Journal: Journal for immunotherapy of cancer

Article Title: PARP inhibitors enhance antitumor immune responses by triggering pyroptosis via TNF-caspase 8-GSDMD/E axis in ovarian cancer.

doi: 10.1136/jitc-2024-009032

Figure Lengend Snippet: Figure 6 Caspase 8 contributes to the pyroptosis induced by PARP inhibition. Ratios of PI+ annexin V+ OVCAR8 (A), A2780 (B), and primary homologous recombination deficiency (HRD) ovarian cancer (OC) tumor cells (C) on niraparib exposure in the presence or absence of caspase inhibitors, including VX765, Z-DEVD-FMK, Z-IETD-FMK, and Z-VAD-FMK. (D) Determination of activated caspase 3 and caspase 8 levels in OVCAR8 and A2780 cells after olaparib and niraparib treatment by flow cytometry. Histological analyses of activated caspase 3 and caspase 8 in OC patient-derived xenograft (PDX) tumors (E) and triple-negative breast cancer (TNBC) PDX tumors (F) with or without niraparib exposure (n=5/6). (G) Western blotting analyses of the cleavage of caspase 8/3 and GSDMD/E in OVCAR8 and A2780 cells on niraparib treatment in the presence or absence of caspase inhibitors, including Z-DEVD-FMK, Z-IETD-FMK, and Z-VAD-FMK. Representative images of OVCAR8 (H) and A2780 cells (I) on niraparib treatment in the presence or absence of caspase inhibitors, including VX765, Z-DEVD-FMK, and Z-IETD-FMK. Mean values±SEM. *P<0.05, **p<0.01, and ***p< 0.001 by Student’s t-test in (A–C) and (E,F).

Article Snippet: VX765 (Inh vx765i) was supplied by Invivogen.

Techniques: Inhibition, Homologous Recombination, Flow Cytometry, Derivative Assay, Western Blot

Journal: Journal of Cell Science

Article Title: Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs

doi: 10.1242/jcs.259002

Figure Lengend Snippet: Trikafta correctors restore F508del-CFTR clustering at the PM. HBE cells transduced with adenoviruses containing wt- or F508del-CFTR. (A,B) PM distribution of wt-CFTR shows clusters (white arrow, N exp =10; n cell =380) and platforms after Thaps (blue arrow, N exp =8; n cell =156). CFTR is also enriched near cell–cell junctions (rim, yellow arrow). (C) F508del-CFTR lacks cluster and does not accumulate near junctions ( N exp =14; n cell =315). (D) There is no recruitment of F508del-CFTR into platforms after Thaps treatment ( N exp =5; n cell =147). (E,F) Lumacaftor (VX-809, 24 h) does not restore F508del-CFTR clustering or entry into platforms ( N exp =6; n cell =213). (G,H) VX-445 plus VX-661 (24 h) restores F508del-CFTR membrane expression, clustering (white arrow) and rim formation (yellow arrow, N exp =19; n cell =842, see inset). Thaps treatment triggers formation of F508del-CFTR-enriched platforms (blue arrow, N exp =6; n cell =264, see inset), further evidence that corrected F508del-CFTR partitions inside lipid rafts. Magnified views of the indicated area are shown in G′,H′.

Article Snippet: CF HBE cells were infected with adenoviruses (wt-CFTR, F508del-CFTR or S13F-CFTR, 50 MOI), cultured to ∼90% confluency, and exposed to the Trikafta correctors VX-445 (3 μM, MedChemExpress) and VX-661 (3 μM, MedChemExpress), or maintained at low temperature (29°C) for 24 h (in OptiMEM).

Techniques: Transduction, Membrane, Expressing

Journal: Journal of Cell Science

Article Title: Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs

doi: 10.1242/jcs.259002

Figure Lengend Snippet: Trikafta correctors restore F508del-CFTR clustering and confinement inside microdomains. HBE cells transduced with wt- or F508del-CFTR adenoviruses. (A) Degree of aggregation (DA ratio) showing that wt-CFTR clusters are five times larger than F508del-CFTR. Thaps increases the wt-CFTR DA ratio due to incorporation in platforms whereas F508del-CFTR DA is unchanged. VX-445 plus VX-661 correction restores normal F508del-CFTR DA ratio and clustering. Thaps treatment increased the DA ratio, indicating entry of the mutant into platforms. (mean±s.e.m.; N exp =2; n cell , n wt =40, n wt+Thaps =37, n ΔF =108, n ΔF+Thaps =72, n ΔF(VX) =98, n ΔF(VX)+Thaps =95). **** P <0.0005; ns, not significant. (B) D micro from k-space image correction spectroscopy analysis indicates confined mobility of CFTR inside microdomains and is higher for F508del-CFTR than wt-CFTR, indicating weaker confinement. Thaps reduces mobility and increases confinement due to the presence of wt-CFTR inside ceramide-rich platforms but does not affect F508del-CFTR, which is excluded from ceramide-rich platforms. VX-445 plus VX-661 correction partially restores F508del-CFTR mobility and confinement under Ctr and Thaps conditions (mean±s.e.m.; N exp =2; n cell , n wt =40, n wt+Thaps =37, n ΔF =91, n ΔF+Thaps =51, n ΔF(VX) =85, n ΔF(VX)+Thaps =80). **** P <0.0005; ** P <0.025. (C,D). Differences in F508del-CFTR degree of aggregation (DA ratio) and confined mobility ( D micro ) in the PM and the ER indicate the presence of some weakly confined F508del-CFTR channels at the PM (mean±s.e.m.; N exp =2; n cell , n ΔF(PM) =39, n ΔF(ER) =30). **** P <0.0005. Unpaired one-tailed t -tests were used throughout the analysis, and each cell is an independent biological sample.

Article Snippet: CF HBE cells were infected with adenoviruses (wt-CFTR, F508del-CFTR or S13F-CFTR, 50 MOI), cultured to ∼90% confluency, and exposed to the Trikafta correctors VX-445 (3 μM, MedChemExpress) and VX-661 (3 μM, MedChemExpress), or maintained at low temperature (29°C) for 24 h (in OptiMEM).

Techniques: Transduction, Mutagenesis, Spectroscopy, One-tailed Test

Journal: Journal of Cell Science

Article Title: Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs

doi: 10.1242/jcs.259002

Figure Lengend Snippet: Trikafta correctors restore S13F-CFTR folding. HBE cells transduced with adenovirus containing S13F-CFTR. (A) Like F508del-CFTR, S13F-CFTR does not form clusters or a rim near junctions ( N exp =11; n cell =341). (B) Mid-section through cell reveals that most S13F-CFTR is intracellular (green arrow). (C,D) VX-445 plus VX-661 correction reduces S13F-CFTR intracellular retention and restores membrane expression, clustering (white arrow) and rim formation (yellow arrow, N exp =16; n cell =582). The green arrow in D highlights the decrease in intracellular S13F-CFTR level after correction therapy. (E,F) S13F-CFTR fails to form platforms after Thaps treatment ( N exp =3; n cell =90) (E), and Trikafta correctors ameliorate this defect ( N exp =7; n cell =306) (F). The blue arrow in F highlights the restoration of S13F-CFTR incorporation in ceramide-rich platforms after correction therapy. (G) As shown by co-immunoprecipitation, VX-445 plus VX-661 (and low temperature, 29°C for 24 h) restores interaction of S13F-CFTR and F508del-CFTR with FLNA suggesting both mutants are misfolded and rescued by these correctors ( N exp =3). 20 μg of protein were used for the lysate blot and 750 μg for the IP blot (2.7% of the IP).

Article Snippet: CF HBE cells were infected with adenoviruses (wt-CFTR, F508del-CFTR or S13F-CFTR, 50 MOI), cultured to ∼90% confluency, and exposed to the Trikafta correctors VX-445 (3 μM, MedChemExpress) and VX-661 (3 μM, MedChemExpress), or maintained at low temperature (29°C) for 24 h (in OptiMEM).

Techniques: Transduction, Membrane, Expressing, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs

doi: 10.1242/jcs.259002

Figure Lengend Snippet: Reciprocal effects of CFTR on membrane lipids. Cells were exposed to CTXB conjugated to the fluorophore Alexa Fluor 594 (CTXB–594), which binds to the ganglioside GM1 and labels GM1-positive lipid rafts, at 0.5 μg/ml for 30 min before imaging. (A,B) CTXB–594 distribution at the PM of non-CF (A) and CF (B) cells showing GM1 clustering. (C–E) ICS analysis comparing distribution of GM1 clusters on HBE cells that overexpress wt- or F508del-CFTR (ΔF). (C) Total fluorescence due to CTXB–594 binding is similar on cells expressing wt- and F508del-CFTR. (D,E) GM1 aggregation (DA ratio) is 3-fold higher and cluster density (CD ratio) (# cluster per μm 2 ) is 3-fold lower for wt-CFTR than F508del-CFTR, suggesting it promotes lipid raft formation. I wt (×10 3 )=2.73±0.04 arbitrary units (AU), I ΔF (×10 3 )=2.49±0.04 AU, DA wt ratio=1.00±0.05, DA ΔF ratio=0.35±0.02, CD wt ratio=1.00±0.06, CD ΔF ratio=2.9±0.2 (mean±s.e.m.; N exp =3, n cell , n wt =52, n ΔF =89). **** P <0.0005. (F–H) Similar results were obtained using untransduced cells expressing endogenous wt- (nonCF) or F508del-CFTR (CF). Overall CTXB fluorescence was similar (F); however, CTXB clusters were 5-fold larger (H) and their number per unit area was ∼5-fold lower on non-CF cells (G). I nonCF (×10 3 )=0.77±0.02 AU, I CF (×10 3 )=0.81±0.01 AU, DA nonCF ratio=1.00±0.07, DA CF ratio=0.19±0.01, CD nonCF ratio=1.00±0.05, CD CF ratio=5.0±0.2 (mean±s.e.m.; N exp =3; n cell , n nonCF =100, n CF =62). **** P <0.0005; ns, not significant. (I,J) Trikafta correctors (VX) increase aggregation state of GM1-positive rafts (CTXB clusters) by ∼2.5-fold when HBE cells overexpress F508del-CFTR or S13F-CFTR [mean±s.e.m. N exp =2, n cell : n S13F =30, n S13F(VX) =30, n ΔF =30, n ΔF(VX) =25]. **** P <0.0005; ns, not significant. Each point on the histogram represents a cell, and each cell is an independent biological sample. Unpaired one-tailed t -tests were used throughout the analysis.

Article Snippet: CF HBE cells were infected with adenoviruses (wt-CFTR, F508del-CFTR or S13F-CFTR, 50 MOI), cultured to ∼90% confluency, and exposed to the Trikafta correctors VX-445 (3 μM, MedChemExpress) and VX-661 (3 μM, MedChemExpress), or maintained at low temperature (29°C) for 24 h (in OptiMEM).

Techniques: Membrane, Imaging, Fluorescence, Binding Assay, Expressing, One-tailed Test

Journal: Frontiers in Pharmacology

Article Title: Investigating the Implications of CFTR Exon Skipping Using a Cftr Exon 9 Deleted Mouse Model

doi: 10.3389/fphar.2022.868863

Figure Lengend Snippet: Cftr Δ9/Δ9 epithelial cell CFTR function. (A,B) . Ussing chamber tracing showing change in short circuit current ( Isc ) in response to drug stimulation on Cftr Δ9/Δ9 ALI cultures pre-treated for 24 h with DMSO. (C,D) . Ussing chamber tracing showing change in Isc in response to drug stimulation on homozygote Cftr exon 9 deleted ALI cultures pre-treated for 24 h with Lumacaftor. (E) . Summary of changes in Isc in response to amiloride, forskolin and CFTR inhibitor-172 for Cftr Δ9/Δ9 ALI cultures pre-treated with DMSO (Black bars) or Lumacaftor (Grey bars) ( Isc , n = 2). Pre-treatment with DMSO (black bar) pre-treatment with Lumacaftor (blue bar), Amiloride addition (green bar), Forskolin stimulation (yellow bar), Ivacaftor stimulation (orange bar), CFTR inhibitor-172 (CI-red bar).

Article Snippet: Inserts were pre-treated with either Lumacaftor (3 μM VX-809, Selleckchem) or DMSO (0.001%, Sigma-Aldrich) 24 h prior to Ussing Chamber analysis.

Techniques:

Journal: PLoS ONE

Article Title: Conditional Inducible Triple-Transgenic Mouse Model for Rapid Real-Time Detection of HCV NS3/4A Protease Activity

doi: 10.1371/journal.pone.0150894

Figure Lengend Snippet: (A) Two groups of triple-transgenic mice (n = 5 per group) were treated with telaprevir (200 mg/kg) or DMSO via oral gavage twice daily for 10 days. During the treatment period, the mice were continuously induced with Dox (1 mg/mL Dox and 50 g/L sugar were dissolved in their drinking water). (B) Triple-transgenic mice were induced with Dox for 10 days (n = 5 per group). At the third day after Dox induction, the mice were treated with boceprevir (100 mg/kg) or DMSO via oral gavage twice daily for 7 days. Blood samples were collected daily from the caudal vein to measure plasma Gluc activity. Each data point represents the mean ± SD. *** = p<0.001 compared with the vehicle control.

Article Snippet: Telaprevir and boceprevir were purchased from MedChem Express (Pumai Biotechnology Co., LTD, Shanghai, China).

Techniques: Transgenic Assay, Activity Assay, Control