Journal: The Journal of Clinical Investigation

Article Title: ANKRD55 is a key regulator of T cell inflammation in multiple sclerosis

doi: 10.1172/JCI195214

Figure Lengend Snippet: ( A ) Overexpression of ANKRD55 and TCP1 in HEK293T cells followed by co-IP analysis to determine their interaction. ( B ) Protein extraction from Jurkat cells with subsequent co-IP assay to determine the interaction between ANKRD55 and TCP1. ( C ) PLA experiment in Jurkat cells to assess the interaction between ANKRD55 and TCP1. Cells were fixed and incubated with primary antibodies against ANKRD55 and TCP1, followed by PLA probe ligation and amplification. Red fluorescent puncta indicate close proximity (<40 nm) between ANKRD55 and TCP1, suggesting a direct or complex-mediated interaction. Nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bars: 2 μm (top), 5 μm (bottom). ( D ) Immunofluorescent staining of Jurkat cells to analyze the colocalization of ANKRD55 and TCP1. Scale bars: 2 μm. ( E – K ) Co-IP assays were performed using HEK293T cells to investigate the interactions between ANKRD55 and individual CCT subunits, including CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, and CCT8. ( L ) Immunofluorescent staining of Jurkat cells to assess colocalization between ANKRD55 and specific CCT subunits (CCT2, CCT3, CCT4, and CCT7). Scale bars: 2 μm (first 4 panels), 1 μm (fifth panel). ( M ) Immunofluorescent staining of Jurkat cells to visualize the subcellular localization of ANKRD55, TCP1, and pericentrin. Scale bars: 5 μm. ( N – P ) Immunoblotting analysis of TCP1 ( N ), CCT3 ( O ), and CCT6 ( P ) expression levels in Jurkat cells following ANKRD55 knockdown.

Article Snippet: TCP1 antibody (catalog 10320 and 68183), CCT2 antibody (catalog 68214), CCT3 antibody (catalog 60264), CCT4 antibody (catalog 67455), CCT5 antibody (catalog 67400), CCT6 antibody (catalog 19793), CCT7 antibody (catalog 68214), DYKDDDDK tag polyclonal antibody (catalog 20543), HA tag polyclonal antibody (catalog 51064), phospho-ERK1/2 (Thr202/Tyr204) polyclonal antibody (catalog 28733), CD247 polyclonal antibody (catalog 12837), and phospho-LCK-Y394 rabbit antibody (catalog AP0182) were purchased from Proteintech.

Techniques: Over Expression, Co-Immunoprecipitation Assay, Protein Extraction, Incubation, Ligation, Amplification, Staining, Western Blot, Expressing, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: ANKRD55 is a key regulator of T cell inflammation in multiple sclerosis

doi: 10.1172/JCI195214

Figure Lengend Snippet: ( A and B ) α-Tubulin immunoblotting in lysates and pellet determined by microtubule sedimentation assay in Jurkat cells with TCP1 ( A ) or ANKRD55 ( B ) knocked down. ( C ) TCP1 degradation rate analyzed via immunoblot after cycloheximide (CHX; 70 μM) treatment for 0–48 hours in control and Jurkat cells overexpressing ANKRD55. ( D – F ) Co-IP detection of interactions between CCT5 and TCP1 ( D ), CCT3 ( E ), or CCT6 ( F ) at varying concentrations of ANKRD55 in HEK293T. ( G ) Immunofluorescence analysis of immune synapse formation between Jurkat and Raji cells. Jurkat cells were prelabeled with CMAC. Raji cells were stimulated with SEE for 30 minutes. The 2 cell types were then cocultured for 30 minutes. Cells were stained with antibodies against ANKRD55, TCP1, pericentrin, and α-tubulin to visualize protein localization at the immune synapse. Scale bars: 2 μm. BF, bright-field; CMAC, CellTracker blue fluorescent probe. ( H ) Flow cytometry–based immune synapse (IS) pattern analysis. ( I and J ) Raji cells (APCs) stained with CFSE and stimulated with SEE for 30 minutes at 37°C and Jurkat cells (T cells) stained with CMTPX. T cell conjugation with APCs after 20 minutes of contact was analyzed by flow cytometry. Conjugate percentages were determined for Jurkat cells with ANKRD55 or TCP1 knocked down ( I ) and pretreatment with HSF1A (50 μM) for 2 hours ( J ). ( K ) Mean clinical score of EAE in mice injected intraperitoneally with PBS or HSF1A (20 mg/mL) ( n = 7 or 8 mice per group), induced by active immunization with MOG 35–55 . ( L ) Immunoblot analysis of TCR signaling in Jurkat cells. Cells included vector control, a stable ANKRD55-overexpressing cell line, and ANKRD55-overexpressing cells pretreated with HSF1A (50 μM, 2 hours). All groups were stimulated on plates coated with anti-CD3 and anti-CD28. Lysates were collected at the indicated time points (1, 2, 15, and 30 minutes) and probed for TCR signaling–associated proteins. ( M ) H&E and Luxol fast blue (LFB) staining of spinal cord sections at the peak of EAE disease. Arrows indicate areas of demyelination. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, by 2-way ANOVA with Tukey’s multiple-comparison test. Data are shown as mean ± SEM.

Article Snippet: TCP1 antibody (catalog 10320 and 68183), CCT2 antibody (catalog 68214), CCT3 antibody (catalog 60264), CCT4 antibody (catalog 67455), CCT5 antibody (catalog 67400), CCT6 antibody (catalog 19793), CCT7 antibody (catalog 68214), DYKDDDDK tag polyclonal antibody (catalog 20543), HA tag polyclonal antibody (catalog 51064), phospho-ERK1/2 (Thr202/Tyr204) polyclonal antibody (catalog 28733), CD247 polyclonal antibody (catalog 12837), and phospho-LCK-Y394 rabbit antibody (catalog AP0182) were purchased from Proteintech.

Techniques: Western Blot, Microtubule Sedimentation Assay, Control, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Flow Cytometry, Conjugation Assay, Injection, Plasmid Preparation, Comparison

Journal: Cell Death & Disease

Article Title: Tumor-derived exosomal CCT6A serves as a matchmaker introducing chemokines to tumor-associated macrophages in pancreatic ductal adenocarcinoma

doi: 10.1038/s41419-025-07720-y

Figure Lengend Snippet: A Schematic diagram of experimental design assessing the potential PDAC-derived M2 TAM-induced exosomal proteins using proteomic detection and public datasets, which identifying CCT6A, CAP1, CAPZA1 and RHOA as the promising contributors. Created with BioRender.com. B IHC analysis of CCT6A and IF staining of CD68 (green) and CD163 (red) in tumor tissue sections from 33 PDAC patients. Scale bar, 100 μm. C Scatter diagram of the tumoral CCT6A expression levels and M2 macrophage infiltration scores in 33 PDAC patients. Simple linear regression and the Pearson correlation coefficient analysis. D Size distribution of human PDAC-derived exosomal particles, as measured by NTA. Blue represents exosomes derived from serum, and red represents exosomes derived from tumor. E Analysis of the expression of specific exosomal biomarkers and CCT6A in exosomes isolated from serum (left) and tumor (right) via western blotting. F Immunoelectron microscopy analysis of CCT6A in exosomes isolated from serum (left) and tumor (right). Scale bar, 50 nm. G Comparison of serum-originated exosomal CCT6A levels by western blotting. ‘P’ represents exosomes derived from serum of PDAC patients, while ‘N’ represents exosomes derived from serum of healthy individuals. ( n = 5). n.s. no significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: For immunogold labeling, exosomes in Saline at a concentration of 1 × 10 9 particles/mL were placed onto glow-discharged copper grids, and the grids were then blocked and incubated with a rabbit anti-human polyclonal antibody (CCT6A, Proteintech Group, 19793-1-AP) that specifically recognizes the CCT6A protein.

Techniques: Derivative Assay, Staining, Expressing, Isolation, Western Blot, Immuno-Electron Microscopy, Comparison

Journal: Cell Death & Disease

Article Title: Tumor-derived exosomal CCT6A serves as a matchmaker introducing chemokines to tumor-associated macrophages in pancreatic ductal adenocarcinoma

doi: 10.1038/s41419-025-07720-y

Figure Lengend Snippet: A Exosomes at different concentrations were directly added to the macrophage culture medium, resulting in final exosome concentrations of 1 × 10¹⁰ particles/mL and 1 × 10¹¹ particles/mL. Analysis of CCT6A levels in PDAC-derived exosomes-treated macrophages through western blotting (left). Quantification (right) of CCT6A/GAPDH level in macrophages treated with the exosomes from CCT6A-silenced cells (Exo sh-CCT6A ) versus from control cells (Exo sh-NC ). ( n = 3). Two-way ANOVA analysis. B RT-qPCR of M2 and M1 markers in macrophages treated with an equal volume of saline without exosomes (Control), AsPC-1-Exo sh-NC or AsPC-1-Exo sh-CCT6A . ( n = 3). Two-way ANOVA analysis. C ELISA analysis for IL-10, TGF-β1, and TNF-α in macrophages treated with different AsPC-1-Exo groups. ( n = 4). Two-way ANOVA analysis. D RT-qPCR of M2 and M1 markers in macrophages treated with Control, BxPC-3-Exo sh-NC or BxPC-3-Exo sh-CCT6A . n = 4. Two-way ANOVA analysis. E ELISA analysis for IL-10, TGF-β1, and TNF-α in macrophages treated with different BxPC-3-Exo groups. ( n = 3). Two-way ANOVA analysis. F Flow cytometry analysis of M2-type (CD11b + CD163 + ) macrophages’ distribution after exosomes treatment (left). Quantification (right) of M2-type macrophages. ( n = 3). One-way ANOVA analysis. RT-qPCR of M2 and M1 markers in macrophages treated with CCT6A-expressing plasmid ( G ) or CCT6A shRNA-expressing ( H ). ( n = 4). Two-way ANOVA analysis. Data presented as mean ± SD. n.s., no significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: For immunogold labeling, exosomes in Saline at a concentration of 1 × 10 9 particles/mL were placed onto glow-discharged copper grids, and the grids were then blocked and incubated with a rabbit anti-human polyclonal antibody (CCT6A, Proteintech Group, 19793-1-AP) that specifically recognizes the CCT6A protein.

Techniques: Derivative Assay, Western Blot, Control, Quantitative RT-PCR, Saline, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Plasmid Preparation, shRNA

Journal: Cell Death & Disease

Article Title: Tumor-derived exosomal CCT6A serves as a matchmaker introducing chemokines to tumor-associated macrophages in pancreatic ductal adenocarcinoma

doi: 10.1038/s41419-025-07720-y

Figure Lengend Snippet: A KEGG enrichment analysis of DEGs between Exo sh-NC and Exo sh-CCT6A group. B Detection of PI3K-AKT signaling in Control (an equal volume of saline without exosomes), Exo sh-NC , and Exo sh-CCT6A treated macrophages via western blotting (left). Quantification (right) of p-PI3K/PI3K and p-AKT/AKT levels. ( n = 3). Two-way ANOVA analysis. C Detection of PI3K-AKT signaling in Control, BxPC-3-Exo from control cells (BxPC-3-Exo OE-NC ), BxPC-3-Exo isolated from CCT6A-overexpressing cells (BxPC-3-Exo OE-CCT6A ), or BxPC-3-Exo OE-CCT6A with PI3K inhibitor LY294002 administration (BxPC-3-Exo OE-CCT6A + LY294002) treated macrophages via western blotting (left). Quantification (right) of p-PI3K/PI3K and p-AKT/AKT levels. ( n = 3). Two-way ANOVA analysis. D RT-qPCR of M2 and M1 markers in macrophages treated with different BxPC-3-Exo groups. ( n = 3). Two-way ANOVA analysis. E Representative images of IF staining for CD68 (red) and CD163 (green) in macrophages treated with different BxPC-3-Exo groups. Scale bar, 20 μm. F Flow cytometry analysis for distribution of M2-type (CD11b + CD163 + ) and M1-type (CD11b + CD86 + ) macrophages after different BxPC-3-Exo treatments (left). Quantification (right) of M2-type and M1-type macrophages. n = 3. One-way ANOVA analysis. Data presented as mean ± SD. n.s. no significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: For immunogold labeling, exosomes in Saline at a concentration of 1 × 10 9 particles/mL were placed onto glow-discharged copper grids, and the grids were then blocked and incubated with a rabbit anti-human polyclonal antibody (CCT6A, Proteintech Group, 19793-1-AP) that specifically recognizes the CCT6A protein.

Techniques: Control, Saline, Western Blot, Isolation, Quantitative RT-PCR, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Tumor-derived exosomal CCT6A serves as a matchmaker introducing chemokines to tumor-associated macrophages in pancreatic ductal adenocarcinoma

doi: 10.1038/s41419-025-07720-y

Figure Lengend Snippet: A Volcano plot of DEPs between Exo sh-NC and Exo sh-CCT6A groups. B KEGG enrichment analysis of DEPs between Exo sh-NC and Exo sh-CCT6A group. C Validation of the exosomal chemokines knockdown efficacy in AsPC-1-Exo (AsPC-1-Exo sh-Chemo ) via western blotting. D Detection of PI3K-AKT signaling in Control (an equal volume of saline without exosomes), AsPC-1-Exo sh-NC , AsPC-1-Exo sh-Chemo , AsPC-1-Exo OE-CCT6A , or exosomes isolated from AsPC-1 cells with downregulated chemokines and overexpressed CCT6A (AsPC-1-Exo sh-Chemo+OE-CCT6A ) treated macrophages via western blotting (left). Quantification (right) of p-PI3K/PI3K and p-AKT/AKT levels. ( n = 3). Two-way ANOVA analysis. E Flow cytometry analysis for distribution of M2-type (CD11b + CD163 + ) and M1-type (CD11b + CD86 + ) macrophages after different AsPC-1-Exo treatments (left). Quantification (right) of M2-type and M1-type macrophages. ( n = 3). One-way ANOVA analysis. F RT-qPCR of M2 and M1 markers in macrophages treated with different AsPC-1-Exo groups. ( n = 3). Two-way ANOVA analysis. Data presented as mean ± SD. n.s. no significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: For immunogold labeling, exosomes in Saline at a concentration of 1 × 10 9 particles/mL were placed onto glow-discharged copper grids, and the grids were then blocked and incubated with a rabbit anti-human polyclonal antibody (CCT6A, Proteintech Group, 19793-1-AP) that specifically recognizes the CCT6A protein.

Techniques: Biomarker Discovery, Knockdown, Western Blot, Control, Saline, Isolation, Flow Cytometry, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Tumor-derived exosomal CCT6A serves as a matchmaker introducing chemokines to tumor-associated macrophages in pancreatic ductal adenocarcinoma

doi: 10.1038/s41419-025-07720-y

Figure Lengend Snippet: A Schematic diagram of experimental design utilizing interactome analysis to identify proteins potentially binding to CCT6A. Created with BioRender.com. B Detection of endogenous interaction between CCT6A and chemokines in AsPC-1-Exo (left) and AsPC-1 cells (right) via Co-IP and western blotting. C Representative Confocal micrographs of CCT6A (green) and chemokines (red) in AsPC-1 cells. Scale bar, 10 μm. D GST pulldown assays performed by western blotting or Coomassie Brilliant Blue (CBB) staining to detect direct binding of CCT6A and chemokines in vitro. E Molecular docking and HDOCK scores of CCT6A with four chemokines.

Article Snippet: For immunogold labeling, exosomes in Saline at a concentration of 1 × 10 9 particles/mL were placed onto glow-discharged copper grids, and the grids were then blocked and incubated with a rabbit anti-human polyclonal antibody (CCT6A, Proteintech Group, 19793-1-AP) that specifically recognizes the CCT6A protein.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Staining, In Vitro

Journal: Cell Death & Disease

Article Title: Tumor-derived exosomal CCT6A serves as a matchmaker introducing chemokines to tumor-associated macrophages in pancreatic ductal adenocarcinoma

doi: 10.1038/s41419-025-07720-y

Figure Lengend Snippet: A Schematic diagram showing the tail vein injection of anti-CD47 nanobody into mice after subcutaneous injection of CCT6A-silenced or control KPC cells. ( n = 6). Created with BioRender.com. B Tumor volume curve. ( n = 6). Black: subcutaneous injection of control KPC cells and tail vein injection of saline (sh-NC + Saline), red: subcutaneous injection of control KPC cells and tail vein injection of anti-CD47 nanobody (sh-NC + anti-CD47), blue: subcutaneous injection of CCT6A-silenced KPC cells and tail vein injection of saline (sh-CCT6A + Saline), and green: subcutaneous injection of CCT6A-silenced KPC cells and tail vein injection of anti-CD47 nanobody (sh-CCT6A + anti-CD47). ( n = 6). General images ( C ) and tumor weights ( D ) of subcutaneous tumors. ( n = 6). E IHC analysis of CCT6A and tyramide signal amplification (TSA) -multiplex IF (mIF) staining (left) of F4/80 (green), CD163 (red) and iNOS (yellow) in the tumor sections. Scale bar, 20 μm. M2 (F4/80 + CD163 + ) and M1 (F4/80 + iNOS + ) -positive cell count statistics (right) of the tumor sections. ( n = 3). F Graphical abstract. Created with BioRender.com. Data presented as mean ± SD. n.s. no significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. One-way ANOVA analysis.

Article Snippet: For immunogold labeling, exosomes in Saline at a concentration of 1 × 10 9 particles/mL were placed onto glow-discharged copper grids, and the grids were then blocked and incubated with a rabbit anti-human polyclonal antibody (CCT6A, Proteintech Group, 19793-1-AP) that specifically recognizes the CCT6A protein.

Techniques: Injection, Control, Saline, Amplification, Multiplex Assay, Staining, Cell Counting