Journal: iScience

Article Title: CPNE5 overexpression inhibits cardiomyocytes apoptosis by promoting the degradation of FAS receptor

doi: 10.1016/j.isci.2025.113302

Figure Lengend Snippet: Overexpression of CPNE5 inhibits the activation of FAS receptor mediated apoptotic pathway in vitro and in vivo (A) Representative image showing immunofluorescence of TUNEL (red, arrows), DAPI (blue, for nuclei), and cardiac troponin (green; bar = 50 μm) in overexpression (Flag- Cpne5 ) or knockdown (si- Cpne5 ) CPNE5 NMCMs under FASL protein treatment. Flag-vector: empty vectors, NC: negative control. (B) Statistical analysis of TUNEL-positive cells in (A). (C and D) After treated with FASL protein, NMCMs overexpression CPNE5 (C) or knockdown CPNE5 group (D) were detected CPNE5, FAS, FADD, cleaved-Caspase8, and cleaved-Caspase3 protein levels by western blot. Bar graph on the right quantification protein level of (C) and (D), normalized to β-actin ( n = 5). (E and G) After TAC (E) or I/R (G) model, AAV9-ctrl and AAV9-OE- Cpne5 mice were detected for CPNE5, FAS, FADD, cleaved-Caspase8, and cleaved-Caspase3 protein levels by Western blot. Bar graph on the right quantification protein level of (E) and (G), normalized to β-actin ( n = 5). (F and H) After TAC (F) or I/R (H) model, WT and KO mice were detected for CPNE5, FAS, FADD, cleaved-Caspase8, and cleaved-Caspase3 protein level by Western blot. Bar graph on the right quantification protein level of (F) and (H), normalized to β-actin ( n = 5). All data are presented as mean ± SD; unpaired two-tailed Student’s t test (two groups) was used to determine statistical significance of experimental data. ∗∗ p < 0.01.

Article Snippet: Following this transfection step, the cells were added with 5μM CHX (MCE, cat. no. HY-12320) for protein stability study, 1 μM Chloroquine (MCE, cat. no. HY-17589A) for lysosome inhibition study, 4 μM MG132 (Selleck Chemicals, cat. no. S2619) for proteasome inhibition study, 500ng/ml FASL protein (MCE, cat. no. HY-P74157) to activate the FAS receptor, 5 μM puromycin 2HCl (Selleck Chemicals, cat. no. S7417) for protein synthesis study.

Techniques: Over Expression, Activation Assay, In Vitro, In Vivo, Immunofluorescence, TUNEL Assay, Knockdown, Plasmid Preparation, Negative Control, Western Blot, Two Tailed Test

Journal: iScience

Article Title: CPNE5 overexpression inhibits cardiomyocytes apoptosis by promoting the degradation of FAS receptor

doi: 10.1016/j.isci.2025.113302

Figure Lengend Snippet: Overexpression of CPNE5 in cardiomyocytes reduces FAS mediated apoptosis of cardiomyocytes (A) Representative image showing immunofluorescence of TUNEL (red, arrows), DAPI (blue, for nuclei), and cardiac troponin (green; bar = 80 μm) in NMCMs, which infected with Flag- Cpne5 and HA- FAS plasm id under FASL protein treatment. Flag-vector: empty vectors. (B) Statistical analysis of TUNEL-positive cells in (A). (C) Representative Western blot in cardiomyocytes infected with Flag- Cpne5 and HA- FAS plasmid under FASL protein treatment. (D–G) Quantification of FAS, FADD, cleaved-Caspase8, and cleaved-Caspase3 protein level of (C), normalized to β-actin ( n = 5). (H) Apoptotic cells were detected using the TUNEL assay after CPNE5 and FAS knockdown under FASL protein stimulation. NC: negative control. bar = 80 μm. (I) Statistical analysis of TUNEL-positive cells in (H). (J) After treated with FASL protein, NMCMs, which treated with si- Cpne5 and si- Fas were detected CPNE5, FAS, FADD, cleaved-Caspase8, and cleaved-Caspase3 protein level by Western blot. (K–N) Quantification FAS, FADD, cleaved-Caspase8, and cleaved-Caspase3 protein levels of (J), normalized to β-actin ( n = 5). All data are presented as mean ± SD; unpaired two-tailed Student’s t test (two groups) and one-way ANOVA with Dunnett’s post hoc test (more than two groups) were used to determine statistical significance of experimental data. ∗∗ p < 0.01.

Article Snippet: Following this transfection step, the cells were added with 5μM CHX (MCE, cat. no. HY-12320) for protein stability study, 1 μM Chloroquine (MCE, cat. no. HY-17589A) for lysosome inhibition study, 4 μM MG132 (Selleck Chemicals, cat. no. S2619) for proteasome inhibition study, 500ng/ml FASL protein (MCE, cat. no. HY-P74157) to activate the FAS receptor, 5 μM puromycin 2HCl (Selleck Chemicals, cat. no. S7417) for protein synthesis study.

Techniques: Over Expression, Immunofluorescence, TUNEL Assay, Infection, Plasmid Preparation, Western Blot, Knockdown, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A CD95L can exist either in a membrane-bound or in a soluble form, the latter resulting from protease cleavage. Key domains include the CD95 binding domain (CD95 Bind), the TNF homology domain (THD) for its homotrimerization, and the proline-rich domain (PRD). B Workflow of the CD95L-IZ construct design, secretory expression, affinity purification, and modification. The recombinant protein consists of a signaling peptide (Sig), an affinity tag (Tag), a cleavage site (Clv), a Cysteine (Cys), an isoleucine zipper domain (IZ) fused via a linker to the extracellular CD95L domain. In method 1 transient CD95-IZ expression was used. Here, a His-tag was used as Tag and a TEV protease sequence as Clv. Affinity purification was done by anti-His-tag antibody coupled agarose beads. In method 2 a cell line stably expressing CD95-IZ was used. Here, a Twin-Strep-Tag was used as Tag and a PreScission cleavage site as Clv. Affinity purification was done by Strep-TactinXT4Flow beads. C Upper panel: examination of cell morphology before and up to three days after transfection. Lower panel: photos of cell culture and transfection using a 10-layer cell factory in a cell culture hood (left) or incubator (right). A T175 cell culture flask was seeded with the same cell density as in a 10-layer cell factory to check the cell condition and confluency. D CD95L-IZ protein expression and secretion was verified by a Western blot of the cell culture medium using anti-CD95L antibody. The image contrast was adjusted for better visibility.

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques: Membrane, Binding Assay, Construct, Expressing, Affinity Purification, Modification, Recombinant, Sequencing, Stable Transfection, Strep-tag, Transfection, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A ELISA analysis of CD95L-IZ-biotin and CD95L-Enzo binding to CD95. B Comparison of representative bright field images showing morphological changes of cells treated with CD95L-IZ-X or soluble CD95L (Enzo Life Science) after 2, 6, and 15 h. Red asterisks indicate the position of apoptotic cell. Scale bars are 50 µm. C Apoptosis dynamics analysis of CD95L-IZ apoptosis induction at different stages: post-purification, post-His-tag cleavage, post-biotinylation, and comparison with CD95L-Enzo. D The efficiency of CD95L-IZ-His apoptosis induction is increased by anti-His-tag antibody crosslinking. E The presence of CD95-His (in 10x, 50x, 100x, and 200x molar excess in comparison to CD95L-IZ-biotin) as competitive binders to the natural CD95 on the cell membrane, successively inhibits CD95L-IZ-biotin apoptosis induction. In panels C, D, and E data points are marked with dots. Hill equation fit curves are represented by solid lines, and standard deviations are indicated with shaded regions.

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Comparison, Purification, Membrane

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A CD95L can exist either in a membrane-bound or in a soluble form, the latter resulting from protease cleavage. Key domains include the CD95 binding domain (CD95 Bind), the TNF homology domain (THD) for its homotrimerization, and the proline-rich domain (PRD). B Workflow of the CD95L-IZ construct design, secretory expression, affinity purification, and modification. The recombinant protein consists of a signaling peptide (Sig), an affinity tag (Tag), a cleavage site (Clv), a Cysteine (Cys), an isoleucine zipper domain (IZ) fused via a linker to the extracellular CD95L domain. In method 1 transient CD95-IZ expression was used. Here, a His-tag was used as Tag and a TEV protease sequence as Clv. Affinity purification was done by anti-His-tag antibody coupled agarose beads. In method 2 a cell line stably expressing CD95-IZ was used. Here, a Twin-Strep-Tag was used as Tag and a PreScission cleavage site as Clv. Affinity purification was done by Strep-TactinXT4Flow beads. C Upper panel: examination of cell morphology before and up to three days after transfection. Lower panel: photos of cell culture and transfection using a 10-layer cell factory in a cell culture hood (left) or incubator (right). A T175 cell culture flask was seeded with the same cell density as in a 10-layer cell factory to check the cell condition and confluency. D CD95L-IZ protein expression and secretion was verified by a Western blot of the cell culture medium using anti-CD95L antibody. The image contrast was adjusted for better visibility.

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques: Membrane, Binding Assay, Construct, Expressing, Affinity Purification, Modification, Recombinant, Sequencing, Stable Transfection, Strep-tag, Transfection, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A CD95L-IZ purification and functionalization. Lane 1 Affinity purification of CD95L-IZ-His using anti-His-tag antibody coupled agarose beads followed by 10 % SDS-PAGE analysis of the purified protein. Lane 2-3 Western blot analysis using anti-His-tag antibody: 100 ng CD95L-IZ-His before (lane 2) and 100 ng CD95L-IZ after His-tag cleavage (lane 3). Lane 4-5 Western blot analysis of site-specific biotinylation using the cysteine maleimide reaction: 300 ng CD95L-IZ before (lane 4) and after (lane 5) biotinylation were loaded, probed with AF488-streptavidin. B Size exclusion chromatography (SEC) analysis of CD95L-IZ-His after His-tag affinity purification compared with a protein standard mix run under identical conditions. The SEC data exhibits peaks corresponding to the trimeric, the dimeric, and the monomeric form of CD95L-IZ-His. C From the SEC data, a standard curve of the protein standard mix is calculated and peak positions of the CD95L-IZ-His data are plotted to derive the corresponding molecular weight. D Droplet assay to verify biotinylation. Top: a sketch of the assay. Bottom: Fluorescence colocalization images of the 488 nm and the 594 nm channel. CD95L-IZ-biotin was non-specifically labeled with ATTO594 NHS-ester and incubated with AF488-streptavidin on a BSA passivated glass coverslip. E DNA origami functionalized with CD95L-IZ-ssDNA. The DNA handle on the CD95L-IZ hybridizes to a complementary anti-handle on the DNA origami, attaching the ligand site-specifically to the DNA origami. The TEM micrograph on the lower right is duplicated and origami outlines and proteins are indicated for better interpretability.

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques: Purification, Affinity Purification, SDS Page, Western Blot, Size-exclusion Chromatography, Molecular Weight, Fluorescence, Labeling, Incubation

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet:

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques:

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A ELISA analysis of CD95L-IZ-biotin and CD95L-Enzo binding to CD95. B Comparison of representative bright field images showing morphological changes of cells treated with CD95L-IZ-X or soluble CD95L (Enzo Life Science) after 2, 6, and 15 h. Red asterisks indicate the position of apoptotic cell. Scale bars are 50 µm. C Apoptosis dynamics analysis of CD95L-IZ apoptosis induction at different stages: post-purification, post-His-tag cleavage, post-biotinylation, and comparison with CD95L-Enzo. D The efficiency of CD95L-IZ-His apoptosis induction is increased by anti-His-tag antibody crosslinking. E The presence of CD95-His (in 10x, 50x, 100x, and 200x molar excess in comparison to CD95L-IZ-biotin) as competitive binders to the natural CD95 on the cell membrane, successively inhibits CD95L-IZ-biotin apoptosis induction. In panels C, D, and E data points are marked with dots. Hill equation fit curves are represented by solid lines, and standard deviations are indicated with shaded regions.

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Comparison, Purification, Membrane