epr28633  (Cell Signaling Technology Inc)

Journal: MedComm - Oncology

Article Title: X-Linked Inhibitor of Apoptosis Protein (XIAP) Contributes to ERK1/2-Mediated Anoikis Resistance in Hepatocellular Carcinoma

doi: 10.1002/mog2.70059

Figure Lengend Snippet: HCC cells exhibit a higher resistance to anoikis compared to normal hepatocytes. THLE-2, PLC/PRF/5, and HCCLM3 were cultured in normal plates to allow attachment for 24 h (for MTS assay, trypan blue exclusion test, and Annexin V-PE/7-AAD staining) or 12 h (for Western blotting), or transferred to ultra-low-attachment plates to induce detachment for 24 h or 12 h. (A) Representative pictures demonstrating XIAP staining in NL, CL, and HCC in situ/MVTT by IHC (scale bar: 200 μm). (B) The representative morphological images of cells cultured in detachment condition for 12 h (scale bar: 200 μm). (C) The relative number of aggregated colonies of cells in each field was counted after detachment. (D) The proportion of viable cells was quantified by MTS assay. (E) The relative number of live cells was determined by trypan blue exclusion test. (F) Western blot analysis of indicated proteins in cells. β-actin was probed as a loading control. (G) The relative density of XIAP, cleaved PARP, or cleaved caspase-3 was normalized against that of β-actin, with semi-quantitative analysis performed using NIH ImageJ software. (H) The proportions of viable (LL), dead (UL), early apoptotic (LR), and late apoptotic (UR) cells were quantified by FACS utilizing Annexin V-PE/7-AAD staining. (I, J) Quantitative analysis of live and apoptotic cells by FACS assay. All data were presented as mean ± SE, n = 3–5. a p < 0.05, difference versus attached group; b p < 0.05, difference versus detached THLE-2 cells group.

Article Snippet: To validate the relationship between XIAP and ERK1/2 at the protein level, the IHC images of XIAP and ERK1/2 in HCC tissues and normal liver tissues were obtained from the Human Protein Atlas.

Techniques: Cell Culture, MTS Assay, Staining, Western Blot, In Situ, Control, Software

Journal: MedComm - Oncology

Article Title: X-Linked Inhibitor of Apoptosis Protein (XIAP) Contributes to ERK1/2-Mediated Anoikis Resistance in Hepatocellular Carcinoma

doi: 10.1002/mog2.70059

Figure Lengend Snippet: Inhibition of ERK1/2 by U0126 attenuates XIAP-evoked anoikis resistance of HCC cells. HCCLM3 and PLC/PRF/5 cells or XIAP-overexpressing HCCLM3 and PLC/PRF/5 cells, pretreated with/without U0126 (5 μM) at indicated concentrations for 1 h, and then detached for 12 h (for Western blotting), 24 h (for trypan blue exclusion assay and DAPI staining), or 2 weeks (soft agar colony formation assay). (A) Western blot analysis of indicated proteins in cells. β-actin was probed as a loading control. (B) The relative densities for p-ERK1/2 (Thr202/Tyr204) and XIAP to β-actin were subjected to semi-quantitative analysis utilizing NIH ImageJ. (C) The relative number of live cells was determined by trypan blue exclusion test. (D) The cell proliferation was estimated by the soft agar colony formation assay. (E) The percentage of cells with fragmented nuclei was quantified by DAPI staining. All data were presented as mean ± SE, n = 3–5. a p < 0.05, + U0126 group versus − U0126 group; b p < 0.05, FLAG-XIAP group versus EGFP-control group.

Article Snippet: To validate the relationship between XIAP and ERK1/2 at the protein level, the IHC images of XIAP and ERK1/2 in HCC tissues and normal liver tissues were obtained from the Human Protein Atlas.

Techniques: Inhibition, Western Blot, Trypan Blue Exclusion Assay, Staining, Soft Agar Assay, Control

Journal: MedComm - Oncology

Article Title: X-Linked Inhibitor of Apoptosis Protein (XIAP) Contributes to ERK1/2-Mediated Anoikis Resistance in Hepatocellular Carcinoma

doi: 10.1002/mog2.70059

Figure Lengend Snippet: Overexpression of mutant XIAP (S87D) suppresses ERK1/2 deficiency-induced anoikis of HCC cells. HCCLM3 and PLC/PRF/5 cells or mutant XIAP (S87D)-overexpressing PLC/PRF/5 and HCCLM3 cells, infected with lentiviral shRNA to ERK1/2 or GFP (as control), respectively, were detached for 12 h (for Western blotting), 24 h (for trypan blue exclusion assay and DAPI staining), or 2 weeks (soft agar colony formation assay). (A) Western blot analysis of indicated proteins in cells. β-actin was probed as a loading control. (B) The relative densities for p- ERK1/2 (Thr202/Tyr204) and p-XIAP (Ser87) to β-actin were subjected to semi-quantitative analysis utilizing NIH ImageJ. (C) The relative number of live cells was evaluated by trypan blue exclusion test. (D) The cell proliferation was estimated by the soft agar colony formation assay. (E) The percentage of cells with fragmented nuclei was quantified by DAPI staining. All data were presented as mean ± SE, n = 3–5. a p < 0.05, shRNA-ERK1/2 group versus shRNA-GFP group; b p < 0.05, FLAG-XIAP (S87D) group versus EGFP-control group.

Article Snippet: To validate the relationship between XIAP and ERK1/2 at the protein level, the IHC images of XIAP and ERK1/2 in HCC tissues and normal liver tissues were obtained from the Human Protein Atlas.

Techniques: Over Expression, Mutagenesis, Infection, shRNA, Control, Western Blot, Trypan Blue Exclusion Assay, Staining, Soft Agar Assay

Journal: MedComm - Oncology

Article Title: X-Linked Inhibitor of Apoptosis Protein (XIAP) Contributes to ERK1/2-Mediated Anoikis Resistance in Hepatocellular Carcinoma

doi: 10.1002/mog2.70059

Figure Lengend Snippet: ERK1/2-mediated expression of XIAP controls anoikis resistance and intrahepatic metastasis in vivo . HCCLM3 cells, XIAP-deficient HCCLM3 cells, ERK1/2-deficient HCCLM3 cells, and ERK1/2-deficient HCCLM3 cells infected with lentiviral FLAG-XIAP (S87D), FLAG-XIAP (H467A), or FLAG-XIAP (D148A/W3110A), respectively, were intraperitoneally injected into BALB/c nude mice (for peritoneal cavities model) or intrahepatically injected into BALB/c nude mice (for hepatic orthotopic transplantation metastatic model). (A) The representative photographs of mouse peritoneal cavity model from the indicated groups obtained 7 days after intraperitoneal injection (scale bar: 10 mm). (B) HCCLM3 cells were collected from ascites, and the live cells were evaluated by trypan blue exclusion test. (C) The procedure for orthotopic implantation of indicated HCCLM3 cells into the liver parenchyma of mouse (scale bar: 10 mm). (D) The typical pictures of liver tissues and H&E staining in the different groups 4 weeks after orthotopic implantation with indicated HCCLM3 cells. The arrows indicate visible intrahepatic metastatic tumors. (E) The intrahepatic metastatic nodules from mouse orthotopic implantation model were quantified. All data were presented as mean ± SE, n = 3–5. a p < 0.05, difference versus control group; b p < 0.05, difference versus shRNA-ERK1/2 group.

Article Snippet: To validate the relationship between XIAP and ERK1/2 at the protein level, the IHC images of XIAP and ERK1/2 in HCC tissues and normal liver tissues were obtained from the Human Protein Atlas.

Techniques: Expressing, In Vivo, Infection, Injection, Transplantation Assay, Staining, Control, shRNA

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