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: 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

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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Chondroitin Sulfate A-Adhering Plasmodium falciparum -Infected Erythrocytes Express Functionally Important Antibody Epitopes Shared by Multiple Variants

doi: 10.4049/jimmunol.1002390

Figure Lengend Snippet: Phagocytosis of opsonized P. falciparum IEs. Erythrocytes infected by EtBr-labeled P. falciparum FCR3-BeWo (A), HB3-BeWo (B), and FCR3-Ctrl (C) were opsonized with human mAbs (B cell supernatants at 1:5 dilution) either alone (PAM1.4, PAM2.8, PAM3.10, PAM4.7, PAM5.2, PAM6.1, PAM7.5, or PAM8.1), in combination (All), or with plasma from P. falciparum-exposed men, P. falciparum-exposed multigravid women, or unexposed controls and coincubated with THP-1 cells. The difference in phagocytosis in the absence (open curve) and presence of Abs (shaded curves) is shown in A. Each experiment was performed at least twice. The results of a typical experiment are shown in as the percentage of THP-1 cells that became EtBr-positive after coincubation with EtBr-labeled IEs. EM, exposed men; EMW, exposed multigravid women; UC, unexposed controls.

Article Snippet: After two additional washes, the opsonized IEs were incubated at a 20:1 ratio with the human monocytic leukemia line THP-1 (TIB-202; LGC Standards, Borås, Sweden) on a rocking table (30 min, 37°C).

Techniques: Infection, Labeling