wild type (WT) mice with or without mouse hepatitis virus (MHV) infection 3 days post-infection. ( D – I ) Immunoblot analysis of ( D , G ) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; ( E , H ) pro- (P55) and cleaved CASP8 (P18), pro- (P35) and cleaved CASP3 (P19 and P17) and pro- (P35) and cleaved CASP7 (P20); ( F) pMLKL, tMLKL, pRIPK3 and tRIPK3; and ( I ) pRIPK3 and tRIPK3 in mock- or IFN-β–treated bone marrow-derived macrophages (BMDMs) or THP-1 cells during MHV or SARS-CoV-2 infection, respectively. Actin was used as the internal control. Molecular weight marker sizes in kDa are indicated in small font on the left of each blot. Asterisk denotes non-specific bands ( A, G ). Data are representative of at least three independent experiments.

Article Snippet: Human THP-1 macrophages were stimulated with 50 ng/mL of IFN-β (PBL Assay, 11410-2) for the indicated time.

Techniques: Western Blot, Virus, Infection, Derivative Assay, Control, Molecular Weight, Marker

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a-c , NINJ1 oligomerization (Native-PAGE) and LDH-release from WT and NINJ1 KO iPSDMs pretreated with glycine or inhibitors as indicated and infected with Mtb mc²6206, MOI 20 for 4h ( a, b ), Mtb H37Rv, 7.5 MOI for 24h ( c ), or treated with cell death stimuli for 4h: d , pyroptosis – LPS and nigericin (LPS+N) w/wo NLRP3 inhibitor MCC950; e , apoptosis – venetoclax; f , necroptosis – Z-VAD-FMK+BV-6+TNFa (zBT) w/wo RIPK3 inhibitor GSK’872; g , ferroptosis – RSL-3 w/wo Ferrostatin-1. Data are means (bars) ± s.e.m. of at least three independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with Tukey’s multiple comparisons test of treatments within each cell line and stimuli between cell lines. For gel source data, see Supplementary Figure 1.

Article Snippet: THP-1 NINJ1 KO monocytes (Cyagen, CA, USA) were transduced by resuspending approximately 750 000 cells in 1 ml filtered and concentrated virus supernatant, adding 8 μg/ml polybrene, and thereafter spinoculating at 1200 g and 32 °C for 90 minutes.

Techniques: Clear Native PAGE, Infection

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, Lysates of WT, NINJ1 KO and GSDMD KO iPSC-derived monocytes (m) and macrophages (M) were subjected to SDS-PAGE and immunoblotted for NINJ1, GSDMD and β-actin. A3 and C6 are NINJ1 KO clones; A7, B8 are GSDMD KO clones. b, Human monocyte-derived macrophages were transfected with siRNA targeting NINJ1 or a non-targeted control and infected with Mtb mc²6206, MOI 20, for 4h. Shown is LDH-release and NINJ1 expression level by qPCR. Data are presented as mean ± s.e.m of four donors (shown with individual symbols). *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with uncorrected Fisher’s LSD. c-j, LDH-release from iPSDM WTs and NINJ1 KO clones A3, C6 ( c-f ) or GSDMD KO clones A7, B8 ( g-j ) pretreated with glycine or inhibitors as indicated and treated with cell death stimuli for 4h: pyroptosis – LPS and nigericin (LPS+N) w/wo NLRP3 inhibitor MCC950 ( c, g ); apoptosis – venetoclax ( d, h ); necroptosis – Z-VAD-FMK+BV-6+TNFα (zBT) w/wo RIPK3 inhibitor GSK’872 ( e, i ); ferroptosis – RSL-3 w/wo Ferrostatin-1 ( f, j ). ( c-f ) Data are means (bars) ± s.e.m. of three (A3) or two (C6) independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with Tukey’s multiple comparisons test. ( g-j ) Data are means (bars) ± s.d. of two or three technical replicates (circles) from 1 independent experiment.

Article Snippet: THP-1 NINJ1 KO monocytes (Cyagen, CA, USA) were transduced by resuspending approximately 750 000 cells in 1 ml filtered and concentrated virus supernatant, adding 8 μg/ml polybrene, and thereafter spinoculating at 1200 g and 32 °C for 90 minutes.

Techniques: Derivative Assay, SDS Page, Clone Assay, Transfection, Control, Infection, Expressing

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a , Representative images of NINJ1 KO THP-1 cells expressing hNINJ1-mNG (THP1-NINJ1-mNeonGreen) pretreated with glycine ( a , right column) before cell death stimuli venetoclax (apoptosis), LPS + nigericin (LPS+N, pyroptosis), RSL-3 (ferroptosis) for 4h, or infection with Mtb mc26206-BFP for 20h. Time-lapse TIRF (mNG), widefield (DRAQ7 and BFP) and brightfield microscopy images of NINJ1-mNG (green), DRAQ7 (red) and Mtb (blue), and morphology, respectively. Scale bars 10 mm. b , Quantification of NINJ1-mNG puncta densities at 4h (LPS+N, RSL-3) or 20h (Mtb) as represented in ( a ). Data are means (bars) ± s.e.m from 1-4 independent experiments (circles), each including analysis of 4-15 cells per condition. Significant differences are indicated as *P<0,05 by RM two-way Anova with Tukey’s multiple comparisons test. c , Representative time-lapse images of NINJ1 clustering (TIRF, green) and DRAQ7 uptake (red) upon Mtb mc26206-BFP infection (blue) temporally aligned by start of NINJ1 clustering (t=0). Time in min. Scale bars 10 mm. d, e , Fitted curves ( d ) and t50 values (x value where y=50% max.) of sigmoidal fits ( e ) of NINJ1 oligomerization kinetics during Mtb-infection, apoptosis (venetoclax), pyroptosis (LPS+N) or ferroptosis (RSL-3) monitored with frame rates of 8-10 sec (Venetoclax, RSL-3, LPS+N) or 5 min (Mtb-infection). 3-5 cells analyzed per condition. Individual kinetic curves and representative images for RCD induction are shown in Extended Data Figure 3.

Article Snippet: THP-1 NINJ1 KO monocytes (Cyagen, CA, USA) were transduced by resuspending approximately 750 000 cells in 1 ml filtered and concentrated virus supernatant, adding 8 μg/ml polybrene, and thereafter spinoculating at 1200 g and 32 °C for 90 minutes.

Techniques: Expressing, Infection, Microscopy

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, b, Quantification of NINJ1-mNG puncta densities monitored by TIRF microscopy ( a ) and phagocytosed bacteria monitored by intracellular BFP intensities ( b ) before NINJ1 inhibition by glycine and after 4h of RCD induction (Venetoclax, LPS+N, RSL-3) or after 3h and 20h of Mtb mc²6206-BFP infection. Data are means (bars) ± s.e.m from 1-4 independent experiments (circles), each including analysis of 4-15 cells per condition. Significant differences are indicated as *P<0,05 by RM two-way Anova with Tukey’s multiple comparisons test. c,d, Kinetics of NINJ1 puncta densities and DRAQ7 intensities monitored with frame rates of 8-10 sec (Venetoclax, RSL-3, LPS+N) with (c) representative Time-lapse images of NINJ1 clustering (TIRF, green) and DRAQ7 (WF, red) aligned by start of NINJ1 clustering. Time in sec. Scale bars 10 μm. Intensity values were normalized to the highest value for each cell and aligned by the start of NINJ1 oligomerization. Data are means ± s.d. for 3-5 cells per condition with sigmoidal fits for NINJ1-mNG puncta density increase (green) and mono-exponential increase for DRAQ7 intensities (red).

Article Snippet: THP-1 NINJ1 KO monocytes (Cyagen, CA, USA) were transduced by resuspending approximately 750 000 cells in 1 ml filtered and concentrated virus supernatant, adding 8 μg/ml polybrene, and thereafter spinoculating at 1200 g and 32 °C for 90 minutes.

Techniques: Microscopy, Bacteria, Inhibition, Infection

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, b, Time-lapse microscopy of NINJ1 KO immortalized mouse BMDMs reconstituted with mNG-tagged human NINJ1 treated with LPS + nigericin ( a ) or RSL-3 ( b ). Image panels show NINJ1-mNG oligomerization (TIRF, green) and DRAQ7 influx (red, WF) and BF with quantification of NINJ1-mNG puncta density kinetics and increase of DRAQ7 intensities aligned by start of NINJ1 clustering. Time points shown in upper left corner in min. Scale bar 10 μm. Quantification shown to the right, data are presented as mean ± 95% CI. c, d, NINJ1 oligomerization (Native-PAGE) ( c ) and LDH release ( d ) from mouse NINJ1 KO iBMDMs expressing human NINJ1-mNG or NINJ1-mSc treated with LPS + nigericin in the presence or absence of glycine.

Article Snippet: THP-1 NINJ1 KO monocytes (Cyagen, CA, USA) were transduced by resuspending approximately 750 000 cells in 1 ml filtered and concentrated virus supernatant, adding 8 μg/ml polybrene, and thereafter spinoculating at 1200 g and 32 °C for 90 minutes.

Techniques: Time-lapse Microscopy, Clear Native PAGE, Expressing

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, b, LDH-release ( a ) and IL-1β release ( b ) from iPSDM WTs pretreated with glycine and cell death inhibitors (MCC950, Z-VAD-FMK, GSK’872 and Ferrostatin-1) and infected with Mtb mc²6206, MOI 20 for 4h. a, Data are means (bars) ± s.e.m. of 6 independent experiments (circles), each with three replicates per condition. b, Data are normalized against the Mtb mc26206 infected sample presented as means ± s.e.m. of 3 independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM one-way ANOVA with Dunnett’s multiple comparisons test against the infected sample. c, d, LDH-release from WT iPSDMs pretreated with glycine or a cocktail of inhibitors (zFG) including Z-VAD-FMK, Ferrostatin-1 and GSK’872 and infected with Mtb mc²6206, MOI 20 for 4h ( c ) or Mtb H37Rv, 7.5 MOI for 24h ( d ). Data are means (bars) ± s.e.m. of at least two independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM two-way ANOVA with Tukey’s multiple comparisons test of reatments within each cell line and stimuli between cell lines. e, NINJ1 oligomerization (Native-PAGE) in WT PSDMs pretreated with glycine or a cocktail of inhibitors (zMFG) including Z-VAD-FMK, MCC950, Ferrostatin-1 and GSK’872 and infected with Mtb mc²6206, MOI 20 for 4h. Only significant results are shown.

Article Snippet: THP-1 NINJ1 KO monocytes (Cyagen, CA, USA) were transduced by resuspending approximately 750 000 cells in 1 ml filtered and concentrated virus supernatant, adding 8 μg/ml polybrene, and thereafter spinoculating at 1200 g and 32 °C for 90 minutes.

Techniques: Infection, Clear Native PAGE

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, LDH release ( a ) from WT iPSDMs pretreated with glycine and cell death inhibitors (MCC950, Z-VAD-FMK, GSK’872 and Ferrostatin-1) for 30 min and infected with Mtb H37Rv, MOI 7,5 for 24h. Data are means (bars) ± s.e.m. of 2 independent experiments (circles), each with three replicates per condition. b, LDH release ( b ) from WT and NINJ1 KO iPSDMs pretreated with 20 µM 2-Bromohexadecanoic acid (2-BP) for 30 min and infected with Mtb mc²6206, MOI 20 for 4h. Data are means (bars) ± s.e.m. of 3 independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with Šídák’s multiple comparisons test of treatments within each cell line and stimuli between cell lines. All comparisons are shown. c,d, IL-1β release from WT iPSDMs pretreated with glycine and cell death inhibitors (MCC950, Z-VAD-FMK, GSK’872 and Ferrostatin-1) for 30 min and infected with Mtb H37Rv, MOI 7,5 for 24h ( c ) or Mtb mc²6206, MOI 20 for 4h ( d ). Data are means (bars) ± s.e.m. of 2-3 independent experiments (circles), each with three replicates per condition.

Article Snippet: THP-1 NINJ1 KO monocytes (Cyagen, CA, USA) were transduced by resuspending approximately 750 000 cells in 1 ml filtered and concentrated virus supernatant, adding 8 μg/ml polybrene, and thereafter spinoculating at 1200 g and 32 °C for 90 minutes.

Techniques: Infection

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, b, Cytokine release from WT and NINJ1 KO iPSDMs pretreated with glycine or a cocktail of inhibitors (zFG) including Z-VAD-FMK, Ferrostatin-1 and GSK’872 and infected with Mtb mc²6206, MOI 20 for 4h ( a ) or Mtb H37Rv, 7.5 MOI for 24h ( b ). Data are means (bars) ± s.e.m. of 3 independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ***P<0,0001 by Two-way ANOVA with Šídák’s multiple comparisons test of treatments within each cell ine and infection between cell lines. All comparisons performed are shown.

Article Snippet: THP-1 NINJ1 KO monocytes (Cyagen, CA, USA) were transduced by resuspending approximately 750 000 cells in 1 ml filtered and concentrated virus supernatant, adding 8 μg/ml polybrene, and thereafter spinoculating at 1200 g and 32 °C for 90 minutes.

Techniques: Infection

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, b, NINJ1-mNG oligomerization in immortalized mouse BMDMs expressing fluorescently tagged human NINJ1 upon infection with Mtb mc2 6206-BFP and treatment with 5 mM Cytochalasin D to prevent phagocytosis of Mtb mc2 6206-BFP with ( a ) representative TIRF microscopy images of hNINJ1-mNG (green), DRAQ7 uptake (red, WF) and bacteria (blue, WF) after 20h of infection, and ( b ) quantification of NINJ1 puncta density before and after 1, 4 and 20h of infection and treatment with Cytochalasin D. c, d, NINJ1-mNG oligomerization upon infection with Mtb mc2 6206-BFP and the RD-1 deficient mutant Mtb mc2 6230-BFP with ( c ) representative TIRF microscopy images before and after 20h of infection of hNINJ1-mNG (green), DRAQ7 uptake (red, WF) and bacteria (blue, WF) and ( d ) quantification of NINJ1 puncta density before and after 1, 4 and 20h of infection. e, LDH-release (e) from WT iPSDMs nfected with Mtb mc²6206 or Mtb mc²6230, MOI 20 for 4h. f, g, NINJ1 oligomerization after treatment with 10µM EsxA, 10µM EsxB and 70µM PDIM individually or in combination, with ( f ) representative TIRF microscopy images of hNINJ1-mNG (green) and DRAQ7 uptake (red, WF) after 20h of treatment and ( g ) quantification of NINJ1 puncta density before and after 1, 4 and 20h of treatment. h,i, NINJ1 oligomerization after treatment with 5 mM EsxA with and without pretreatment with glycine with ( h ) representative TIRF microscopy images of hNINJ1-mNG (green) and DRAQ7 uptake (red, WF) after 4h of treatment and ( i ) quantification of NINJ1 puncta density before and after 1 and 4h of treatment. j, k, LDH-release from WT iPSDMs treated with 1 mM EsxA for 4h with or without pretreatment with glycine ( j ) and treated with 5 and 10 mM EsxB for 4h ( k ). Data are means (bars) ± s.e.m. from 3 experiments with 4-8 cells per condition each. Significant differences are indicated as *P<0,05 by RM two-way Anova with Tukey’s multiple comparisons test. Scale bars 10 mm.

Article Snippet: THP-1 NINJ1 KO monocytes (Cyagen, CA, USA) were transduced by resuspending approximately 750 000 cells in 1 ml filtered and concentrated virus supernatant, adding 8 μg/ml polybrene, and thereafter spinoculating at 1200 g and 32 °C for 90 minutes.

Techniques: Expressing, Infection, Microscopy, Bacteria, Mutagenesis

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, b, Intracellular DRAQ7 ( a ) or BFP ( b ) intensities of iBMDMs untreated or infected with Mtb mc²6206-BFP, Mtb mc²6230-BFP or Mtb mc²6230-BFP pre-coated with 10 μM EsxA before and after 1, 4 and 20h of infection. c, d, NINJ1 oligomerization after infection with Mtb mc²6230-BFP or Mtb mc²6230-BFP pre-coated with 10 μM EsxA before and after 1, 4 and 20h of infection with ( c ) representative TIRF microscopy images of hNINJ1-mNG (green), DRAQ7 uptake (red, WF) and Mtb mc²6230-BFP (blue, WF) after 20h of infection and ( d ) quantification of NINJ1 puncta density before and after 1, 4 and 20h of treatment. e,f, NINJ1 oligomerization after treatment with 1, 5 or 10 μM EsxA before and after 1, 4 and 20h with ( e ) representative TIRF microscopy images of hNINJ1-mNG (green) and DRAQ7 uptake (red, WF) after 20h of treatment and ( f ) quantification of INJ1 puncta density before and after 1, 4 and 20h of treatment. g, Quantification of NINJ1 puncta density after treatment with 0.1, 1, 5 or 10 μM EsxB before and after 1, 4 and 20h. h, Quantification of intracellular DRAQ7 intensities after treatment with 10 μM EsxA, 10 μM EsxB and 70 μM PDIM individually or in combination. Data are means (bars) ± s.e.m. from 3-4 independent experiments. significant differences are indicated as *P<0,05 by RM two-way Anova with Tukey’s multiple comparisons test. Scale bars 10 μm.

Article Snippet: THP-1 NINJ1 KO monocytes (Cyagen, CA, USA) were transduced by resuspending approximately 750 000 cells in 1 ml filtered and concentrated virus supernatant, adding 8 μg/ml polybrene, and thereafter spinoculating at 1200 g and 32 °C for 90 minutes.

Techniques: Infection, Microscopy

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, b, LDH release ( a ) and NINJ1 oligomerization (native-PAGE) ( b ) from WT iPSDMs in DMEM with or without calcium or pretreated with EGTA (4 mM) or BAPTA-AM (5 µM) in calcium-free DMEM for 30 min before infection with Mtb mc²6206, MOI 20 for 4h. Data are means (bars) ± s.e.m. of 5-7 independent experiments (circles), each with three eplicates per condition. c, d, LDH release ( c ) and NINJ1 oligomerization (native-PAGE) ( d ) from WT PSDMs in DMEM with or without calcium or pretreated with EGTA (4 mM) or BAPTA-AM (5 µM) in calcium-free DMEM before stimulation with LPS + Nigericin. Data are means (bars) ± s.e.m. of 4-6 ndependent experiments (circles), each with three replicates per condition. e, LDH release from WT PSDMs in DMEM with or without calcium or pretreated with EGTA (4 mM) or BAPTA-AM (5 µM) in calcium-free DMEM before stimulation with RSL-3 for 4h. Data are means (bars) ± s.e.m. of 3 ndependent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM one-way ANOVA with Dunnett’s multiple comparisons test against the nfected/stimulated sample with calcium. f, LDH release from WT iPSDMs pretreated with 300 mM sucrose for 30 min before infection with Mtb mc²6206, MOI 20 or stimulation with RSL-3 for 4h. Data are means (bars) ± s.e.m. of 1-4 independent experiments (circles), each with three replicates per condition. g-i, LDH release ( g,h ) and NINJ1 oligomerization (native-PAGE) ( i ) from WT iPSDMs pretreated with PEG8000 (5 mM or 10 mM) for 30 min before infection with Mtb mc²6206, MOI 20 ( g ) or stimulation with RSL-3 ( h ) for 4h. Data are means (bars) ± s.e.m. of 7-10 independent experiments (circles), each with hree replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM one-way ANOVA with Dunnett’s multiple comparisons test against the infected or RSL-3 stimulated sample. All comparisons performed are shown. j, Relative light units (RLU) from WT iPSDMs pretreated with PEG8000 (5 mM or 10 mM) for 30 min before infection with Mtb mc²6206-luciferase for 4h. Data are means (bars) ± s.e.m. of 2 ndependent experiments (circles), each with three replicates per condition. k, LDH release ( k ) from WT PSDMs pretreated with PEG8000 (5 mM or 10 mM) for 30 min before stimulation with LPS + N. Data are means (bars) ± s.e.m. of 3 independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM one-way ANOVA with Dunnett’s multiple comparisons est against the LPS and nigericin stimulated sample. All comparisons performed are shown. l, NINJ1 oligomerization (native-PAGE) from WT iPSDMs treated with glycine or PEG8000 30 min before stimulation with LPS + Nigericin or RSL-3 for 4h.

Article Snippet: THP-1 NINJ1 KO monocytes (Cyagen, CA, USA) were transduced by resuspending approximately 750 000 cells in 1 ml filtered and concentrated virus supernatant, adding 8 μg/ml polybrene, and thereafter spinoculating at 1200 g and 32 °C for 90 minutes.

Techniques: Clear Native PAGE, Infection, Luciferase