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: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

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: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

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-d , Cytokine release from WT iPSDMs pretreated with glycine or inhibitors as indicated for 30 min and treated with cell death stimuli for 4h: pyroptosis – LPS and nigericin (LPS+N) w/wo NLRP3 inhibitor MCC950 ( a ); apoptosis – venetoclax ( b ); necroptosis – zVAD+BV-6+TNFα (zBT) w/wo RIPK3 inhibitor GSK’872 ( c ); ferroptosis – RSL-3 w/wo Ferrostatin-1 ( d ). Cytokine levels were assessed by multiplex ELISA. Data are means (bars) of two individual experiments (circles), each with two replicates.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Multiplex Assay, Enzyme-linked Immunosorbent Assay

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: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

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: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Microscopy, Bacteria, Inhibition, Infection

Journal: NPJ Precision Oncology

Article Title: Microfluidic-based dynamic BH3 profiling predicts anticancer treatment efficacy

doi: 10.1038/s41698-022-00333-0

Figure Lengend Snippet: a FACS-based DBP curves after incubation for 16 h with DMSO, imatinib, ripretinib, and dactolisib in primary cancer cells. Results are represented as the percentage of cells with cytochrome c retained inside the mitochondria after incubation with increasing concentrations of BIM peptide. b Quantification of % priming (percentage of cells with cytochrome c scape) in primary cancer cells after incubation with BIM, BAD, HRK, and MS1 peptides. c Quantification of % priming after BIM peptide incubation in primary cancer cells incubated for 16 h with DMSO, dactolisib, and the combination of dactolisib and venetoclax. d Visualization of primary cancer cells seeded inside the cell chambers of the microfluidic chip after treatment with DMSO, dactolisib and the combination of dactolisib and venetoclax for 16 h and exposure to increasing concentrations of BIM peptide generated using microfluidics. Alive cells at the start of the assay are marked in green (calcein AM) and TMRE (red field) is used to identify initiation of MOMP (marking the start of apoptosis). Scale bars, 100 μm. e Microfluidic-based DBP curves after incubation of the primary cancer cells for 16 h with DMSO, dactolisib, and the combination of dactolisib and venetoclax. Results are represented as the percentage of cells with positive TMRE signal after incubation with increasing concentrations of BIM peptide produced by the microfluidic gradient generator. f Quantification of % priming (percentage of cells with negative TMRE signal) in primary cancer cells inside the microfluidic chip. Values were chosen in the BIM peptide condition where the DMSO-treated cells started to engage MOMP and lose TMRE staining.

Article Snippet: Imatinib, dactolisib, and gefitinib were obtained from LC Laboratories (Woubourn, MA, USA); venetoclax was purchased at MedChemExpress (Monmouth Junction, NJ, USA), and repretinib from Selleckchem (Munich, Germany).

Techniques: Incubation, Generated, Produced, Staining

Journal: Leukemia

Article Title: Potent combination benefit of the AKT inhibitor capivasertib and the BCL-2 inhibitor venetoclax in diffuse large B cell lymphoma

doi: 10.1038/s41375-024-02401-9

Figure Lengend Snippet: A Base line Western blot biomarker profiling of ABC-, GCB-, and unclassified-DLBCL cell models with the OCI-LY1 cell line serving as reference control. B Capivasertib monotherapy anti-proliferative activity (GI 50 ) (μM) and venetoclax monotherapy apoptotic response (Log AC 50 ) (M) across GCB- and ABC-DLBCL cell lines. Order of cell lines is identical to that shown at the bottom of ( C ). C Loewe synergy scores for the combination of capivasertib and venetoclax in GCB- and ABC-DLBCL cell lines. + and – indicates each cell line’s monotherapy sensitivity to either capivasertib or venetoclax. Order of cell lines identical to that shown in ( B ). D Time course of caspase-3/7 activity in a cell model unresponsive to the either monotherapy or combination (TMD8), and two responsive models: WSU-DLCL2 (PTEN-mutant; GCB) and SUDHL-4 (PTEN wild-type; GCB) ( n = 3/group). E Relative cell proliferation of SUDHL4, WSU-DLCL2 and TMD8 cell lines pretreated overnight with the pan-caspase inhibitor Q-VD-OPH (50 µM) or vehicle and then dosed with compounds for 24 h ( n = 5/group; 2-way ANOVA with Sidak’s multiple comparisons **** p < 0.0001). Staurosporine (5 µM) was a positive control for caspase induction and cell killing.

Article Snippet: Venetoclax was purchased from Medkoo Biosciences (Durham, NC).

Techniques: Western Blot, Biomarker Assay, Control, Activity Assay, Mutagenesis, Positive Control

Journal: Leukemia

Article Title: Potent combination benefit of the AKT inhibitor capivasertib and the BCL-2 inhibitor venetoclax in diffuse large B cell lymphoma

doi: 10.1038/s41375-024-02401-9

Figure Lengend Snippet: A Western blot profiling of the AKT signalling pathway, known AKT substrates, and ( B ) apoptotic markers in WSU-DLCL2 and SUDHL-4 when treated with capivasertib and venetoclax monotherapies and in combination at 2and 4 h. C Western blot profiling of apoptotic biomarkers time course of WSU-DLCL2 and SUDHL4 cell lines pretreated with vehicle (DMSO) or Q-VD-OPH (50 µM) then dosed with indicated compounds at 1, 2, and 4 h. D CRISPR/Cas9-mediated of single and double knockout (DKO) of BAK and BAX in WSU-DLCL2 and SUDHL4 cell lines. E Comparison of the induction of cell death markers between BAK/BAX wild-type and DKO WSU-DLCL2 and SUDHL4 cell lines when treated with compounds for 4 h.

Article Snippet: Venetoclax was purchased from Medkoo Biosciences (Durham, NC).

Techniques: Western Blot, CRISPR, Double Knockout, Comparison

Journal: Leukemia

Article Title: Potent combination benefit of the AKT inhibitor capivasertib and the BCL-2 inhibitor venetoclax in diffuse large B cell lymphoma

doi: 10.1038/s41375-024-02401-9

Figure Lengend Snippet: A Tumour growth curves of CB.17 SCID mice bearing the GCB-DLBCL cell line SuDHL4 xenograft tumours treated with capivasertib (130 mg/kg BID 10/14 4-day on/3-day off) monotherapy, venetoclax (100 mg/kg QD) monotherapy and in combination in accordance with schedules captured in Supplementary Table . B Assessment of sustained tumour regression (>60 days post dosing). Both capivasertib and venetoclax were administered on a 4-day on/3-day off schedule relative to combinations using reduced frequency of venetoclax treatment. C Assessment of tumour activity of capivasertib dose reduction versus venetoclax dose reduction on anti-tumour response or tumour regression. Data is represented as geometric mean of tumour volumes and standard error of the mean. * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: Venetoclax was purchased from Medkoo Biosciences (Durham, NC).

Techniques: Activity Assay

Journal: Leukemia

Article Title: Potent combination benefit of the AKT inhibitor capivasertib and the BCL-2 inhibitor venetoclax in diffuse large B cell lymphoma

doi: 10.1038/s41375-024-02401-9

Figure Lengend Snippet: A Western blot analysis of PTEN expression in the VFN-D7 and VFN-D1_KTC human DLBCL PDX models. B Tumour growth curves of NSG mice bearing tumours with the double-hit GCB DLBCL PDX model, VFN-D7 or ( C ) the non-GCB DLCBL PTEN deficient PDX model, VFN-D1_KTC treated with capivasertib on a slightly modified schedule (130 mg/kg BID 10/14 3-day on/4-day off) monotherapy, venetoclax (100 mg/kg QD) and in combination with both agents on 3-day on/4-day off schedule. Comparison of combination treatment tumour regression compared to either monotherapy groups. Data is represented as arithmetic mean and standard error of the mean. * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: Venetoclax was purchased from Medkoo Biosciences (Durham, NC).

Techniques: Western Blot, Expressing, Modification, Comparison

Journal: Leukemia

Article Title: Potent combination benefit of the AKT inhibitor capivasertib and the BCL-2 inhibitor venetoclax in diffuse large B cell lymphoma

doi: 10.1038/s41375-024-02401-9

Figure Lengend Snippet: Assessment of tumour regressions following addition of once weekly rituximab to capivasertib and venetoclax relative to the capivasertib and venetoclax combination on 4-day on/3-day off schedule. A , B Tumour growth curves of CB.17 SCID mice bearing with the GCB-DLBCL cell line ( A ) SUDHL4 and ( B ) SUDHL5 xenograft models treated with capivasertib (130 mg/kg BID 10/14 4-day on/3-day off) monotherapy, venetoclax (100 mg/kg QD) monotherapy or rituximab (10 mg/kg 2QW) monotherapy and in combination in accordance with schedules shown in Supplementary Table . C Comparison of initial tumour response and tumour regression following the triple combination of capivasertib, venetoclax and rituximab in the WSU-DLCL2 xenograft model relative to the doublet combinations and RCHOP (10 mg/kg rituximab 2QW, 25 mg/kg cyclophosphamide once IP, 3 mg/kg doxorubicin hydrochloride once IV, 0.25 mg/kg vincristine sulphate once IV and 0.5 mg/kg prednisone QD5 PO) treated as indicated (see Supp. Table ). D Assessment of the sustained tumour regressions in the capivasertib venetoclax versus capivasertib venetoclax rituximab groups. E Tumours from the RCHOP treated group shown in ( C ) that were resistant to treatment (purple dots) were retreated with the capivasertib, venetoclax and rituximab triplet (yellow dots) and tumour regression assessed. All data is represented as geometric mean of tumour volumes and standard error of the mean (* p < 0.05, ** p < 0.001, *** p < 0.0001).

Article Snippet: Venetoclax was purchased from Medkoo Biosciences (Durham, NC).

Techniques: Comparison