Journal: eLife

Article Title: The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission

doi: 10.7554/elife.84515

Figure Lengend Snippet: Figure 2. Crystal structure of the CAPN7(MIT)2-IST1322-366 complex. (A) Ribbon representation of CAPN7(MIT)2 (blue) in complex with IST1322-366 (green, with buried core interface sidechains shown) (PDB 8UC6). Locations of residues that were mutated in CAPN7(MIT)2 are shown in magenta, turquoise, and red. Dashed lines show CAPN7 and IST1 residues that lack well-defined electron density (see ‘Materials and methods’). (B) Structure of the CAPN74-68– IST1324-332 complex (blue and green) overlaid with the structure of VPS4A3-75-CHMP6168-179 MIM complex (gray and light blue, PDB 2K3W). Note the similar

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) Hela- N Maureen Powers Lab HeLa cells selected for transfectability Cell line (Homo sapiens) HEK293T ATCC CRL- 3216 Antibody Anti- FLAG (M2, mouse monoclonal) Sigma F1804 WB (1:5000) Antibody Anti- MYC (4A6, mouse monoclonal) Millipore 05- 724 WB (1:2500) Antibody Anti- RFP (rat monoclonal) ChromoTek 5F8 IF (1:500) Antibody Anti- RFP (mouse monoclonal) ChromoTek 6G6 WB (1:1000) Antibody Anti- alpha- tubulin (DM1A, mouse monoclonal) Cell Signaling Technologies DM1A IF (1:2000) Antibody Anti- alpha- Tubulin (chicken polyclonal) Synaptic Systems 302 206 IF (1:1000) Antibody Anti- CAPN7 (rabbit polyclonal) Proteintech Cat# 26985- 1- AP IF (1:500) WB (1:4000) Antibody Anti- IST1 (rabbit polyclonal) Sundquist Lab/Covance UT560 IF (1:1000) Paine et al. eLife 2023;12:e84515.

Techniques:

Journal: eLife

Article Title: The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission

doi: 10.7554/elife.84515

Figure Lengend Snippet: Figure 3. Mutational analyses of the CAPN7-IST1 complex. (A) Fluorescence polarization anisotropy binding isotherms showing CAPN7(MIT)2 constructs binding to IST1316-366. Isotherm data points and dissociation constants are means from three independent experiments ± standard error of the mean. WT, V18D, and F98D binding isotherms are reproduced from Wenzel et al., 2022 for comparison. (B) Size-exclusion chromatography binding analyses of free proteins or 1:1 mixtures of full-length CAPN7 with WT (black) or L328D/L355A mutant (green) full-length IST1. Note that the IST1 mutations disrupt

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) Hela- N Maureen Powers Lab HeLa cells selected for transfectability Cell line (Homo sapiens) HEK293T ATCC CRL- 3216 Antibody Anti- FLAG (M2, mouse monoclonal) Sigma F1804 WB (1:5000) Antibody Anti- MYC (4A6, mouse monoclonal) Millipore 05- 724 WB (1:2500) Antibody Anti- RFP (rat monoclonal) ChromoTek 5F8 IF (1:500) Antibody Anti- RFP (mouse monoclonal) ChromoTek 6G6 WB (1:1000) Antibody Anti- alpha- tubulin (DM1A, mouse monoclonal) Cell Signaling Technologies DM1A IF (1:2000) Antibody Anti- alpha- Tubulin (chicken polyclonal) Synaptic Systems 302 206 IF (1:1000) Antibody Anti- CAPN7 (rabbit polyclonal) Proteintech Cat# 26985- 1- AP IF (1:500) WB (1:4000) Antibody Anti- IST1 (rabbit polyclonal) Sundquist Lab/Covance UT560 IF (1:1000) Paine et al. eLife 2023;12:e84515.

Techniques: Fluorescence, Binding Assay, Construct, Comparison, Size-exclusion Chromatography, Mutagenesis

Journal: eLife

Article Title: The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission

doi: 10.7554/elife.84515

Figure Lengend Snippet: Figure 4. IST1 binding is required for CAPN7 midbody localization. (A) Representative immunofluorescence images showing the extent of midbody colocalization of mCherry-CAPN7 constructs (or the mCherry control), with endogenous IST1 in synchronous, NoCut checkpoint-active cells. Endogenous CAPN7 was depleted with siRNA while siRNA-resistant CAPN7-mCherry constructs were inducibly expressed. (B) Quantification of the colocalization of mCherry-CAPN7 constructs with endogenous IST1 at midbodies (corresponding to the images in A). Colocalization was scored blinded as described in ‘Materials and methods.’ Bars represent the mean and standard error of the mean from three independent experiments where > 50 IST1-positive midbody-stage cells were counted per experiment. Statistical analyses were performed using unpaired t-tests that compared the percentage of rescue constructs that colocalized with IST1 at midbodies to the mCherry alone control. ****p<0.0001, ns (not significant) p>0.05.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) Hela- N Maureen Powers Lab HeLa cells selected for transfectability Cell line (Homo sapiens) HEK293T ATCC CRL- 3216 Antibody Anti- FLAG (M2, mouse monoclonal) Sigma F1804 WB (1:5000) Antibody Anti- MYC (4A6, mouse monoclonal) Millipore 05- 724 WB (1:2500) Antibody Anti- RFP (rat monoclonal) ChromoTek 5F8 IF (1:500) Antibody Anti- RFP (mouse monoclonal) ChromoTek 6G6 WB (1:1000) Antibody Anti- alpha- tubulin (DM1A, mouse monoclonal) Cell Signaling Technologies DM1A IF (1:2000) Antibody Anti- alpha- Tubulin (chicken polyclonal) Synaptic Systems 302 206 IF (1:1000) Antibody Anti- CAPN7 (rabbit polyclonal) Proteintech Cat# 26985- 1- AP IF (1:500) WB (1:4000) Antibody Anti- IST1 (rabbit polyclonal) Sundquist Lab/Covance UT560 IF (1:1000) Paine et al. eLife 2023;12:e84515.

Techniques: Binding Assay, Immunofluorescence, Construct, Control

Journal: eLife

Article Title: The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission

doi: 10.7554/elife.84515

Figure Lengend Snippet: Figure 5. IST1-binding and catalytic activity are required for CAPN7 abscission and NoCut functions. (A, C) Quantification of midbody-stage and multinucleate HeLa cells from unperturbed asynchronous cultures (A), or cells in which NoCut checkpoint activity was sustained by Nup153 depletion (C). (B, D) Representative images from the quantified datasets in (A and C), respectively. Midbodies are marked with magenta arrows, and multinucleate cells are marked with white arrowheads. In all cases, cells were depleted of endogenous CAPN7, followed by expression of the designated DOX- inducible ‘rescue’ construct. Bars represent the mean and standard error of the mean from three independent experiments where >300 cells were counted per experiment. Statistical analyses were performed using unpaired t-tests comparing the sum of midbody-stage and multinucleate cells for each individual treatment to the same sum in siNT (non-targeting) control-treated cells. ***p<0.001, **p<0.01, ns (not significant) p>0.05.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) Hela- N Maureen Powers Lab HeLa cells selected for transfectability Cell line (Homo sapiens) HEK293T ATCC CRL- 3216 Antibody Anti- FLAG (M2, mouse monoclonal) Sigma F1804 WB (1:5000) Antibody Anti- MYC (4A6, mouse monoclonal) Millipore 05- 724 WB (1:2500) Antibody Anti- RFP (rat monoclonal) ChromoTek 5F8 IF (1:500) Antibody Anti- RFP (mouse monoclonal) ChromoTek 6G6 WB (1:1000) Antibody Anti- alpha- tubulin (DM1A, mouse monoclonal) Cell Signaling Technologies DM1A IF (1:2000) Antibody Anti- alpha- Tubulin (chicken polyclonal) Synaptic Systems 302 206 IF (1:1000) Antibody Anti- CAPN7 (rabbit polyclonal) Proteintech Cat# 26985- 1- AP IF (1:500) WB (1:4000) Antibody Anti- IST1 (rabbit polyclonal) Sundquist Lab/Covance UT560 IF (1:1000) Paine et al. eLife 2023;12:e84515.

Techniques: Binding Assay, Activity Assay, Expressing, Construct, Control

Journal: eLife

Article Title: The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission

doi: 10.7554/elife.84515

Figure Lengend Snippet: Figure 6. CAPN7 and SPAST are required to maintain the NoCut checkpoint in response to DNA bridges and replication stress. Quantification of midbody-stage and multinucleate HeLa cells from asynchronous cultures in which NoCut checkpoint activity was sustained by inducing DNA bridges with ICRF-193 treatment (A) or replication stress with aphidicolin treatment (B), following siRNA treatments to deplete the indicated proteins. Bars represent the mean and standard error of the mean from three independent experiments where >300 cells were counted per experiment. Statistical analyses were performed using unpaired t-tests comparing the sum of midbody-stage and multinucleate cells for each siRNA treatment with or without NoCut checkpoint maintenance with the indicated drug. **p<0.01, ns (not significant) p>0.05.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) Hela- N Maureen Powers Lab HeLa cells selected for transfectability Cell line (Homo sapiens) HEK293T ATCC CRL- 3216 Antibody Anti- FLAG (M2, mouse monoclonal) Sigma F1804 WB (1:5000) Antibody Anti- MYC (4A6, mouse monoclonal) Millipore 05- 724 WB (1:2500) Antibody Anti- RFP (rat monoclonal) ChromoTek 5F8 IF (1:500) Antibody Anti- RFP (mouse monoclonal) ChromoTek 6G6 WB (1:1000) Antibody Anti- alpha- tubulin (DM1A, mouse monoclonal) Cell Signaling Technologies DM1A IF (1:2000) Antibody Anti- alpha- Tubulin (chicken polyclonal) Synaptic Systems 302 206 IF (1:1000) Antibody Anti- CAPN7 (rabbit polyclonal) Proteintech Cat# 26985- 1- AP IF (1:500) WB (1:4000) Antibody Anti- IST1 (rabbit polyclonal) Sundquist Lab/Covance UT560 IF (1:1000) Paine et al. eLife 2023;12:e84515.

Techniques: Activity Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Calpain inhibitor MDL28170 alleviates cerebral ischemia‑reperfusion injury by suppressing inflammation and autophagy in a rat model of cardiac arrest

doi: 10.3892/etm.2023.11895

Figure Lengend Snippet: Western blotting analysis for calpastatin, calpain-1 and calpain-2. (A) Representative western blots of calpastatin, calpain-1 and calpain-2. (B) Western blotting analysis of calpastatin. (C) Western blotting analysis of calpain-1. (D) Western blotting analysis of calpain-2. Band intensities were normalized to those of GAPDH. (n=3). * P<0.05 vs. the sham group; # P<0.05 vs. the NS group; & P<0.05 vs. the DMSO group. NS, normal saline; MDL-l, low-dose MDL28170 group; MDL-h, high-dose MDL28170 group.

Article Snippet: The specimens were initially incubated at 4˚C with rabbit polyclonal anti-calpain-2 (1:2,000; cat. no. 11472-1-AP; ProteinTech Group, Inc.).

Techniques: Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Calpain inhibitor MDL28170 alleviates cerebral ischemia‑reperfusion injury by suppressing inflammation and autophagy in a rat model of cardiac arrest

doi: 10.3892/etm.2023.11895

Figure Lengend Snippet: Double immunofluorescence staining. (A) Dual-immunofluorescence for calpain-2 and LC3 in the cerebral cortex 24 h post- cardiac arrest/cardiopulmonary resuscitation. LC3 positive cells were stained red. Calpain-2 positive cells were stained green. The nucleus was stained using DAPI (blue). Scale bar, 20 µm (white bar). The white frame in merge images are enlarged into detail images (enlarge to 4x the original image size). (B) Calpain-2-positive cell rate in groups (calpain-2-positive cell number/DAPI-positive cell number). (C) LC3-positive cell rate in groups (LC3-positive cell number/DAPI-positive cell number). (D) Calpain-2 + LC3-positive cell rate in groups (calpain-2 + LC3-positive cell number/DAPI-positive cell number). (n=3). * P<0.05 vs. the sham group; # P<0.05 vs. the NS group. NS, normal saline; MDL-h, high-dose MDL28170 group.

Article Snippet: The specimens were initially incubated at 4˚C with rabbit polyclonal anti-calpain-2 (1:2,000; cat. no. 11472-1-AP; ProteinTech Group, Inc.).

Techniques: Double Immunofluorescence Staining, Immunofluorescence, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Calpain inhibitor MDL28170 alleviates cerebral ischemia‑reperfusion injury by suppressing inflammation and autophagy in a rat model of cardiac arrest

doi: 10.3892/etm.2023.11895

Figure Lengend Snippet: Graph abstract. (A) CA/CPR can induce CIRI and activate inflammation and autophagy. (B) Calpain inhibitor MDl28170 suppressed calpain-2 and improved CIRI by allieviating inflammation and autophagy in a rat model of CA. CA, cardiac arrest; CPR, cardiopulmonary resuscitation; CIRI, cerebral ischaemia reperfusion injury. TNF-α, tumour necrosis factor-a; IL-1β, interleukin 1β; IL-6, interleukin 1.

Article Snippet: The specimens were initially incubated at 4˚C with rabbit polyclonal anti-calpain-2 (1:2,000; cat. no. 11472-1-AP; ProteinTech Group, Inc.).

Techniques:

Journal: iScience

Article Title: Calpain-mediated cleavage generates a ZBTB18 N-terminal product that regulates HIF1A signaling and glioblastoma metabolism

doi: 10.1016/j.isci.2022.104625

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CAPN2 , Cell Signaling Technology , Cat#2539S; RRID: AB_2069843.

Techniques: Virus, Recombinant, Purification, Mutagenesis, Caspase-Glo Assay, Reverse Transcription, Sequencing, Extraction, Gene Expression, Western Blot, Software, Microscopy