Journal: The Journal of Biological Chemistry

Article Title: Hsp90 co-chaperone FKBP4 facilitates CCT8 folding and connects Hsp90 to chaperonin-dependent proteostasis

doi: 10.1016/j.jbc.2025.110914

Figure Lengend Snippet: FKBP4 prevents CCT8 aggregation. A , following the knockdown of FKBP4 in HCT116 cells for 72 h, the aggregation of CCT1 and CCT8 proteins within cells was assessed using a filter trap assay. Cell lysates were filtered through a nitrocellulose membrane, and the retained proteins on the membrane were detected using specific antibodies against CCT1 and CCT8. B , quantitative analysis of CCT1 and CCT8 protein aggregation, normalized to GAPDH, in shFKBP4 HCT116 cells. C , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of FKBP4, CCT8, CCT1, CDK2, α-tubulin, and GAPDH within the cells. D , quantitative analysis of CCT8 and CCT1 protein expression, normalized to GAPDH, in shFKBP4 HCT116 cells. E , following the knockdown of FKBP4 in HCT116 cells for 72 h, the aggregation of CCT2 and CCT3 proteins within cells was assessed using a filter trap assay. The experimental procedure mirrored that of ( A ). F , quantitative analysis of CCT2 and CCT3 protein aggregation, normalized to GAPDH, in shFKBP4 HCT116 cells. G , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of FKBP4, CCT2, CCT3, and GAPDH within the cells. The experimental procedure mirrored that of ( C ). H , quantitative analysis of CCT2 and CCT3 protein expression, normalized to GAPDH, in shFKBP4 HCT116 cells. I , shFKBP4 HCT116 cells were harvested after 3 days of puromycin selection. The cell lysates were biochemically fractionated into Triton-soluble and -insoluble fractions as described in “ ”. The FKBP4 expression levels and Triton-soluble and -insoluble fractions of CCT8 were analyzed by western blotting. The GAPDH was used as a loading control. J , The ratio of the CCT8 Triton-insoluble form in ( I ). K , The primary structures of FKBP4 and FKBP5. L , after 48 h of transfection, vector, FLAG-tagged FKBP4 or FLAG-tagged FKBP5 expressing HEK293T cell lysates were precipitated by anti-FLAG antibodies, and the products were detected for the co-purification of the endogenous proteins. M , comparison of the interaction between Hsp90 and FLAG-tagged FKBP4 or FLAG-tagged FKBP5 in ( L ). N , following the knockdown of FKBP5 in HCT116 cells for 72 h, the aggregation of CCT8 proteins within cells was assessed using a filter trap assay. The experimental procedure mirrored that of ( A ). O , quantitative analysis of the protein aggregation of CCT8, normalized to GAPDH, in shFKBP5 HCT116 cells. P , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of FKBP5, CCT8, and GAPDH within the cells. The experimental procedure mirrored that of ( C ). Q , quantitative analysis of CCT8 protein expression, normalized to GAPDH, in shFKBP5 HCT116 cells. R , shFKBP5 HCT116 cells were harvested after puromycin selection for 3 days. The experimental procedure mirrored that of ( I ). S , the ratio of the CCT8 Triton-insoluble form in ( R ). T , HCT116 cells were treated with 17-AAG (20 μM) or ganetespib (2 μM) for 24 h, and CCT8 aggregation was assessed by filter trap assay. U , quantitative analysis of CCT8 protein aggregation with GAPDH serving as an internal control in ( T ). V , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of HSPA1A, CCT8, HSP90, and GAPDH within the cells. The experimental procedure mirrored that of ( C ). W , quantitative analysis of CCT8 protein expression was normalized with GAPDH in ( V ). All data were analyzed with an unpaired two-tailed Student's t test. Each dataset is expressed as mean ± SD for n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Primary antibodies were used to detect FKBP4 (1:1000, ab97306, Abcam), GAPDH (1:1000, GTX100118, GeneTex, USA), E-cadherin (1:1000, 610182, BD), FASN (1:1000, GTX109833, GeneTex), FLNA (1:1000, GTX112939, GeneTex), TALIN-1 (1:500, GTX102215, GeneTex), MRE11 (1:1000, PC388, Calbiochem), DDX3 (1:1000, GTX110614, GeneTex), TCP1 theta (CCT8) (1:1000, GTX105725, GeneTex), CDK2 (1:500, 2546, Cell Signaling), CCT1 (1:250, sc-53454, Santa-cruz Biotechnology), HSP90 (1:1000, sc-13119, Santa-cruz Biotechnology), HSPA1A (1:1000, sc-66048, Santa-cruz Biotechnology), FKBP5 (1:1000, GTX113438, GeneTex), α-tubulin (1:1000, MCA78G, Bio-Rad Laboratories Inc), FLAG (1:2000, F3165, Sigma-Aldrich), c-Myc (1:1000, #11667203001, Roche), CCT2 (1:500, sc-374152, Santa-cruz Biotechnology), CCT3 (1:500, sc-271336, Santa-cruz Biotechnology), HA (1:5000, MMS-101R, Covance), LexA (1:1000, sc-7544, Santa-cruz Biotechnology), and Pgk1 (1:10000, 459250, Invitrogen).

Techniques: Knockdown, TRAP Assay, Membrane, Western Blot, Expressing, Selection, Control, Transfection, Plasmid Preparation, Copurification, Comparison, Two Tailed Test

Journal: iScience

Article Title: CCT3-mediated regulation of XPO1/RB1 axis stability promotes cellular senescence and tumor progression in clear cell renal carcinoma

doi: 10.1016/j.isci.2026.114840

Figure Lengend Snippet: CCT3 expression is associated with unfavorable clinical outcomes in clear cell renal carcinoma (ccRCC) (A) Bar graph depicts the protein expression levels of CCT3 in selected cancer types and their respective normal tissues. Data were obtained from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) unpaired sample analysis. (B) Upregulated expression of CCT3 in clear cell renal carcinoma (ccRCC) compared to normal kidney tissues, as determined by the UALCAN online database analysis. (C) Analysis of the impact of CCT3 expression on the p53/Rb pathway in ccRCC, derived from the CPTAC database. (D) Correlation analysis between CCT3 mRNA expression levels and the mRNA expression levels of key genes involved in the p53/Rb pathway (TP53, Rb1, CDK4, and E2F3) using Spearman correlation, as analyzed by GEPIA. (E and F) Kaplan-Meier survival curves illustrating the association between CCT3 expression and overall survival (OS) and progression-free survival (PFS) in patients with ccRCC. Patients were stratified into low and high CCT3 expression groups. Statistical significance is indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗ p < 0.001, and ns for non-significant differences. Data in A are presented as mean ± standard deviation (SD). Two-tailed Student’s t test.

Article Snippet: CCT3 , Proteintech , Cat#10571-1-AP; RRID: AB_2073658.

Techniques: Expressing, Derivative Assay, Standard Deviation, Two Tailed Test

Journal: iScience

Article Title: CCT3-mediated regulation of XPO1/RB1 axis stability promotes cellular senescence and tumor progression in clear cell renal carcinoma

doi: 10.1016/j.isci.2026.114840

Figure Lengend Snippet: The depletion of CCT3 impairs the abilities of proliferation, migration, and invasion in ccRCC cells (A) Western blot analysis was performed to assess the protein expression of CCT3 in four pairs of clear cell renal cell carcinoma tissues (T) and their corresponding adjacent normal tissues (N). ( n = 4). (B) The immunohistochemical results of CCT3 in ccRCC and normal renal tissue in the HPA database. (C) Western blot was used to assess the effects of two distinct CCT3 knockdown sequences in 786-O and 769-P cells. (D and E) Wound-healing assay indicates that CCT3 knockdown suppresses the viability of 786-O and 769-P cells. (F and G) Transwell assay indicates impaired abilities of migration and invasion of 769-P and 786-O cells. (H and I) The proliferative abilities of stably CCT3-depleted 769-P and 786-O cells were measured with an EdU staining assay. (Scale bars, 100 μm. Data in C-I are presented as the means ± SDs ( n = 3 per group), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). Two-tailed Student’s t test for A, Student’s t test for C-I.

Article Snippet: CCT3 , Proteintech , Cat#10571-1-AP; RRID: AB_2073658.

Techniques: Migration, Western Blot, Expressing, Immunohistochemical staining, Knockdown, Wound Healing Assay, Transwell Assay, Stable Transfection, Staining, Two Tailed Test

Journal: iScience

Article Title: CCT3-mediated regulation of XPO1/RB1 axis stability promotes cellular senescence and tumor progression in clear cell renal carcinoma

doi: 10.1016/j.isci.2026.114840

Figure Lengend Snippet: The reduction of CCT3 induces cellular senescence in ccRCC cells (A) Western blot analysis was performed to assess the protein expression of CDK4, CyclinD, p-Rb (Ser807/811), and p21 in CCT3 knockdown cells. (B) The mRNA expression levels of senescence-associated genes, including interleukin 1 A, TNF, CDK2, CDK4, and CDK6 in CCT3-depleted cells were examined by qRT-PCR. (C) Changes of SA-β-gal activity in CCT3-knockdown cells. Data represent the percentage of cells staining positive for SA-β-gal ±SD. (D) Cell cycle analysis calculated the distribution of the cells in G1, S, and G2/M phases. (E) Representative immunofluorescent staining of p21 and p-Rb (Ser807/811) in each cell. (F) Western blot analysis was performed to assess the protein expression of CDK4, CyclinD, p-Rb (Ser807/811), and p21 in CCT3 overexpressed cells. (G) Wound-healing assay indicates that the overexpression of CCT3 enhances the proliferation capacity of A498 cells. (H) The Transwell assay demonstrated that the overexpression of CCT3 enhances the migratory and invasive capabilities of A498 cells. (Scale bars, 100 μm, all data were shown as the mean ± SD ( n = 3 per group), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001). Student’s t test.

Article Snippet: CCT3 , Proteintech , Cat#10571-1-AP; RRID: AB_2073658.

Techniques: Western Blot, Expressing, Knockdown, Quantitative RT-PCR, Activity Assay, Staining, Cell Cycle Assay, Wound Healing Assay, Over Expression, Transwell Assay

Journal: iScience

Article Title: CCT3-mediated regulation of XPO1/RB1 axis stability promotes cellular senescence and tumor progression in clear cell renal carcinoma

doi: 10.1016/j.isci.2026.114840

Figure Lengend Snippet: Knockdown of XPO1 inhibited the invasion and migration abilities of ccRCC cells (A) The Venn diagram obtained by taking the intersection of the relevant gene sets of CCT3 and RB1 after screening. (B) Expression level of XPO1 in clear cell RCC from the TCGA database. (C) The immunohistochemical results of XPO1 in ccRCC and normal renal tissue in the HPA database. (D) A Kaplan-Meier curve illustrates OS in patients with ccRCC from low and high XPO1 expression groups. (E) The promoter methylation level of XPO1 in KIRC from the TCGA database. (F) Western blot analysis was performed to assess the protein expression of XPO1 in CCT3-depleted cells. (G) Using Spearman correlation analysis, the correlation between Gene CCT3 and XPO1 expression, and the correlation between XPO1 and RB1 expression. (H) Wound-healing assay indicates that XPO1 knockdown suppresses the viability of 786-O and 769-P cells. (I) Transwell assay indicates impaired abilities of migration and invasion of 769-P and 786-O cells. (Scale bars, 100 μm, all data were shown as the mean ± SD ( n = 3 per group), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). Two-tailed Student’s t test for B and E. Student’s t test for F, H, and I.

Article Snippet: CCT3 , Proteintech , Cat#10571-1-AP; RRID: AB_2073658.

Techniques: Knockdown, Migration, Expressing, Immunohistochemical staining, Methylation, Western Blot, Wound Healing Assay, Transwell Assay, Two Tailed Test

Journal: iScience

Article Title: CCT3-mediated regulation of XPO1/RB1 axis stability promotes cellular senescence and tumor progression in clear cell renal carcinoma

doi: 10.1016/j.isci.2026.114840

Figure Lengend Snippet: CCT3 regulates RB1 activity by influencing the stability of the XPO1 protein (A) The Co-IP was performed to analyze the endogenous interaction between CCT3 and XPO1 in ccRCC cells. protein pellets were analyzed by Western blot with anti-CCT3 and anti-XPO1 antibodies. (B) Pull-down of CCT3, XPO1, and CCT3 with GST-tagged was analyzed by Western Blot. (C) Western blot analysis of XPO1 in control or CCT3–knockdown cells treated with CHX for the times indicated. (D) Western blot analysis of XPO1 in control or CCT3–knockdown cells treated with BafA1. (E) Western blot analysis of XPO1 in control or CCT3–knockdown cells treated with MG132. (F) Visualization results of the CCT3 and XPO1 molecular docking. (All data were shown as the mean ± SD ( n = 3 per group), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001). One-way ANOVA for C, Student’s t test for D and E.

Article Snippet: CCT3 , Proteintech , Cat#10571-1-AP; RRID: AB_2073658.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Western Blot, Control, Knockdown

Journal: iScience

Article Title: CCT3-mediated regulation of XPO1/RB1 axis stability promotes cellular senescence and tumor progression in clear cell renal carcinoma

doi: 10.1016/j.isci.2026.114840

Figure Lengend Snippet: Rescue experiments demonstrate the effects of CCT3 overexpression and XPO1 knockdown on cell function (A) Western blot was used to detect the expression of RB1 and Cyclin D in nuclear and cytoplasmic fractions of cells. (B) Western blot analysis was performed to assess the protein expression of Cyclin D, p-Rb (Ser807/811) in the rescue assay. (C) Senescence β-Galactosidase Staining in rescue assay. (D) A healing assay was performed in the rescue assay. (E) Transwell assay was performed in the rescue assay. (Scale bars, 100 μm, all data were shown as the mean ± SD ( n = 3 per group), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001). One-way ANOVA for D, E.

Article Snippet: CCT3 , Proteintech , Cat#10571-1-AP; RRID: AB_2073658.

Techniques: Over Expression, Knockdown, Cell Function Assay, Western Blot, Expressing, Rescue Assay, Staining, Transwell Assay

Journal: iScience

Article Title: CCT3-mediated regulation of XPO1/RB1 axis stability promotes cellular senescence and tumor progression in clear cell renal carcinoma

doi: 10.1016/j.isci.2026.114840

Figure Lengend Snippet: Knockdown of CCT3 combined with a selective XPO1 inhibitor can promote cellular senescence to suppress tumor progression in vivo (A) Nude mice were used to establish a tumor xenograft model utilizing CCT3 knockdown cell lines and the selective XPO1 inhibitor, Selinexor. (B and C) The tumor volumes and tumor weight of each group. (D) Western blot analysis was performed to assess the protein expression of p53, Cyclin D, CDK4, and PCNA in tissue. (E and F) The immunohistochemistry and immunofluorescence assays of Ki67. (Scale bars, 100 μm. All data were shown as the mean ± SD ( n = 3 per group), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001). Repeated measures ANOVA for B and C. One-way ANOVA for D and E.

Article Snippet: CCT3 , Proteintech , Cat#10571-1-AP; RRID: AB_2073658.

Techniques: Knockdown, In Vivo, Western Blot, Expressing, Immunohistochemistry, Immunofluorescence