human caspase 8  (Addgene inc)

Journal: bioRxiv

Article Title: Challenging a role for ceramide channels and microdomains in apoptosis induction using a bottom-up approach

doi: 10.1101/2025.11.14.688508

Figure Lengend Snippet: (a) Schematic outline of calcein release assay for measuring pore-forming activity of cBid-activated recombinant Bax in large unilamellar vesicles. (b) Real-time chromatograms of calcein released from PC:CL (80:20) vesicles co-incubated with the indicated amount of unlabeled Bax or DY-647P1-labeled Bax and 50 nM cBid. Leakiness of vesicles was tested by omitting Bax and cBid (no addition). (c) Real-time chromatograms of calcein released from PC (100) and PC:CL (80:20) vesicles incubated with different combinations of 100 nM Bax and 50 nM cBid. Calcein release was measured over 120 min at 2 min intervals using a fluorescence plate reader. For each time point, the mean values of three independent measurements were determined and normalized to the value measured after Triton X100 (TX) addition, which served as a reference for 100% permeabilization.

Article Snippet: Dextran Alexa Fluor 647 10 kDa (cat. no. D22914) and Dextran fluorescein 70 kDa (cat. no. D1823) were from Invitrogen. cBid (cat. no. 882-B8) was from R&D Systems. α-Bax rabbit monoclonal antibody (cat. no. 5023s) was from Cell signaling, α-rabbit HRP (cat. no. 170-6515) was from BioRad.

Techniques: Release Assay, Activity Assay, Recombinant, Incubation, Labeling, Fluorescence

Journal: bioRxiv

Article Title: Challenging a role for ceramide channels and microdomains in apoptosis induction using a bottom-up approach

doi: 10.1101/2025.11.14.688508

Figure Lengend Snippet: (a) Schematic outline of calcein permeabilization assay for measuring pore-forming activity of recombinant Bax in giant unilamellar vesicles (GUVs). (b) GUVs prepared from PC (100) or PC:CL (80:20) were stained with DiI ( red ) and incubated with calcein ( green ) in the absence or presence of 400 nM Bax 647 ( magenta ) and 50 nM cBid. GUVs were imaged at the indicated time points using a LSM Airyscan microscope. Scale bar 10 µm. (c) Quantification of the permeability of GUVs for Bax 647 ( magenta ) and calcein ( green ) at the indicated incubation times. GUVs with a degree of filling of 40 % or more were considered permeable. For GUVs prepared from PC:CL (80:20), a total number of 70-100 GUVs were analyzed in three independent experiments. For GUVs prepared from PC (100), a total of 50 GUVs were analyzed in one experiment. Data are means ± SD. (d) GUVs prepared from PC (100; DiO; green ) or PC:CL (80:20; DiI; red ) were incubated in the presence of 400 nM Bax 647 ( magenta ) and 50 nM cBid. GUVs were imaged as in (b). Line scans along the path of the arrows show the degree of leakage of Bax 647 inside PC and PC:CL-containing GUVs. Scale bar 10 µm.

Article Snippet: Dextran Alexa Fluor 647 10 kDa (cat. no. D22914) and Dextran fluorescein 70 kDa (cat. no. D1823) were from Invitrogen. cBid (cat. no. 882-B8) was from R&D Systems. α-Bax rabbit monoclonal antibody (cat. no. 5023s) was from Cell signaling, α-rabbit HRP (cat. no. 170-6515) was from BioRad.

Techniques: Activity Assay, Recombinant, Staining, Incubation, Microscopy, Permeability

Journal: bioRxiv

Article Title: Challenging a role for ceramide channels and microdomains in apoptosis induction using a bottom-up approach

doi: 10.1101/2025.11.14.688508

Figure Lengend Snippet: (a) GUVs prepared with egg PC (100) and stained with DiI ( red ) were incubated with Alexa-fluor647 10kDa dextran (Dex10; magenta ) and fluorescein 70 kDa dextran (Dex70; green ) and then imaged using a LSM Airy scan microscope. At the indicated incubation time, the permeability of the GUVs for Dex10 and Dex70 was quantified. GUVs with a filling degree of 40% or more were considered permeable. In addition, the filling degree of the GUVs was determined at 35 min of incubation. (b) GUVs prepared with PC:CL (90:10) were processed as in (a). (c) GUVs prepared with PC:CL (90:10) were incubated in the presence of 400 nM Bax and 50 nM cBid and processed as in (a). (d) GUVs prepared with PC:Cer 16 (90:10) were processed as in (a). (e) GUVs prepared with PC:Cer brain (90:10) were processed as in (a). A total of 200-1050 GUVs were analyzed per condition in at least three independent experiments. PC (100), n=5; PC:Cer 16 (90:10), n=3; PC:Cer brain (90:10), n=4; PC:CL (90:10), n=5; PC:CL (90:10) + Bax, n=4. Data are means ± SD. Scale bar, 10 µm.

Article Snippet: Dextran Alexa Fluor 647 10 kDa (cat. no. D22914) and Dextran fluorescein 70 kDa (cat. no. D1823) were from Invitrogen. cBid (cat. no. 882-B8) was from R&D Systems. α-Bax rabbit monoclonal antibody (cat. no. 5023s) was from Cell signaling, α-rabbit HRP (cat. no. 170-6515) was from BioRad.

Techniques: Staining, Incubation, Microscopy, Permeability

Journal: bioRxiv

Article Title: Challenging a role for ceramide channels and microdomains in apoptosis induction using a bottom-up approach

doi: 10.1101/2025.11.14.688508

Figure Lengend Snippet: (a) Silica beads coated with membranes prepared from PC (100; green ) were mixed with uncoated beads and then incubated with 200 nM DY-647P1-labeled Bax (Bax 647 ; magenta ) and 50 nM cBid. At the indicated incubation times, beads were imaged by confocal fluorescence and differential interference contrast (DIC) microscopy. (b) Silica beads coated with membranes prepared from PC:CL (90:10; green ) were mixed with uncoated beads and then incubated with 200 nM Bax 647 (magenta) and 50 nM cBid. At the indicated incubation times, beads were imaged as in (a). Scale bar, 10 µm. (c) Bax 647 bound to uncoated beads or beads coated with membranes prepared from PC (100) or PC:CL (90:10) was quantified at the indicated incubation times and expressed as relative fluorescence intensity. Data shown are based on a total of 600 to 900 individual measurements per condition in 3 independent experiments.

Article Snippet: Dextran Alexa Fluor 647 10 kDa (cat. no. D22914) and Dextran fluorescein 70 kDa (cat. no. D1823) were from Invitrogen. cBid (cat. no. 882-B8) was from R&D Systems. α-Bax rabbit monoclonal antibody (cat. no. 5023s) was from Cell signaling, α-rabbit HRP (cat. no. 170-6515) was from BioRad.

Techniques: Incubation, Labeling, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Challenging a role for ceramide channels and microdomains in apoptosis induction using a bottom-up approach

doi: 10.1101/2025.11.14.688508

Figure Lengend Snippet: (a) Silica beads coated with membranes prepared from PC (100; red ) or PC:CL (90:10; green ) were mixed and then incubated with 200 nM Bax 647 ( magenta ) and 50 nM cBid. At the indicated incubation times, beads were imaged by confocal fluorescence and DIC microscopy. (b) Silica beads coated with membranes prepared from PC:CL (90:10; red ) or PC:Cer 16 (90:10; green ) were mixed, incubated with Bax 647 ( magenta ) and cBid, and then visualized as in (a). (c) Silica beads coated with membranes prepared from PC (100; red ) or PC:Cer 16 (90:10; green ) were mixed, incubated with Bax 647 ( magenta ) and cBid, and then visualized as in (a). Scale bar, 10 µm. (d) Bax 647 bound to beads coated with membranes prepared from PC (100), PC:CL (90:10) or PC:Cer 16 (90:10) was quantified at the indicated incubation times and expressed as relative fluorescence intensity. Data shown are based on a total of 350 to 900 individual measurements per condition in at least 3 independent experiments.

Article Snippet: Dextran Alexa Fluor 647 10 kDa (cat. no. D22914) and Dextran fluorescein 70 kDa (cat. no. D1823) were from Invitrogen. cBid (cat. no. 882-B8) was from R&D Systems. α-Bax rabbit monoclonal antibody (cat. no. 5023s) was from Cell signaling, α-rabbit HRP (cat. no. 170-6515) was from BioRad.

Techniques: Incubation, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Challenging a role for ceramide channels and microdomains in apoptosis induction using a bottom-up approach

doi: 10.1101/2025.11.14.688508

Figure Lengend Snippet: (a) GUVs prepared from PC (100) were stained with DiI ( red ) and incubated with 10 kDa dextran (Dex10; magenta ) and 70 kDa dextran (Dex70; green ) in the presence of 400 nM Bax and 50 nM cBid and then imaged using a LSM Airy scan microscope. At the indicated incubation time, the permeability of the GUVs for Dex10 and Dex70 was quantified. GUVs with a filling degree of 40% or more were considered permeable. In addition, the filling degree of the GUVs was determined at 35 min of incubation. (b) GUVs prepared with PC:CL (90:10) were processed as in (a). (c) GUVs prepared with PC:Cer 16 (90:10) were processed as in (a). A total of 310-850 GUVs were analyzed per condition over at least three independent experiments. PC (100), n=4; PC:CL (90:10), n=4; PC:Cer 16 (90:10). Data are means ± SD. Scale bar, 10 µm.

Article Snippet: Dextran Alexa Fluor 647 10 kDa (cat. no. D22914) and Dextran fluorescein 70 kDa (cat. no. D1823) were from Invitrogen. cBid (cat. no. 882-B8) was from R&D Systems. α-Bax rabbit monoclonal antibody (cat. no. 5023s) was from Cell signaling, α-rabbit HRP (cat. no. 170-6515) was from BioRad.

Techniques: Staining, Incubation, Microscopy, Permeability

Journal: bioRxiv

Article Title: Challenging a role for ceramide channels and microdomains in apoptosis induction using a bottom-up approach

doi: 10.1101/2025.11.14.688508

Figure Lengend Snippet: Real-time chromatograms of calcein released from PC:CL (80:20) vesicles incubated in the absence or presence of 100 nM Bax or 50 nM cBid. Leakiness of vesicles was tested by omitting Bax and cBid (no addition). Calcein release was measured over 120 min at 2 min intervals using a fluorescence plate reader. For each time point, the mean values of three independent measurements were determined and normalized to the value measured after Triton X100 (TX) addition, which served as a reference for 100% permeabilization.

Article Snippet: Dextran Alexa Fluor 647 10 kDa (cat. no. D22914) and Dextran fluorescein 70 kDa (cat. no. D1823) were from Invitrogen. cBid (cat. no. 882-B8) was from R&D Systems. α-Bax rabbit monoclonal antibody (cat. no. 5023s) was from Cell signaling, α-rabbit HRP (cat. no. 170-6515) was from BioRad.

Techniques: Incubation, Fluorescence