α casp 1  (Santa Cruz Biotechnology)

Journal: PLOS Pathogens

Article Title: African swine fever virus pEP364R acts as an important inflammatory-inducing factor to activate NLRP3 inflammasome-mediated pyroptosis by regulating DDX3X

doi: 10.1371/journal.ppat.1013874

Figure Lengend Snippet: (A–C) BMDMs were infected with ASFV (MOI = 0.1/1/10) for 24 h. Cell viability (A) , Casp-1 activity (B) , and LDH release ( C ) were measured using CCK-8 assay kit, Casp-1 activity assay kit, and LDH assay kit, respectively. (D) PAMs were infected with ASFV-GFP (MOI = 1) for 24 h, followed by propidium iodide (PI) staining to observe cell death. A positive control (LPS [60 ng/mL for 8 h] + nigericin [Nig, 2 μM for 2 h] ) was included. (E) BMDMs were infected with ASFV (MOI = 1) for 24 h. Expression of pyroptosis marker proteins (Casp-1, IL-1β, and GSDMD-N) was analyzed by WB. (F) PAMs were infected with ASFV at an MOI of 1 for 24 h, fixed with 2% glutaraldehyde, and processed for transmission electron microscopy (TEM). Arrows indicate plasma membrane rupture. ( G ) iPAM cells were infected with ASFV at an MOI of 1. Formation of ASC specks and membrane localization of N-GSDMD were observed by immunofluorescence assay (IFA). ( H ) iPAM cells were infected with ASFV (MOI = 1) or positive control (LPS + Nig). ASC oligomerization was detected by WB using disuccinimidyl suberate (DSS) crosslinking. (I) PBMCs were infected with ASFV (MOI = 1) for 24 h. Expression of pyroptosis markers (Casp-1, IL-1β, N-GSDMD) and phosphorylation of IκBα and p65 in the NF-κB pathway were analyzed by WB. (J) PBMCs were infected with ASFV (MOI = 1) for 24 h, followed by PI staining to observe cell death. (K) PBMCs were infected with ASFV (MOI = 1) for 24 hours. Cell viability, Casp-1 activity, IL-1β transcription, and ASFV p72 mRNA level were assessed by CCK-8 assay, Casp-1 activity kit, qPCR, and qPCR, respectively. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; statistical significance was determined by one-way ANOVA.

Article Snippet: IL-1β (D3U3E) Rabbit mAb, Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb, Casp-1 (D7F10) Rabbit mAb, Cleaved Casp-1 (Asp297) (D57A2) Rabbit mAb, Gasdermin D (E9 X) Rabbit mAb, Cleaved Gasdermin D (Asp276) Crosslinker, and fluorescent secondary antibodies were purchased from CST.

Techniques: Infection, Activity Assay, CCK-8 Assay, Lactate Dehydrogenase Assay, Staining, Positive Control, Expressing, Marker, Transmission Assay, Electron Microscopy, Clinical Proteomics, Membrane, Immunofluorescence, Phospho-proteomics

Journal: PLOS Pathogens

Article Title: African swine fever virus pEP364R acts as an important inflammatory-inducing factor to activate NLRP3 inflammasome-mediated pyroptosis by regulating DDX3X

doi: 10.1371/journal.ppat.1013874

Figure Lengend Snippet: (A and B) PAMs were pretreated with the Casp-1 inhibitor VX765 (10 μM, 2 h) ( A ) or the NLRP3 inhibitor MCC950 (10 μM, 1 h) (B) , followed by ASFV infection (MOI = 1) for 24 h. DMSO was used as a control. IL-1β secretion was measured by ELISA, and pyroptosis biomarkers (Casp-1, IL-1β, N-GSDMD) expression were determined by WB. (C) PAMs were pretreated with VX765 or MCC950 as in (A and B) , infected with ASFV (MOI = 1) for 24 h, and subjected to PI staining. ASFV-induced cell death was observed. (D–F) PAMs were transfected with shASC (D) , shCasp-1 (E) , or shNLRP3 ( F ) for 72 h, followed by ASFV infection (MOI = 1) for 24 h. shNC was used as a negative control. ELISA used for IL-1β secretion detection, and pyroptosis biomarkers expression were determined by WB. (G) PAMs were transfected with shASC, shCasp-1, or shNLRP3 for 72 h, infected with ASFV (MOI = 1) for 24 h, and subjected to PI staining. LPS + Nig served as a positive control. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; statistical significance was determined by one-way ANOVA.

Article Snippet: IL-1β (D3U3E) Rabbit mAb, Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb, Casp-1 (D7F10) Rabbit mAb, Cleaved Casp-1 (Asp297) (D57A2) Rabbit mAb, Gasdermin D (E9 X) Rabbit mAb, Cleaved Gasdermin D (Asp276) Crosslinker, and fluorescent secondary antibodies were purchased from CST.

Techniques: Infection, Control, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Transfection, Negative Control, Positive Control

Journal: PLOS Pathogens

Article Title: African swine fever virus pEP364R acts as an important inflammatory-inducing factor to activate NLRP3 inflammasome-mediated pyroptosis by regulating DDX3X

doi: 10.1371/journal.ppat.1013874

Figure Lengend Snippet: (A) PK-15 cells, with activated NLRP3 inflammasome components, were transfected with 2 μg of each of the 185 plasmids encoding ASFV proteins. IL-1β secretion was screened by ELISA to identify proteins inducing high IL-1β levels. ( B ) iPAMs cells were transfected with 2 μg of EP364R plasmid. At 24 h post-transfection (h.p.t), cells were fixed and the subcellular localization of EP364R was observed by confocal microscopy. Scale bars, 20 μm. (C) BMDMs cells were transfected with 2 μg of EP364R plasmid. IL-1β secretion in the supernatant was measured by ELISA at 24 h. ( D ) iPAMs were transfected with increasing concentrations (0.25, 0.5, 1.0 μg) of EP364R plasmid, with 1 μg of empty vector (EV) plasmid as a control. At 24 h, 10 μl of CCK-8 reagent was added, followed by incubation at 37°C for 1 hour. Absorbance was measured at 450 nm. ( E ) iPAMs were transfected with 2 μg of EP364R plasmid. Lactate dehydrogenase (LDH) release was detected using an LDH assay kit at 0, 12, and 24 h. ( F ) iPAMs were transfected with 2 μg of EP364R plasmid, alongside blank control, positive control [LPS (60 ng/mL for 8 h) + Nigericin (Nig, 2 μM for 2 h) ], liposome (4 μl) control, and negative control (EV, 2 μg). At 24 h, cells were stained with propidium iodide (PI) and cell death was observed by fluorescence microscopy. (G) Cell morphology was observed by microscopy 24 h after transfection of iPAMs with 2 μg of EP364R plasmid. ( H ) iPAMs were co-transfected with plasmids for NLRP3 (3 μg), pro-IL-1β (3 μg), ASC (1 μg), pro-Casp-1 (1 μg), and increasing amounts of EP364R (0, 2, 4, 6 μg). Cells were harvested and lysed 24 h p.t., and IL-1β secretion was analyzed by WB. ( I ) iPAMs were transfected with 2 μg of EP364R plasmid, with LPS + Nig treatment as a positive control. Cells were lysed 24 h p.t., and the expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB. ( J ) iPAMs were transfected with 2 μg of EP364R plasmid, with LPS + Nig as a positive control. At 24 h p.t., cell lysates were centrifuged. The supernatant was prepared as the ‘input’ sample. The cell pellet was resuspended in 100 μl PBS, cross-linked with 2 mM DSS at 37°C for 30 min, and then directly mixed with protein loading buffer to prepare the ‘pellet’ sample. ASC and GSDMD-N oligomerization were detected by WB. ( K ) iPAMs were transfected with 2 μg of EP364R plasmid. At 24 h p.t., cells were fixed, and ASC speck formation and GSDMD-N localization were observed by confocal microscopy. Scale bars, 20 μm. A P value less than 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IL-1β (D3U3E) Rabbit mAb, Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb, Casp-1 (D7F10) Rabbit mAb, Cleaved Casp-1 (Asp297) (D57A2) Rabbit mAb, Gasdermin D (E9 X) Rabbit mAb, Cleaved Gasdermin D (Asp276) Crosslinker, and fluorescent secondary antibodies were purchased from CST.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Confocal Microscopy, Control, CCK-8 Assay, Incubation, Lactate Dehydrogenase Assay, Positive Control, Negative Control, Staining, Fluorescence, Microscopy, Expressing

Journal: PLOS Pathogens

Article Title: African swine fever virus pEP364R acts as an important inflammatory-inducing factor to activate NLRP3 inflammasome-mediated pyroptosis by regulating DDX3X

doi: 10.1371/journal.ppat.1013874

Figure Lengend Snippet: ( A ) The pHBLV-EP364R lentiviral overexpression plasmid (10 μg) was co-transfected with lentiviral packaging helper plasmids psPAX2 (10 μg) and pMD2.G (10 μg) into HEK293T cells. Supernatants containing lentivirus were collected at 48 and 72 h p.t., and purified, and used to infect iPAMs. Stable puromycin-resistant cells expressing green fluorescent protein (GFP) were selected. Stable EP364R expression was confirmed by WB and PCR. ( B ) Casp-1, IL-1β, and GSDMD-N in EP364R-overexpressed iPAMs (LV-EP364R) were detected by WB, iPAMs expressing only GFP and resistance genes served as negative controls (LV-Vector), and Mock cells were normal iPAMs. ( C-D ) Transcriptional and secretion levels of cytokines IL-1β, IL-6, TNF-α, CXCL10, and IFN-β were measured by qPCR and ELISA in EP364R-overexpressed iPAMs. ( E ) Successful EP364R overexpressed lentivirus was purified and determined by WB at 72 h p.t. ( F ) Mice were intravenously injected twice with 5 × 10 7 TU of purified EP364R overexpression lentivirus, with a 48-h interval. Mice were randomly selected from each group and euthanized, and pathological changes in the heart, liver, spleen, lung, and kidney were examined. ( G ) Mice from the LV-EP364R and LV-Vector groups were randomly selected and euthanized. Tissues (heart, liver, spleen, lung, kidney; 1 g each) were harvested, homogenized, and proteins were extracted using tissue lysis buffer. Expression of IL-1β was detected by WB. And p value less than 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IL-1β (D3U3E) Rabbit mAb, Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb, Casp-1 (D7F10) Rabbit mAb, Cleaved Casp-1 (Asp297) (D57A2) Rabbit mAb, Gasdermin D (E9 X) Rabbit mAb, Cleaved Gasdermin D (Asp276) Crosslinker, and fluorescent secondary antibodies were purchased from CST.

Techniques: Over Expression, Plasmid Preparation, Transfection, Purification, Expressing, Enzyme-linked Immunosorbent Assay, Injection, Lysis

Journal: PLOS Pathogens

Article Title: African swine fever virus pEP364R acts as an important inflammatory-inducing factor to activate NLRP3 inflammasome-mediated pyroptosis by regulating DDX3X

doi: 10.1371/journal.ppat.1013874

Figure Lengend Snippet: ( A ) iPAMs were transfected with 2 μg of small hairpin RNA (shEP364R-1, -2, or -3) plasmids. At 72 h p.t., cells were transfected with 2 μg of EP364R plasmid for 24 h. small hairpin RNA Nonspecific shRNA (shNC) as a negative control. RNA was extracted for qPCR, and cells were lysed for WB analysis to screen for the most effective shEP364R. ( B ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV (MOI = 1). EP364R transcription levels were validated by qPCR 24 h p.i (hours post infection). ( C ) iPAMs were transfected with 50 nM of the selected siEP364R. At 72 hours post-transfection, cells were infected with ASFV at an MOI of 1. The culture supernatants were collected 24 hours post-infection, processed through repeated freeze-thaw cycles and lyophilization, and then analyzed by ELISA to quantify secreted IL-1β. ( D ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV (MOI = 1) for 24 h. IL-1β secretion was measured by ELISA, and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB. ( E ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. CCK-8 reagent (10 μl) was added, followed by incubation at 37°C for 1 h, and absorbance was measured at 450 nm. LPS and Nigericin stimulation group as a positive control. ( F ) iPAMs were transfected with 2 μg of shEP364R-3 or shNC plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Intracellular Casp-1 activity was measured using a Caspase-1 activity assay kit. ( G ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. LDH release was detected using an LDH assay kit. ( H ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Cells were stained with PI (10 μl) and cell death was observed by fluorescence microscopy. ( I ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Cell lysates were centrifuged; the supernatant was prepared as the ‘input’ sample. The pellet was resuspended in PBS, cross-linked with DSS, incubated at 37°C for 30 min, and mixed with loading buffer to prepare the ‘pellet’ sample. ASC oligomerization was detected by WB. ( J ) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. RNA was extracted, and transcriptional levels of IL-1β, IL-6, TNF-α, CXCL10, and EP364R were measured by qPCR. A P value less than 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IL-1β (D3U3E) Rabbit mAb, Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb, Casp-1 (D7F10) Rabbit mAb, Cleaved Casp-1 (Asp297) (D57A2) Rabbit mAb, Gasdermin D (E9 X) Rabbit mAb, Cleaved Gasdermin D (Asp276) Crosslinker, and fluorescent secondary antibodies were purchased from CST.

Techniques: Transfection, Plasmid Preparation, shRNA, Negative Control, Infection, Lyophilization, Enzyme-linked Immunosorbent Assay, Expressing, CCK-8 Assay, Incubation, Positive Control, Activity Assay, Lactate Dehydrogenase Assay, Staining, Fluorescence, Microscopy

Journal: PLOS Pathogens

Article Title: African swine fever virus pEP364R acts as an important inflammatory-inducing factor to activate NLRP3 inflammasome-mediated pyroptosis by regulating DDX3X

doi: 10.1371/journal.ppat.1013874

Figure Lengend Snippet: ( A ) iPAMs were pretreated with the NLRP3 inhibitor MCC950 (10 μM) for 1 h, then transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h p.t. ( B ) iPAMs were pretreated with the Casp-1 inhibitor VX765 (10 μM) for 2 h, then transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h p.t. ( C-E ) iPAMs were transfected with 3 μg of shNLRP3 (C) , shASC (D) , or shCasp-1 ( E ) plasmids. At 72 h p.t., cells were transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h later. ( F ) iPAMs were transfected with 3 μg of shNLRP3, shASC, or shCasp-1 plasmids. At 72 h p.t., cells were transfected with 3 μg of EP364R plasmid. At 24 h p.t., cells were stained with PI (10 μl) and cell death was observed by microscopy. (G) Bone marrow cells were isolated from NLRP3-/- knockout mice and differentiated for 7 days in medium containing L929 cell-conditioned supernatant. Cells were then transfected with 3 μg of EP364R plasmid. Transcriptional levels of EP364R and NLRP3 were measured by qPCR 24 h p.t., showing no significant difference in EP364R transcription but confirmed absence of NLRP3. (H) Bone marrow-derived macrophages (BMDMs) from NLRP3-/- mice (prepared as in G) were transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h p.t. A P value less than 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IL-1β (D3U3E) Rabbit mAb, Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb, Casp-1 (D7F10) Rabbit mAb, Cleaved Casp-1 (Asp297) (D57A2) Rabbit mAb, Gasdermin D (E9 X) Rabbit mAb, Cleaved Gasdermin D (Asp276) Crosslinker, and fluorescent secondary antibodies were purchased from CST.

Techniques: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Microscopy, Isolation, Knock-Out, Derivative Assay

Journal: PLOS Pathogens

Article Title: African swine fever virus pEP364R acts as an important inflammatory-inducing factor to activate NLRP3 inflammasome-mediated pyroptosis by regulating DDX3X

doi: 10.1371/journal.ppat.1013874

Figure Lengend Snippet: Upon infection of domestic swine tissues, the ASFV-encoded protein EP364R binds to a specific spatial structure of DDX3X, which triggers the oligomerization of the NLRP3 inflammasome. This activation leads to the cleavage and activation of Casp-1, which in turn executes pyroptotic cell death and facilitates the release of pro‑inflammatory cytokines and damage‑associated molecular patterns (DAMPs). This cascade is likely a key driver of the cytokine storm‑mediate tissues damage during infection. HAMNO, a small molecule targeting EP364R, inhibits ASFV replication through disruption of the EP364R-DDX3X interaction.

Article Snippet: IL-1β (D3U3E) Rabbit mAb, Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb, Casp-1 (D7F10) Rabbit mAb, Cleaved Casp-1 (Asp297) (D57A2) Rabbit mAb, Gasdermin D (E9 X) Rabbit mAb, Cleaved Gasdermin D (Asp276) Crosslinker, and fluorescent secondary antibodies were purchased from CST.

Techniques: Infection, Activation Assay, Disruption

Journal: iScience

Article Title: Functional characterization of CASP, a CUX1 isoform, reveals its tumor-promoting role in colorectal cancer via TRIM21-mediated signaling

doi: 10.1016/j.isci.2026.114783

Figure Lengend Snippet: Altered CASP expression in CRC patients (A) Identification of CASP as a key prognostic marker via integrative analysis of expression profiles and TCGA survival data. Venn diagram showing the overlap of differentially expressed proteins between CRCs and normal tissues, as well as colorectal adenomas and normal tissues. A total of 248 candidates were analyzed for prognosis in TCGA, and 21 candidates were subsequently identified. (B) Protein expression of CASP in normal colon tissue ( n = 20), polyps ( n = 18), adenoma ( n = 22), and CRC ( n = 30) tissue based on MS proteomics data. (C) mRNA expression of CASP (probe 202367_at) in normal colon tissue ( n = 54), polyps ( n = 48), primary tumors ( n = 185), CRC liver metastasis ( n = 46), and CRC lung metastasis ( n = 20) tissue from the GSE41258 dataset. (D) WB analysis of CASP expression in different CRC cell lines and the normal intestinal epithelial cell line NCM460. (E) The band intensities from WB analysis were quantified using ImageJ software and target protein bands was normalized to the corresponding control (β-actin or GAPDH) to account for variations in sample loading. (F) CASP expression was analyzed by IHC in 97 paired CRC/adjacent tissues (top right) and in 107 CRC samples plus 98 adjacent tissues (bottom right). Representative images are shown on the left. (G) Proportion of case distribution of MMR function/MSI status and mutations in TP53 according to CASP expression in GSE41258 (left) dataset and GSE39582 (right) dataset. (H and I) Kaplan-Meier survival curves of CRC patients according to CASP expression using datasets from GSE41258 ( n = 120) and GSE14333 ( n = 226). (J) Co-staining of the Golgi marker GM130 and CASP in DLD-1 and HT-29 cells revealed clear evidence of colocalization in the Golgi apparatus; Scale bars: 10 μm. Data are expressed as mean ± standard deviation (SD). Statistical analyses were performed on GraphPad Prism. (B, C, E, and F- bottom right) Unpaired t test. (F- top right) Paired t test. (G) Chi-square test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies against CASP (1:200, 11733-1-AP, Proteintech) or β-catenin (1:200, EM0306, Huabio).

Techniques: Expressing, Marker, Software, Control, Staining, Standard Deviation

Journal: iScience

Article Title: Functional characterization of CASP, a CUX1 isoform, reveals its tumor-promoting role in colorectal cancer via TRIM21-mediated signaling

doi: 10.1016/j.isci.2026.114783

Figure Lengend Snippet: CASP impedes MAPK signaling by negatively regulating TRIM21 protein stability (A) Immunoblot analysis of TRIM21 immunoprecipitated using an FLAG tag fused with CASP in 293T cells. (B) Immunoblot analysis of CASP immunoprecipitated using a His tag fused with TRIM21 in 293T cells. (C) Immunoblot validation of endogenous co-precipitation of TRIM21 with CASP antibody in DLD-1and LoVo cells. (D) Co-IP analysis with an anti-His antibody was performed in DLD-1 cells 48h after transfection with constructs expressing the His-tagged full-length, N-terminal cytoplasmic, or C-terminal Golgi domain of CASP (or an empty vector), to assess their respective interactions with TRIM21. (E and F) qPCR (E) and WB (F) analyses of TRIM21 expression at the mRNA and protein levels, respectively, in DLD-1 cells following CASP knockdown or overexpression compared to their respective controls. (G) Quantification of TRIM21 protein expression from the WB shown ( C), which displays TRIM21 and CASP levels in protein extracts from subcutaneous xenograft tumors of nude mice. Band intensities for TRIM21 were normalized to β-actin (loading control) using ImageJ software. (H) 293T cells were co-transfected with plasmids encoding HA-Ub, His-TRIM21, and control or shCASP-1. Cells were treated with MG132 for 6 h prior to lysis. His-TRIM21 was immunoprecipitated from cell lysates using Ni-NTA beads, and its ubiquitination level was detected by immunoblotting with anti-HA antibody. (I and J) Turnover of TRIM21 protein was assessed in scrambled and CASP-KD DLD-1 cells treated with 60 μg/mL CHX for the indicated times. Representative blots (I) and quantification (J) demonstrating that CASP depletion significantly slows the degradation rate of TRIM21. (K and L) c-Myc turnover was analyzed in parallel under the similar conditions. Representative blots (K) and quantification (L) revealing that CASP knockdown accelerates the degradation kinetics of c-Myc. Protein levels were normalized to GAPDH and are presented as the percentage remaining relative to time 0. (M) DLD-1 cells were treated with the proteasome inhibitor MG132 (20 μM) for the indicated times (0, 1, 2, 4, 6, and 8 h). WB analysis was performed to monitor the stabilization of TRIM21 protein. (N) Alterations in the proliferative capacity of CASP-KD DLD-1 cells and their control counterparts after transient transfection with TRIM21 siRNA. Immunoblot analysis showing the knockdown efficiency of TRIM21(up-left); Cell viability measured on days 1, 3, 5, and 7 using the CCK-8 assay(up-right); Schematic representation of the cell colony formation assay (down-left); Statistical graph showing the number of colonies for each group (down-right). (O) Alterations in key molecules of the MAPK pathway in control and CASP-KD DLD-1 cells following TRIM21 downregulation. Data are expressed as mean ± SD. Statistical analyses were performed on GraphPad Prism. (E, G, and N) Unpaired t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns = no significant difference.

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies against CASP (1:200, 11733-1-AP, Proteintech) or β-catenin (1:200, EM0306, Huabio).

Techniques: Western Blot, Immunoprecipitation, FLAG-tag, Biomarker Discovery, Co-Immunoprecipitation Assay, Transfection, Construct, Expressing, Plasmid Preparation, Knockdown, Over Expression, Control, Software, Lysis, Ubiquitin Proteomics, CCK-8 Assay, Colony Assay

Journal: iScience

Article Title: Functional characterization of CASP, a CUX1 isoform, reveals its tumor-promoting role in colorectal cancer via TRIM21-mediated signaling

doi: 10.1016/j.isci.2026.114783

Figure Lengend Snippet: Altered CASP expression in CRC patients (A) Identification of CASP as a key prognostic marker via integrative analysis of expression profiles and TCGA survival data. Venn diagram showing the overlap of differentially expressed proteins between CRCs and normal tissues, as well as colorectal adenomas and normal tissues. A total of 248 candidates were analyzed for prognosis in TCGA, and 21 candidates were subsequently identified. (B) Protein expression of CASP in normal colon tissue ( n = 20), polyps ( n = 18), adenoma ( n = 22), and CRC ( n = 30) tissue based on MS proteomics data. (C) mRNA expression of CASP (probe 202367_at) in normal colon tissue ( n = 54), polyps ( n = 48), primary tumors ( n = 185), CRC liver metastasis ( n = 46), and CRC lung metastasis ( n = 20) tissue from the GSE41258 dataset. (D) WB analysis of CASP expression in different CRC cell lines and the normal intestinal epithelial cell line NCM460. (E) The band intensities from WB analysis were quantified using ImageJ software and target protein bands was normalized to the corresponding control (β-actin or GAPDH) to account for variations in sample loading. (F) CASP expression was analyzed by IHC in 97 paired CRC/adjacent tissues (top right) and in 107 CRC samples plus 98 adjacent tissues (bottom right). Representative images are shown on the left. (G) Proportion of case distribution of MMR function/MSI status and mutations in TP53 according to CASP expression in GSE41258 (left) dataset and GSE39582 (right) dataset. (H and I) Kaplan-Meier survival curves of CRC patients according to CASP expression using datasets from GSE41258 ( n = 120) and GSE14333 ( n = 226). (J) Co-staining of the Golgi marker GM130 and CASP in DLD-1 and HT-29 cells revealed clear evidence of colocalization in the Golgi apparatus; Scale bars: 10 μm. Data are expressed as mean ± standard deviation (SD). Statistical analyses were performed on GraphPad Prism. (B, C, E, and F- bottom right) Unpaired t test. (F- top right) Paired t test. (G) Chi-square test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The following primary antibodies were used: β-actin (1:5000, M1210-2, Huabio), GAPDH (1:50000, 60004-1-Ig, Proteintech), CASP (1:2000, 11733-1-AP, Proteintech), c-Myc (1:5000, 67447-1-Ig, Proteintech), Cyclin D1 (1:1000, 55506, Cell Signaling Technology), p21 (1:1000, 2947, Cell Signaling Technology), N-cadherin (1:5000, 22018-1-AP, Proteintech), Vimentin (1:5000, 10366-1-AP, Proteintech), MMP-9 (1:1000, 10375-2-AP, Proteintech) and β-catenin (1:1000, EM0306, Huabio).

Techniques: Expressing, Marker, Software, Control, Staining, Standard Deviation

Journal: iScience

Article Title: Functional characterization of CASP, a CUX1 isoform, reveals its tumor-promoting role in colorectal cancer via TRIM21-mediated signaling

doi: 10.1016/j.isci.2026.114783

Figure Lengend Snippet: CASP impedes MAPK signaling by negatively regulating TRIM21 protein stability (A) Immunoblot analysis of TRIM21 immunoprecipitated using an FLAG tag fused with CASP in 293T cells. (B) Immunoblot analysis of CASP immunoprecipitated using a His tag fused with TRIM21 in 293T cells. (C) Immunoblot validation of endogenous co-precipitation of TRIM21 with CASP antibody in DLD-1and LoVo cells. (D) Co-IP analysis with an anti-His antibody was performed in DLD-1 cells 48h after transfection with constructs expressing the His-tagged full-length, N-terminal cytoplasmic, or C-terminal Golgi domain of CASP (or an empty vector), to assess their respective interactions with TRIM21. (E and F) qPCR (E) and WB (F) analyses of TRIM21 expression at the mRNA and protein levels, respectively, in DLD-1 cells following CASP knockdown or overexpression compared to their respective controls. (G) Quantification of TRIM21 protein expression from the WB shown ( C), which displays TRIM21 and CASP levels in protein extracts from subcutaneous xenograft tumors of nude mice. Band intensities for TRIM21 were normalized to β-actin (loading control) using ImageJ software. (H) 293T cells were co-transfected with plasmids encoding HA-Ub, His-TRIM21, and control or shCASP-1. Cells were treated with MG132 for 6 h prior to lysis. His-TRIM21 was immunoprecipitated from cell lysates using Ni-NTA beads, and its ubiquitination level was detected by immunoblotting with anti-HA antibody. (I and J) Turnover of TRIM21 protein was assessed in scrambled and CASP-KD DLD-1 cells treated with 60 μg/mL CHX for the indicated times. Representative blots (I) and quantification (J) demonstrating that CASP depletion significantly slows the degradation rate of TRIM21. (K and L) c-Myc turnover was analyzed in parallel under the similar conditions. Representative blots (K) and quantification (L) revealing that CASP knockdown accelerates the degradation kinetics of c-Myc. Protein levels were normalized to GAPDH and are presented as the percentage remaining relative to time 0. (M) DLD-1 cells were treated with the proteasome inhibitor MG132 (20 μM) for the indicated times (0, 1, 2, 4, 6, and 8 h). WB analysis was performed to monitor the stabilization of TRIM21 protein. (N) Alterations in the proliferative capacity of CASP-KD DLD-1 cells and their control counterparts after transient transfection with TRIM21 siRNA. Immunoblot analysis showing the knockdown efficiency of TRIM21(up-left); Cell viability measured on days 1, 3, 5, and 7 using the CCK-8 assay(up-right); Schematic representation of the cell colony formation assay (down-left); Statistical graph showing the number of colonies for each group (down-right). (O) Alterations in key molecules of the MAPK pathway in control and CASP-KD DLD-1 cells following TRIM21 downregulation. Data are expressed as mean ± SD. Statistical analyses were performed on GraphPad Prism. (E, G, and N) Unpaired t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns = no significant difference.

Article Snippet: The following primary antibodies were used: β-actin (1:5000, M1210-2, Huabio), GAPDH (1:50000, 60004-1-Ig, Proteintech), CASP (1:2000, 11733-1-AP, Proteintech), c-Myc (1:5000, 67447-1-Ig, Proteintech), Cyclin D1 (1:1000, 55506, Cell Signaling Technology), p21 (1:1000, 2947, Cell Signaling Technology), N-cadherin (1:5000, 22018-1-AP, Proteintech), Vimentin (1:5000, 10366-1-AP, Proteintech), MMP-9 (1:1000, 10375-2-AP, Proteintech) and β-catenin (1:1000, EM0306, Huabio).

Techniques: Western Blot, Immunoprecipitation, FLAG-tag, Biomarker Discovery, Co-Immunoprecipitation Assay, Transfection, Construct, Expressing, Plasmid Preparation, Knockdown, Over Expression, Control, Software, Lysis, Ubiquitin Proteomics, CCK-8 Assay, Colony Assay

Journal: iScience

Article Title: Functional characterization of CASP, a CUX1 isoform, reveals its tumor-promoting role in colorectal cancer via TRIM21-mediated signaling

doi: 10.1016/j.isci.2026.114783

Figure Lengend Snippet: Altered CASP expression in CRC patients (A) Identification of CASP as a key prognostic marker via integrative analysis of expression profiles and TCGA survival data. Venn diagram showing the overlap of differentially expressed proteins between CRCs and normal tissues, as well as colorectal adenomas and normal tissues. A total of 248 candidates were analyzed for prognosis in TCGA, and 21 candidates were subsequently identified. (B) Protein expression of CASP in normal colon tissue ( n = 20), polyps ( n = 18), adenoma ( n = 22), and CRC ( n = 30) tissue based on MS proteomics data. (C) mRNA expression of CASP (probe 202367_at) in normal colon tissue ( n = 54), polyps ( n = 48), primary tumors ( n = 185), CRC liver metastasis ( n = 46), and CRC lung metastasis ( n = 20) tissue from the GSE41258 dataset. (D) WB analysis of CASP expression in different CRC cell lines and the normal intestinal epithelial cell line NCM460. (E) The band intensities from WB analysis were quantified using ImageJ software and target protein bands was normalized to the corresponding control (β-actin or GAPDH) to account for variations in sample loading. (F) CASP expression was analyzed by IHC in 97 paired CRC/adjacent tissues (top right) and in 107 CRC samples plus 98 adjacent tissues (bottom right). Representative images are shown on the left. (G) Proportion of case distribution of MMR function/MSI status and mutations in TP53 according to CASP expression in GSE41258 (left) dataset and GSE39582 (right) dataset. (H and I) Kaplan-Meier survival curves of CRC patients according to CASP expression using datasets from GSE41258 ( n = 120) and GSE14333 ( n = 226). (J) Co-staining of the Golgi marker GM130 and CASP in DLD-1 and HT-29 cells revealed clear evidence of colocalization in the Golgi apparatus; Scale bars: 10 μm. Data are expressed as mean ± standard deviation (SD). Statistical analyses were performed on GraphPad Prism. (B, C, E, and F- bottom right) Unpaired t test. (F- top right) Paired t test. (G) Chi-square test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: An anti-CASP rabbit antibody (1:3000, 11733-1-AP, Proteintech) was used for IHC staining.

Techniques: Expressing, Marker, Software, Control, Staining, Standard Deviation

Journal: iScience

Article Title: Functional characterization of CASP, a CUX1 isoform, reveals its tumor-promoting role in colorectal cancer via TRIM21-mediated signaling

doi: 10.1016/j.isci.2026.114783

Figure Lengend Snippet: CASP promoted CRC cell proliferation both in vitro and in vivo (A) WB analysis of CASP protein level in control and CASP-OE DLD-1 and HCT116 cells (left) as well as scramble and CASP-KD DLD-1, LoVo, and HT-29 cells (right). (B) CCK-8 assay for investigating proliferation of the CASP knockdown in DLD-1, LoVo, and HT-29 cells, along with the CASP overexpression in HCT116 cells. (C) Colony formation ability of the CASP knockdown in DLD-1, LoVo, and HT-29 cells, along with the CASP overexpression in HCT116 cells; Quantification analysis is presented in the right histogram. (D) Colony formation rescue ability of CASP expression (shCASP-1 + CASP-OE) in DLD-1 cells; upper histogram represents quantification analysis. (E) Subcutaneous tumor formation in nude mice ( n = 5/group) with CASP-KD cells; tumors derived from DLD-1 cells (left) and tumor growth curve of control (scramble, SCR) and CASP-KD DLD-1 cells in xenograft model (right IHC images of Ki-67 expression in xenograft tumor tissue and the result for Ki-67 positivity (%) is displayed in the lower panel. (F) The cell cycle distribution of CASP knockdown in DLD-1 and LoVo cells was analyzed by flow cytometry. (G) WB analysis of cell cycle related proteins was investigated. Data are expressed as mean ± SD. Statistical analyses were performed on GraphPad Prism. (B–F) Unpaired t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: An anti-CASP rabbit antibody (1:3000, 11733-1-AP, Proteintech) was used for IHC staining.

Techniques: In Vitro, In Vivo, Control, CCK-8 Assay, Knockdown, Over Expression, Expressing, Derivative Assay, Flow Cytometry

Journal: iScience

Article Title: Functional characterization of CASP, a CUX1 isoform, reveals its tumor-promoting role in colorectal cancer via TRIM21-mediated signaling

doi: 10.1016/j.isci.2026.114783

Figure Lengend Snippet: CASP promoted CRC cell metastasis both in vitro and in vivo (A and B) Transwell assay for investigating migration properties CASP knockdown (A) and overexpression (B) in DLD-1, LoVo and HCT116 cells; Scale bars: 100 μm. (C) Representative images of luciferase signals in metastatic foci formed by control and CASP-KD LoVo cells in immunodeficient mice. (D) Quantification of photon flux in metastatic foci formed by control and CASP-KD LoVo cells in immunodeficient mice. (E) Representative images and quantification of photon flux of the stripped liver and lung of from the sacrificed NSG mice in metastatic model. (F) H&E staining in pulmonary metastatic foci from the sacrificed NSG mice in metastatic model. (G) Changes in EMT-related protein markers and MMP-9 in response to CASP alteration in DLD-1 and HCT116 cells. Data are expressed as mean ± SD. Statistical analyses were performed on GraphPad Prism. (A, B, D, E, and F) Unpaired t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns = no significant difference.

Article Snippet: An anti-CASP rabbit antibody (1:3000, 11733-1-AP, Proteintech) was used for IHC staining.

Techniques: In Vitro, In Vivo, Transwell Assay, Migration, Knockdown, Over Expression, Luciferase, Control, Staining