jd633 vector  (Addgene inc)

Journal: bioRxiv

Article Title: Early-peaking caspase-7 activity at the plasma membrane drives apoptotic phosphatidylserine exposure

doi: 10.1101/2025.03.17.643603

Figure Lengend Snippet: (a) Alignment of the amino acid sequence of caspase-3 IDR, caspase-7 IDR, IDR mutants. Acidic amino acids are shown in red. Basic amino acids are shown in blue. Caspase recognition sites are shown in boxes. Mutated amino acids are highlighted in gray. (b, c) Basic and hydrophobic (BH)-scores of caspase-3 and caspase-7. The red line indicates a threshold of 0.6. Caspase-3 IDR and caspase-7 IDR are masked with orange and green blocks, respectively. (d) Δt 1/2 of PM Dual FRET transiently expressed in WT (n = 45 cells), caspase-7 KO (n = 37 cells), with 7IDR-3-FLAG (n = 35 cells), 7IDR ED/A -3-FLAG (n = 17 cells), 7IDR KR/A -3-FLAG (n = 34 cells). (e) Δt 1/2 of PM Dual FRET transiently expressed in WT (n = 15 cells), caspase-7 KO (n = 44 cells), with 7IDR-3-FLAG (n = 24 cells), 7IDR D23A -3-FLAG (n = 31 cells). (f) A schematic image illustrating differential interference contrast (DIC), TIRF, and epifluorescence observation (top). Representative images of mScarlet3 expressing HeLa cells (bottom). Scale bar: 50 μm. (g) The ratio of TIRF fluorescence intensity to epifluorescence intensity, normalized to mScarlet3 (mS3) alone. (h) Lipid overlay assays with recombinant Lact-C2-FLAG, 7IDR D23A -3 CtoG -FLAG, 7IDR D23A, ED/A - 3 CtoG -FLAG, 7IDR Δ23 -3 CtoG -FLAG, and 7IDR Δ23, KR/A -3 CtoG -FLAG. (i) A schematic diagram depicting Lact-C2 interacting with phosphatidylserine (PS) in the inner leaflet of the PM (top). Representative images of Lact-C2 fused with HaloTag7 (bottom). Scale bars: 10 μm. (j) Δt 1/2 of PM Dual FRET transiently expressed in WT (n = 27 cells), caspase-7 KO (n = 19 cells), with 7IDR-3-FLAG (n = 16 cells), 7IDR-3-FLAG + HaloTag7-Lact-C2 (n = 18 cells), 7IDR-3-FLAG + HaloTag7-Lact-C2 AAA (n = 26 cells). (k) Δt 1/2 of PM Dual FRET transiently expressed in WT (n = 27 cells), with HaloTag7-Lact-C2 (n = 29 cells), HaloTag7-Lact-C2 AAA (n = 25 cells). For all figures, dot plots show the mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s comparison test. NS: P > 0.05; *: P < 0.05; **: P < 0.01; ****: P < 0.0001.

Article Snippet: For pcDNA3.1-myc-mScarlet3 , the coding sequence of mScarlet3 was PCR-amplified using myc-tag harboring primers from pmScarlet3_C1 (#189753; Addgene) and ligated to the Eco RⅠ Hin dⅢ-digested pcDNA3.1 vector using In-Fusion.

Techniques: Sequencing, Expressing, Fluorescence, Recombinant, Comparison

Journal: bioRxiv

Article Title: Early-peaking caspase-7 activity at the plasma membrane drives apoptotic phosphatidylserine exposure

doi: 10.1101/2025.03.17.643603

Figure Lengend Snippet: (a, b) Live imaging of mScarlet3 and mSCAT3 transiently expressed in caspase-7 KO HeLa cells during extrinsic apoptosis at the indicated time ((a), mScarlet3; (b) 7IDR-3 C-terminally fused with mScarlet3). Zero min indicates the time at which the mVenus/mECFP ratio reaches half the maximum and minimum. Representative images of mScarlet3 (top) and pseudo-colored FRET ratio images of cytosolic mSCAT3 (bottom: mVenus/mECFP) in HeLa cells during extrinsic apoptosis. Scale bars, 10 μm.

Article Snippet: For pcDNA3.1-myc-mScarlet3 , the coding sequence of mScarlet3 was PCR-amplified using myc-tag harboring primers from pmScarlet3_C1 (#189753; Addgene) and ligated to the Eco RⅠ Hin dⅢ-digested pcDNA3.1 vector using In-Fusion.

Techniques: Imaging