apoptosis protein  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Sympathetic Controls Fate and Function of Adult Sensory Neurons

doi: 10.64898/2026.02.12.702354

Figure Lengend Snippet: (A) Representative immunofluorescence images showing elevated cleaved caspase-3 expression in L4 DRG 16 hours after mSYMPX. (B) Quantification of cleaved caspase-3 fluorescence intensity normalized to the cellular area confirmed increased expression in the mSYMPX group (unpaired t-test: ***p < 0.001 vs. sham, n=6). (C) Proteome profiling identified differentially expressed proteins between sham and mSYMPX groups. (D) Illustration of the cytokine array containing 21 different antibodies with duplicates. The array also contains three positive control (PC) proteins with strong signals in three corners of the membrane (for each membrane was used n=3). (E) Volcano plot highlighting downregulation of anti-apoptotic proteins in DRG tissues with mSYMPX. (F-G) Western blot analysis showing XIAP downregulation at POD2, with band quantification confirming reduced expression level (unpaired t-test: *p < 0.05 vs. sham, n=3). (H) ELISA analysis demonstrated upregulation of the pro-apoptotic proteins BAX (n=5-7) and Smac/Diablo (I) (one-way ANOVA with Tukey’s post hoc test; *p < 0.05 vs. control, n=6). (J) apoptosis pathway summary.

Article Snippet: After blocking with bovine serum albumin (BSA) for 1 hour, membranes were incubated overnight at 4 °C with X-linked inhibitor of apoptosis protein (XIAP) antibody (Rabbit, 1:500, Novus Biologicals, Cat. No. NBP220918).

Techniques: Immunofluorescence, Expressing, Fluorescence, Positive Control, Membrane, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Advanced Science

Article Title: Synthetic Lethality of SHP2 and XIAP Suppresses Proliferation and Metastasis in KRAS ‐mutant Nonsmall Cell Lung Cancer

doi: 10.1002/advs.202411642

Figure Lengend Snippet: Embelin inhibits KRAS ‐mutant tumor development. A) Correlation detection between mRNA expression of PTPN11 and KRAS in patients with LUAD ( n = 706) using IlluminaHiSeq analysis with data derived from TCGA database. B) Correlation and number at risk of SHP2 expression with the overall survival probability of KRAS mutant lung cancer patients ( n = 92) in the TCGA database. C) Protein expression and phosphorylation levels of SHP2 in RAS ‐WT, NRAS , HRAS , and KRAS ‐mutant cells. D) Effect of 3471 bioactive compounds at 10 µM against SHP2, with Na 3 VO 4 serving as a control. Z’ factor values for 69 plates screened, Z’ = 0.81, S/N = 40.8. E) Binding energy between candidate drugs and SHP2 full‐length domain, catalytic region, and ΔSHP2 domain. F) HeLa cells were pretreated with DMSO, SHP099, or various compounds at 20 µM for 2 h before stimulation with EGF (10 ng mL −1 ) for an additional 2 h. Cell lysates were subjected to immunoblotting with SHP2, p‐SHP2, and GAPDH antibodies. Data are from three independent experiments are presented. G) Spearman's correlation coefficient between drug‐related targets and KRAS mRNA in different types of tumors through the TCGA database. H) BCL2 , TPO , RAF1 , MCL1 , MPL , and XIAP mRNA expression levels in LUAD tumors and adjacent tissues through the TCGA database. I) XIAP protein expression in RAS ‐WT, NRAS ‐mutant, HRAS ‐mutant, and KRAS ‐mutant cells. J) Effect of candidate drugs (10 µM) on colony formation in A549, NCI‐H2122, and NCI‐H1944 cells.

Article Snippet: Similarly, the protein expression of SHP2 and XIAP in KRAS wild‐type cells was significantly lower compared to KRAS mutant cells, with p ‐values of 0.0430 and 0.0381, respectively, from the Human Protein Atlas (HPA) database (Figure , Supporting Information).

Techniques: Mutagenesis, Expressing, Derivative Assay, Phospho-proteomics, Control, Binding Assay, Western Blot

Journal: Advanced Science

Article Title: Synthetic Lethality of SHP2 and XIAP Suppresses Proliferation and Metastasis in KRAS ‐mutant Nonsmall Cell Lung Cancer

doi: 10.1002/advs.202411642

Figure Lengend Snippet: Embelin inhibits proliferation and metastasis in KRAS ‐mutant NSCLC cells both in vitro and in vivo. A) The effect of embelin on cell viability across a mini‐panel of KRAS ‐mutant and ‐wild‐type cell lines after 24 h was measured using the CCK‐8. Bars, ± SEM. The curves were plotted using a variable slope (four‐parameter) nonlinear fit. B) Cells were treated with embelin for 48 h in 3D culture cell mode, and the intracellular ATP chemiluminescence was detected. *** p < 0.001 compared to the control group (unpaired two‐tailed Student's t ‐test). C) Effects of embelin and SHP099 inducing senescence shown using β‐galactosidase staining in human NCI‐H2122 cells (×40). D) Effects of colony formation of embelin in KRAS wild‐type and mutant cells. E) Statistical graph of cycle ratio of NCI‐H2122 cells after treating with various concentrations of embelin. ** p < 0.01 and *** p < 0.001 compared to the control group (one‐way ANOVA). F) Western blotting of PCNA protein expression after treating with various concentrations of embelin. G) Effects of SHP099 and GDC‐0152 separately or in combination with SHP099 and embelin for 24 h on the cell viability determined using the CCK‐8. *** p < 0.001 compared to the control group (unpaired two‐tailed Student's t ‐test). H) Cells were instantaneously transfected with si PTPN11 and si XIAP and with both for 24 h, and embelin was added for 24 h. Cell proliferation was detected using the CCK8. *** p < 0.001 and **** p < 0.0001 compared to siCtrl group, and ns p > 0.05 compared to relative groups (unpaired two‐tailed Student's t ‐test). I) NCI‐H2122 cells were inoculated into the upper lumen of a Transwell cell (the upper layer of the cell was coated with Matrigel for invasion analysis) and treated with embelin and SHP099 (10 µM) for 24 h. Images of cell migration (above) and invasion (below) were captured using a microscope. J) After treatment with embelin and SHP099 (10 µM) for 24 h, expression of EMT‐labeled protein in NCI‐H2122 cells was detected using IF. K) Western blotting was performed to assess protein expression of EMT markers in NCI‐H2122 cells, including E‐cadherin, N‐cadherin, vimentin, and fibronectin, and to verify mTOR protein expression. The above experiments were conducted with three independent replicates. L, M) Tumors were excised from mice at the termination of the experiment. Representative images of mice with xenograft tumors were captured using a camera (M) and weights were measured (L) ( n = 6, each group). ** p < 0.01 and *** p < 0.001 compared to control group, and ## p < 0.01 compared to GDC‐0152 + SHP099 groups (one‐way ANOVA). N) Tumor volume of BALb/c nude mice with NCI‐H2122 subcutaneous transplantation tumors were measured every 3 days. GraphPad was used to conduct statistical analysis of data ( n = 6, each group). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control group (unpaired two‐tailed Student's t ‐test). O) IHC analyses of Ki67 expression and H&E staining of tumor tissue (×200). ( n = 6, each group).

Article Snippet: Similarly, the protein expression of SHP2 and XIAP in KRAS wild‐type cells was significantly lower compared to KRAS mutant cells, with p ‐values of 0.0430 and 0.0381, respectively, from the Human Protein Atlas (HPA) database (Figure , Supporting Information).

Techniques: Mutagenesis, In Vitro, In Vivo, CCK-8 Assay, Control, Two Tailed Test, Staining, Western Blot, Expressing, Transfection, Migration, Microscopy, Labeling, Transplantation Assay

Journal: Advanced Science

Article Title: Synthetic Lethality of SHP2 and XIAP Suppresses Proliferation and Metastasis in KRAS ‐mutant Nonsmall Cell Lung Cancer

doi: 10.1002/advs.202411642

Figure Lengend Snippet: Embelin induces apoptosis in human KRAS ‐mutant NSCLC cells. A) Volcano map of DEGs ( p adj value < 0.05, |log2foldchange| > 0). B) Scatter map of GO enrichment analysis of apoptosis‐related biological process obtained by cluster analysis of differential genes between control and embelin group. C) NCI‐H2122 cells were treated with embelin and SHP099 for 24 h and apoptotic cells were detected through Annexin‐V‐FITC/PI double staining. Percentages of apoptotic NCI‐H2122 cells are shown. *** p < 0.001 compared to the control group (unpaired two‐tailed Student's t ‐test). The experiments were conducted with three independent replicates. D) NCI‐H2122 cells were treated with embelin and SHP099 (10 µM) for 24 h. The effect of ROS release and fluorescence intensity of NCI‐H2122 cells were detected. Rosup was used as a positive control. The experiments were conducted with three independent replicates. E) Heatmap hierarchical clustering displayed DEGs in the apoptosis biological processes between the control, SHP099, and embelin groups through RNAseq with p adj < 0.05. F) NCI‐H2122 cells were treated with embelin and SHP099 (10 µM) for 24 h and the expressions of apoptosis‐related proteins were measured using western blotting. The experiments were conducted with three independent replicates. G) GSEA of protein insertion into mitochondrial membrane involved in the apoptotic signaling pathway of embelin induced in NCI‐H2122 cells. NES > 1.5, p < 0.05, q < 0.25. H, I) NCI‐H2122 cells were treated with embelin (1, 10, and 15 µM) and SHP099 (10 µM) for 24 h, and MitoSOX (H) and MitoTracker (I) expressions were measured using an IF assay. The experiments were conducted with three independent replicates. (J, K) NCI‐H2122 cells were instantaneously transfected with si PTPN11 and si XIAP for 24 h, individual or both, and ROS release and fluorescence intensity statistics J) and percentage of late apoptosis cells K) and were assesses, with siNC as the control. ** p < 0.01 and *** p < 0.001 compared to siCtrl group. The experiments were conducted with three independent replicates. L) TUNEL and DAPI fluorescence staining of tumor tissue in NCI‐H2122 subcutaneous transplantation tumor mouse model (× 200). ** p < 0.01 compared to control or siCtrl group; *** p < 0.001 compared to control or siCtrl group.

Article Snippet: Similarly, the protein expression of SHP2 and XIAP in KRAS wild‐type cells was significantly lower compared to KRAS mutant cells, with p ‐values of 0.0430 and 0.0381, respectively, from the Human Protein Atlas (HPA) database (Figure , Supporting Information).

Techniques: Mutagenesis, Control, Double Staining, Two Tailed Test, Fluorescence, Positive Control, Western Blot, Membrane, Transfection, TUNEL Assay, Staining, Transplantation Assay

Journal: Advanced Science

Article Title: Synthetic Lethality of SHP2 and XIAP Suppresses Proliferation and Metastasis in KRAS ‐mutant Nonsmall Cell Lung Cancer

doi: 10.1002/advs.202411642

Figure Lengend Snippet: Embelin inhibits cancer‐related signaling pathways in KRAS ‐mutant NSCLC cells. A) Dot map of KEGG enrichment analysis for the top 20 signaling pathways obtained through cluster analysis of DEGs between the control group and the embelin treated group in transcriptomics data. B) Heatmap hierarchical clustering displayed the DEGs in different signaling pathways between the control, SHP099, and embelin‐treated groups using RNAseq with p adj < 0.05. C, D) GSEA of activation of JUN kinase activity (C) and mTOR signaling pathway (D) of embelin induced in NCI‐H2122 cells. NES >1.5, p < 0.05, q < 0.25. E) Experiment validation of protein expression and phosphorylation of ERK/MAPK, p38/MAPK, JNK/MAPK, PI3K/AKT, Wnt, NF‐κB, and JAK/STAT signaling pathways in A549 and NCI‐H2122 cells after 24 h of treatment with various concentrations of embelin and SHP099. F) NCI‐H2122 cells were transfected with si PTPN11 or si XIAP or both for 24 h, and changes in expression and phosphorylation of ERK/MAPK, p38/MAPK, JNK/MAPK, PI3K/AKT, Wnt, NF‐κB, and JAK/STAT signaling pathways were measured using western blotting. All western blot experiments were conducted with three independent replicates.

Article Snippet: Similarly, the protein expression of SHP2 and XIAP in KRAS wild‐type cells was significantly lower compared to KRAS mutant cells, with p ‐values of 0.0430 and 0.0381, respectively, from the Human Protein Atlas (HPA) database (Figure , Supporting Information).

Techniques: Protein-Protein interactions, Mutagenesis, Control, Activation Assay, Activity Assay, Biomarker Discovery, Expressing, Phospho-proteomics, Transfection, Western Blot

Journal: Advanced Science

Article Title: Synthetic Lethality of SHP2 and XIAP Suppresses Proliferation and Metastasis in KRAS ‐mutant Nonsmall Cell Lung Cancer

doi: 10.1002/advs.202411642

Figure Lengend Snippet: Embelin suppresses negative feedback in RAS ‐mutant cells. A) Effects of embelin on SHP2 and ERK expression and phosphorylation in KRAS‐, NRAS‐ , and HRAS‐ mutant cells at different times. B) Effects of SHP099 on SHP2 and ERK expression and phosphorylation in KRAS, NRAS , and HRAS‐ mutant cells at different times. C) Heatmap hierarchical clustering displays DEGs in KEGG pathway of EGFR tyrosine kinase inhibitor resistance between the control, SHP099, and embelin‐treated groups through transcriptomics with p adj < 0.05. D) Effects of embelin and SHP099 on IL‐6 mRNA expression and STAT3 phosphorylation level of NCI‐H2122 cells were assessed through transcriptomics detection and western blotting. (D) Statistical graph of cycle ratio of NCI‐H2122 cells after treating with embelin and SHP099 at 10 µM for 96 h. *** p < 0.001 compared to control group (unpaired two‐tailed Student's t ‐test). E) Statistical graph of cycle ratio of NCI‐H2122 cells after treating with embelin (15 µM) or SHP099(10 µM) for 72 h. *** p < 0.001 compared to control group (unpaired two‐tailed Student's t ‐test). F) Expression changes in MIG‐6 and SPRY2 in NCI‐H2122 and A549 cells after treatment with embelin and SHP099 (10 µM) for 24 h. G) Changes in MIG‐6 and SPRY2 combined with SHP2 in NCI‐H2122 cells treated with embelin (1, 10, and 15 µM) and SHP099 (10 µM) for 12 h. H) Expression changes in MIG‐6 and SPRY2 protein expression assessed using western blotting in NCI‐H2122 and A549 cells after transfection with si PTPN11 and si XIAP separately or together for 24 h. *** p < 0.001 compared to control group. All experiments were conducted with three independent replicates.

Article Snippet: Similarly, the protein expression of SHP2 and XIAP in KRAS wild‐type cells was significantly lower compared to KRAS mutant cells, with p ‐values of 0.0430 and 0.0381, respectively, from the Human Protein Atlas (HPA) database (Figure , Supporting Information).

Techniques: Mutagenesis, Expressing, Phospho-proteomics, Control, Western Blot, Two Tailed Test, Transfection

Journal: International Journal of Biological Sciences

Article Title: Mechanistic role of metal-responsive transcription factor-1 (MTF1) in cadmium-induced prostate carcinogenesis

doi: 10.7150/ijbs.110174

Figure Lengend Snippet: mRNA seq analysis of MTF1shRNA knockdown and CTBPH1 tumors. A . PCA plot showing MTF1_KD and ZIC2_KO CTBPH1 xenograft tumors against control examined through RNA-seq gene expression data. The data is colored according to condition (CTBPH1_control, MTF1_KD, and ZIC2_KO). B . The volcano plot analysis of differentially expressed genes between MTF1shRNA knockdown against shRNA control is plotted on the X axis, and false discovery rate (FDR) significance is plotted on the Y axis (-log10 scale). The grey dots represent no significant change, the red dots represent logFC of >1 and FDR < 0.05, and the blue dots represent logFC < -1 and FDR < 0.05. C&D . Heat map and gene enrichment analysis showed the crucial genes enriched at the WNT signaling and pluripotency, ZINC homeostasis, stem cell differentiation, apoptosis modulation, EMT, and hedgehog signaling pathway. Gene set enrichment analysis (GSEA) of MTF1shRNA knockdown tumor compared to shRNA control tumors

Article Snippet: Western blotting was performed using specific antibodies against MTF1 (ab236401), ZIC2 (ab150404), ZIC2 (immunohistochemistry) (Sigma AU35821), Shh (ab53281), PARP (CST, #9542), NFκB p65 (CST, #8242), Bcl-2 (CST, #15071), Bcl-xL (CST, #2764), X-linked inhibitor of apoptosis protein (XIAP; Cell signaling #2045), ALDH1A1 (D4R9V) (CST, #12035), SOX2 (CST, #3579), NANOG (cell signaling #3608), CD44 (cell signaling #5604), Slug (CST, #9585), E-Cadherin (CST, #3195), β-catenin (CST, #8480), N-cadherin (CST, #13116), or β-Actin (CST, #4970; loading control).

Techniques: Knockdown, Control, RNA Sequencing, Gene Expression, shRNA, Cell Differentiation

Journal: International Journal of Molecular Sciences

Article Title: Modulation of TNFR 1-Triggered Inflammation and Apoptosis Signals by Jacaranone in Cancer Cells

doi: 10.3390/ijms252413670

Figure Lengend Snippet: The degradation of cIAP2 induced by jacaranone played a crucial role in modulating inflammation and apoptosis. ( A ) The expression of cIAP2 protein was reduced by jacaranone treatment. MCF7 cells were treated with jacaranone (10 μM) for 3 h and 6 h, followed by TNFα (20 ng/mL) treatment for 0.5 h prior to sample collection. Western blot analysis was performed to assess the levels of cIAP1, cIAP2, XIAP, RIPK1, TRAF2, and IκBα. GAPDH was used as a loading control. ( B , C ) The expression of cIAP2 protein was reduced by jacaranone treatment. MDA-MB-231 ( B ) and HeLa ( C ) cells were treated with jacaranone (10 μM) for 3 h and 6 h, followed by TNF-α (20 ng/mL) treatment for 0.5 h prior to sample collection. Western blot analysis was performed to assess the levels of cIAP1 and cIAP2. GAPDH was used as a loading control. ( D ) The impact of jacaranone on cIAP2 mRNA levels was investigated in MCF7 cells. Cells were treated with jacaranone (10 μM) for 3 h, followed by TNFα (20 ng/mL) treatment for 0.5 h. RT-PCR was employed to assess the cellular mRNA levels. ( E ) The downregulation of cIAP2 expression induced by jacaranone can be effectively inhibited by MG132 treatment. MCF7 cells were pre-treated with 10 μM MG132 for 1 h prior to the administration of 10 μM jacaranone for 6 h. TNFα (20 ng/mL) was added 0.5 h before sample collection, and cIAP2 expression levels were assessed using immunoblotting techniques, with GAPDH and β-tubulin used as a reference for sample normalization. ( F ) The degradation of IκBα induced by TNFα is inhibited by jacaranone in a cIAP2-dependent manner. MCF7 cells were transfected with si-cIAP2. After 48 h of transfection, the cells were treated with jacaranone (10 μM) for 3 h and then stimulated with TNFα (20 ng/mL) for 0.5 h before sample collection. Western blotting was performed to analyze the levels of cIAP2 and IκBα, and GAPDH was used as a loading control. ns: no significance, ** p < 0.01, *** p < 0.001, **** p < 0.0001. All blots above are representative of one of three experiments.

Article Snippet: The western blot analysis revealed the expression levels of IKBα (cell signaling technology, #4812) (Danvers, MA, USA), p-IKKα/β (cell signaling technology, #2697) (Danvers, MA, USA), caspase-8 (cell signaling technology, #4790) (Danvers, MA, USA), cleaved-caspase-8 (cell signaling technology, #98134 and #9496) (Danvers, MA, USA), cIAP1 (cell signaling technology, #7065) (Danvers, MA, USA), cIAP2 (cell signaling technology, #3130) (Danvers, MA, USA), XIAP (cell signaling technology, #2042) (Danvers, MA, USA), RIPK1 (BD Biosciences, 551042) (Franklin Lakes, NJ, USA), TRAF2 (cell signaling technology, #4724), TNFR1 (Santa Cruz, sc-374185) (Santa Cruz, CA, USA), and FADD (cell signaling technology, #2782) (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Transfection