human rcc cell lines 786 o  (ATCC)

Journal: Research

Article Title: IFI44 Promotes Clear Cell Renal Cell Carcinoma Progression via PRDX1 and Predicts Poor Prognosis

doi: 10.34133/research.1102

Figure Lengend Snippet: IFI44 knockdown inhibits malignant phenotypes in RCC cells. (A) IFI44 levels in RCC cell lines assessed by Western blot with β-actin as the control. (B) IFI44 protein levels assessed by Western blot in Caki-2 and 786-O cells following IFI44 knockdown (KD) or negative control (NC). (C and D) Densitometric quantification of IFI44 levels in Caki-2 (C) and 786-O (D) cells normalized to β-actin. (E) CCK-8 assay showing Caki-2 and 786-O cell proliferation at 0, 24, 48, and 72 h. (F) Flow cytometric analysis and quantification of Caki-2 and 786-O cell apoptosis following IFI44 KD. (G) Representative wound healing images and quantification of migration rates of Caki-2 and 786-O cells after KD or NC treatment. (H) Representative images of cell migration and invasion in Caki-2 and 786-O after KD or NC treatment, with corresponding quantification of migrated/invaded cells shown below.

Article Snippet: The human RCC cell lines 786-O (TCHu186), Caki-1 (TCHu135), Caki-2 (TCHu251), and ACHN (TCHu199) were sourced from the American Type Culture Collection in Shanghai, China.

Techniques: Knockdown, Western Blot, Control, Negative Control, CCK-8 Assay, Migration

Journal: Advanced Science

Article Title: ITM2B Truncation Promotes Migrasome Formation to Accelerate Renal Cell Carcinoma Growth

doi: 10.1002/advs.202511683

Figure Lengend Snippet: Truncated‐ITM2B promotes the growth of RCC. A) Immunohistochemical analysis of ITM2B expression in clinical RCC tissues and corresponding para‐carcinoma tissues. B) The cleavage of endogenous ITM2B was detected in various cancer cell lines. C,D) ITM2B expression was detected in carcinoma (abbreviated as C) and corresponding para‐carcinoma tissues (abbreviated as P) from RCC patients C). The expression levels of full‐length ITM2B and ITM2B truncation were quantified with ImageJ D) ( n = 18 patients). E) Control (empty vector), ITM2B, ITM2B I115A, and ITM2B 1‐115 were overexpressed in 786‐O cells (top) and ITM2B‐knockdown Renca cells (bottom). F,G) Control and ITM2B‐knockdown luciferase‐expressing Renca cells were conducted to orthotopic allografts. Tumor growth was indicated and quantified by the luciferase signals (F, n = 8 mice). Ki67 expression levels were indicated, and fields ( n = 9) from three independent tumor tissues were randomly choose to quantify Ki67 expression G). H,I) Indicated Renca cells were conducted to orthotopic allografts. Tumor growths (H, n = 8 mice) and the expression levels of Ki67 (I, n = 9 fields) were shown. Data were shown as the mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The human RCC cell lines 786‐O (#CL‐0010), ACHN (#CL‐0021), and the mouse RCC cell line Renca (#CL‐0568) were purchased from Procell Life Science&Technology and maintained according to manufacturer's instructions.

Techniques: Immunohistochemical staining, Expressing, Control, Plasmid Preparation, Knockdown, Luciferase

Journal: Advanced Science

Article Title: ITM2B Truncation Promotes Migrasome Formation to Accelerate Renal Cell Carcinoma Growth

doi: 10.1002/advs.202511683

Figure Lengend Snippet: ITM2B truncation promotes RCC growth through migrasome. A) GFP‐ITM2B‐, GFP‐ITM2B I115A ‐, and GFP‐ITM2B 1‐115 ‐expressing 786‐O cells were stained with WGA. B) GFP‐ITM2B‐mCherry was expressed in 786‐O cells, live‐cell imaging was applied to visualize its subcellular location. C) APEX2‐ITM2B, APEX2‐ITM2B I115A, and APEX2‐ITM2B 1‐115 were expressed in 786‐O cells, and the APEX2 signals in migrasomes of these cells were visualized under transmission electron microscope. Empty vector without APEX2‐tag was used as a negative control. D) Cell body and migrasome of 786‐O cells were prepared, and the endogenous ITM2B was indicated. E) Flag‐tagged ITM2B, ITM2B I115A, and ITM2B 1‐115 were expressed in 786‐O cells, and the cell body and migrasome were prepared. F) Fresh tissues from orthotopic allografts were collected to visualize in situ migrasomes in allografts using electron microscopy. G,H) Renca cells were subcutaneously inoculated into the flank of BALB/c mice. The migrasomes derived from different groups of Renca cells as indicated were injected into peritumoral regions. Tumor growths and weights were indicated (G, n = 8 mice), the expression levels of Ki67 (H, n = 9 fields) in tumor tissues were indicated. I,J) Flag‐ITM2B and Flag‐ITM2B I115A were reintroduced into ITM2B‐knockdown Renca cells, and the cells were subcutaneously inoculated into the flank of BALB/c mice. Migrasomes derived from Flag‐ITM2B‐expressing Renca cells were collected and injected into peritumoral regions of Flag‐ITM2B I115A ‐expressing allografts. Tumor growths (I, n = 8 mice) and the expression levels of Ki67 (J, n = 9 fields) were indicated. K) Migrasomes in 100 mL early morning urine from normal volunteer ( n = 21) and RCC patients ( n = 21) were purified. Indicated proteins were detected in uric migrasomes. Data were shown as the mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The human RCC cell lines 786‐O (#CL‐0010), ACHN (#CL‐0021), and the mouse RCC cell line Renca (#CL‐0568) were purchased from Procell Life Science&Technology and maintained according to manufacturer's instructions.

Techniques: Expressing, Staining, Live Cell Imaging, Transmission Assay, Microscopy, Plasmid Preparation, Negative Control, In Situ, Electron Microscopy, Derivative Assay, Injection, Knockdown, Purification

Journal: Advanced Science

Article Title: ITM2B Truncation Promotes Migrasome Formation to Accelerate Renal Cell Carcinoma Growth

doi: 10.1002/advs.202511683

Figure Lengend Snippet: ITM2B truncation promotes migrasome formation. A) Control (empty vector), ITM2B I115A , ITM2B 1‐115 , and ITM2B 116‐266 were overexpressed in 786‐O cells, and the cells were stained with WGA to show corresponding migrasomes (left). The migrasome numbers with 100 cells were counted in each group (right). B) Super‐resolution SIM was applied to visualize GFP‐positive dot structures in indicated 786‐O cells. C) GFP‐ITM2B‐, GFP‐ITM2B I115A ‐, and GFP‐ITM2B 1‐115 ‐expressing 786‐O cells were separately subjected to widefield imaging and TIRF imaging in the same cells of each group. D) 3D imaging to visualize the spatial distribution of GFP‐ITM2B‐mCherry in living 786‐O cells. E,F) Time‐lapse imaging of GFP‐ITM2B‐expressing 786‐O cells exhibited the transfer process of ITM2B from inside cells to RFs (E, indicated by arrows), and the process of ITM2B‐mediated migrasome swelling (F, indicated by arrows). G) GFP‐ITM2B 1‐115 and SMS2‐mCherry were expressed in 786‐O cells. Time‐lapse imaging was applied to visualize the transfer process of ITM2B 1‐115 (green) and SMS2 (red) from cell body to RFs. H) Time‐lapse imaging was applied to visualize the GFP‐ITM2B truncation‐ and SMS2‐positive migrasome formation. Data were shown as the mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The human RCC cell lines 786‐O (#CL‐0010), ACHN (#CL‐0021), and the mouse RCC cell line Renca (#CL‐0568) were purchased from Procell Life Science&Technology and maintained according to manufacturer's instructions.

Techniques: Control, Plasmid Preparation, Staining, Expressing, Imaging

Journal: Advanced Science

Article Title: ITM2B Truncation Promotes Migrasome Formation to Accelerate Renal Cell Carcinoma Growth

doi: 10.1002/advs.202511683

Figure Lengend Snippet: ITM2B truncation recruits TSPAN4 to facilitate migrasome formation. A) TSPAN4 was knocked down in GFP‐ITM2B 1‐115 ‐expressing 786‐O cells, live‐cell imaging was applied to observe the GFP‐ITM2B 1‐115 migrasomes (left). Arrows indicate the GFP‐ITM2B 1‐115 puncta on RFs. Migrasome numbers were counted with 100 cells in each group (right). B) GFP‐ITM2B 1‐115 and TSPAN4‐mCherry were expressed in 786‐O cells, time‐lapse imaging indicated GFP‐ITM2B 1‐115 enriched at migrasome formation sites prior to TSPAN4‐mCherry. C) ITM2B fragments and TSPAN4 were expressed in 786‐O cells, their interactions were detected. D) Top, predicted structures of ITM2B and TSPAN4 by AlphaFold2. Bottom, molecular modeling of ITM2B binding to TSPAN4. E) ITM2B truncation or its point mutants and TSPAN4 were expressed in 786‐O cells, their interactions were detected. F) TSPAN4, together with ITM2B truncation and their point mutants as indicated, were expressed in 786‐O cells. The His pull‐down assay indicated the covalent binding between ITM2B truncation and TSPAN4. G) Control (empty vector), GFP‐ITM2B 1–115 and GFP‐ITM2B 1–115 C54S were separately transfected into the TSPAN4‐mCherry‐expressing 786‐O cells (left). Arrows indicated GFP‐ITM2B 1–115 C54S puncta without TSPAN4 enrichment on retraction fibers. Migrasome numbers were counted with 100 cells in each group (right). H,I) Flag‐ITM2B 1‐115 was overexpressed in TSPAN4‐knockdown luciferase‐expressing Renca cells. The cells were orthotopically injected into mice. Tumors in kidney (H, n = 8 mice) and the expression levels of Ki67 (I, n = 9 fields) were indicated. J,K) Flag‐ITM2B 1–115 and Flag‐ITM2B 1–115 C54S were reintroduced into ITM2B‐knockdown Renca cells, and the cells were subcutaneously inoculated into the flank of BALB/c mice. Migrasomes derived from Flag‐ITM2B 1‐115‐expressing Renca cells were collected and injected into peritumoral regions of Flag‐ITM2B 1–115 C54S ‐expressing allografts. Tumor growths and weights (J, n = 8 mice) and the expression levels of Ki67 (K, n = 9 fields) were indicated. Data were shown as the mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The human RCC cell lines 786‐O (#CL‐0010), ACHN (#CL‐0021), and the mouse RCC cell line Renca (#CL‐0568) were purchased from Procell Life Science&Technology and maintained according to manufacturer's instructions.

Techniques: Expressing, Live Cell Imaging, Imaging, Binding Assay, Pull Down Assay, Control, Plasmid Preparation, Transfection, Knockdown, Luciferase, Injection, Derivative Assay

Journal: Advanced Science

Article Title: ITM2B Truncation Promotes Migrasome Formation to Accelerate Renal Cell Carcinoma Growth

doi: 10.1002/advs.202511683

Figure Lengend Snippet: ITM2B Truncation Promotes the Migracytosis of Active Caspase‐7. A) The cell body and migrasome were prepared in 786‐O cells with or without caspase‐7 knockdown, the expression levels of c‐CASP7 and CASP7 were detected. B) BFP‐CASP7‐mCherry‐expressing 786‐O cells were stained with WGA, and live‐cell imaging was applied to show the different statuses of caspase‐7 in cell body and migrasome. C) HA‐ITM2B 1‐115 was stably expressed in 786‐O cells, and HA‐ITM2B 1‐115 ‐containg vesicles were isolated via immunocapture and detected by western blotting. D) Flag‐ITM2B 1‐115 ‐HA was stably expressed in 786‐O cells. Anti‐Flag magnetic beads were used to immunocapture Flag‐ITM2B 1‐115 ‐HA‐containing vesicles, and the vesicles were incubated with protease K in vitro. E) GFP‐ITM2B 1‐115 and BFP‐CASP7‐mCherry were expressed in 786‐O cells, and super‐resolution SIM was applied to visualize their subcellular locations. F) The cell body and migrasome from indicated 786‐O cells were prepared, the levels of c‐CASP7 and ITM2B were detected. G) Control (empty vector), ITM2B, ITM2B I115A , and ITM2B 1‐115 were reintroduced into ITM2B‐knockdown 786‐O cells. The cell body and migrasome were prepared. H) GFP‐ITM2B 1‐115 and BFP‐CASP7‐mCherry were expressed in 786‐O cells. Time‐lapse imaging indicated c‐CASP7 accumulation accompanying with GFP‐ITM2B 1‐115 enrichment in migrasomes. I) ITM2B 1‐115 , together with CASP7 or its subunits, was expressed in 786‐O cells, the interaction between ITM2B and CASP7 or its subunits was determined. J,K) Renca cells were subcutaneously inoculated into the flank of BALB/c mice. The migrasomes derived from different groups of Renca cells as indicated were injected into peritumoral regions. Tumor growths and weights (J, n = 8 mice), the expression levels of Ki67 (K, n = 9 fields) were indicated. Data were shown as the mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The human RCC cell lines 786‐O (#CL‐0010), ACHN (#CL‐0021), and the mouse RCC cell line Renca (#CL‐0568) were purchased from Procell Life Science&Technology and maintained according to manufacturer's instructions.

Techniques: Knockdown, Expressing, Staining, Live Cell Imaging, Stable Transfection, Isolation, Western Blot, Magnetic Beads, Incubation, In Vitro, Control, Plasmid Preparation, Imaging, Derivative Assay, Injection

Journal: Advanced Science

Article Title: ITM2B Truncation Promotes Migrasome Formation to Accelerate Renal Cell Carcinoma Growth

doi: 10.1002/advs.202511683

Figure Lengend Snippet: Active caspase‐7 from RCC cells promotes IL‐6 secretion from macrophages. A) Cells as indicated were incubated with DiI‐labeled migrasomes for 12 h, and then the DiI signals were detected by flow cytometry. HL‐60 cells were incubated with 1.3% DMSO for 5 days to differentiate into neutrophils. THP‐1 cells were incubated with PMA to differentiate into macrophages. B) mCherry‐expressing THP‐1 cells were pretreated with PMA (100 ng mL −1 ) for 24 h to differentiate into macrophages, then GFP‐ITM2B 1‐115 ‐expressing 786‐O cells were co‐cultured with macrophages for 16 h. Time‐lapse imaging was applied to visualize the uptake of tumor cell‐derived migrasomes by macrophages. C) Migrasomes derived from BFP‐CASP7‐7‐mCherry‐expressing 786‐O cells were used to incubated with THP‐1‐differentiated macrophages for 24 h. WGA was used to stain the macrophages. D,E) THP‐1‐differentiated macrophages were incubated with migrasomes derived from ITM2B‐overexpressing or CASP7‐knockdown 786‐O cells for 24 h, the expression levels of c‐CASP7 were detected D). The culture mediums of indicated cells were collected to detect the levels of IL‐6 secretion (E, n = 3). F,G) Macrophages were incubated with migrasomes derived from overexpressing different ITM2B mutants as indicated in 786‐O cells. the expression levels of c‐CASP7 F) and corresponding IL‐6 secretion (G, n = 3) were detected. H,I) Tumor tissues from Figure were stained separately with F4/80 (macrophage maker), Flag‐antibody, and DAPI (nucleus) to indicate the uptake of Flag‐ITM2B 1‐115 migrasomes by macrophages H). The activation of IL‐6/STAT3 pathway in tumor tissues from Figure were detected I). J) The activation of IL‐6/STAT3 pathway in tumor tissues from Figure was detected. K) The activation of IL‐6/STAT3 pathway in tumor tissues from Figure was detected. Data were shown as the mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The human RCC cell lines 786‐O (#CL‐0010), ACHN (#CL‐0021), and the mouse RCC cell line Renca (#CL‐0568) were purchased from Procell Life Science&Technology and maintained according to manufacturer's instructions.

Techniques: Incubation, Labeling, Flow Cytometry, Expressing, Cell Culture, Imaging, Derivative Assay, Staining, Knockdown, Activation Assay

Journal: Advanced Science

Article Title: ITM2B Truncation Promotes Migrasome Formation to Accelerate Renal Cell Carcinoma Growth

doi: 10.1002/advs.202511683

Figure Lengend Snippet: Hyperuricemia facilitates ITM2B cleavage in RCC Cells. A) 786‐O cells were treated with MSU crystal (400 µg mL −1 ) for 3 h and then cultured with fresh medium until 12 h, the endogenous ITM2B expression was indicated (top). Gomori's methenamine silver (GMS) was applied to stain the urate crystals in 786‐O cells (bottom). B) Flag‐ITM2B‐ or Flag‐ITM2B I115A ‐expressing 786‐O cells were treated with MSU crystal, ITM2B cleavage was detected. C) Flag‐ITM2B‐ or Flag‐ITM2B I115A ‐expressing 786‐O cells were treated with MSU crystal, and WGA was applied to stain migrasomes (left). Migrasome numbers with 100 cells were counted in each group (right). D) ITM2B‐knockdown 786‐O cells were treated with MSU crystal. The cell body and migrasome were collected and detected. E,F) THP‐1‐differentiated macrophages were incubated with migrasomes derived from indicated 786‐O cells for 24 h. The expression levels of c‐CASP7 E) and corresponding IL‐6 secretion (F, n = 3) were detected. G) In hyperuricemia tumor‐bearing BALB/c mice, urine was collected and incubated on FN‐precoated dishes. WGA was applied to stain migrasomes, and GFP‐positive migrasomes were counted in 200 µm × 200 µm fields ( n = 5 mice). H–J) Flag‐ITM2B and Flag‐ITM2B I115A were reintroduced into ITM2B‐knockdown luciferase‐expressing Renca cells. The cells were conducted to orthotopic allografts with or without hyperuricemia induction. Tumors in kidney were indicated and quantified by luciferase signals H). The expression levels of Ki67 in kidney tumor were indicated and quantify (I, n = 9 fields). ITM2B cleavage and the activation of IL‐6/STAT3 pathway were indicated J). Data were shown as the mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The human RCC cell lines 786‐O (#CL‐0010), ACHN (#CL‐0021), and the mouse RCC cell line Renca (#CL‐0568) were purchased from Procell Life Science&Technology and maintained according to manufacturer's instructions.

Techniques: Cell Culture, Expressing, Staining, Knockdown, Incubation, Derivative Assay, Luciferase, Activation Assay

Journal: Molecular Cancer

Article Title: ACOX2 destabilizes the MRE11-RAD50-NBS1 complex and boosts anticancer immunity via the cGAS-STING pathway in clear cell renal cell carcinoma

doi: 10.1186/s12943-025-02420-9

Figure Lengend Snippet: ACOX2 inhibits the biological characteristics of ccRCC. A , B Quantitative polymerase chain reaction (qPCR) ( A ) and immunoblotting ( B ) of ACOX2 in HK-2 and seven RCC cell lines. C , D Immunoblotting of ACOX2 in the indicated 786-O ( C ) and Caki-1 ( D ) cells. E , F Colony formation assay of the indicated 786-O ( E ) and Caki-1 ( F ) cells. G , H Growth curves of the indicated 786-O ( G ) and Caki-1 ( H ) cells using Cell Counting Kit-8 (CCK-8). I , J Wound healing assay of the indicated 786-O (I) and Caki-1 ( J ) cells. Scale bar: 200 μm. K , L Transwell invasive assay of the indicated 786-O ( K ) and Caki-1 ( L ) cells. Scale bar: 200 μm. M , N Percentage of apoptosis cell of the indicated 786-O ( M ) and Caki-1 ( N ) cells with flow cytometry analysis. O Schematic illustration for the generation of ccRCC cell-derived xenograft (CDX) model. P , Q The growth curves ( P ) and tumor weight ( Q ) of the indicated 786-O CDX. R , S The growth curves ( R ) and tumor weight ( S ) of the indicated Caki-1 CDX. Statistical significance was determined by two-tailed unpaired t-test ( A , E - N , Q , S ) and two-way analysis of variance (ANOVA) ( P , R ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns P ≥ 0.05. Experiments were independently repeated three times with similar results; data of one representative experiment are shown ( B - F , I - N )

Article Snippet: Human embryonic kidney cell HEK293T, human renal cortex proximal tubule epithelial cell HK-2, and human RCC cell lines containing 786-O, 769-P, A-498, Caki-1, Caki-2, ACHN, and SW839 were obtained from ATCC, authenticated by STR profiling, and tested negative for mycoplasma contamination.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Colony Assay, Cell Counting, CCK-8 Assay, Wound Healing Assay, Flow Cytometry, Derivative Assay, Two Tailed Test

Journal: Molecular Cancer

Article Title: ACOX2 destabilizes the MRE11-RAD50-NBS1 complex and boosts anticancer immunity via the cGAS-STING pathway in clear cell renal cell carcinoma

doi: 10.1186/s12943-025-02420-9

Figure Lengend Snippet: ACOX2 sensitizes ccRCC cell, cell-derived xenograft, patient-derived organoid, and patient-derived xenograft to PARPi. A The correlation between ACOX2 expression and half-maximal inhibitory concentration (IC 50 ) of olaparib in seven RCC cell lines. B Immunoblotting of ACOX2 in the indicated 786-O cells. C IC 50 of olaparib in the indicated 786-O cells. D Colony formation assay in the indicated 786-O cells in response to olaparib. E Immunoblotting of ACOX2 in the indicated Caki-1 cells. F IC 50 of olaparib in the indicated Caki-1 cells. G Colony formation assay in the indicated Caki-1 cells in response to olaparib. H Schematic illustration for generation of Caki-1 CDX. I Immunoblotting of ACOX2 in the indicated Caki-1 cells. J , K Growth curves ( J ) and tumor weight ( K ) of the indicated Caki-1 PDX groups. L Schematic illustration for generation of ccRCC patient-derived organoid (PDO). M Representative hematoxylin-eosin (H&E) and IHC staining for ccRCC PDO. Scale bar: 40 μm. N Representative mIHC staining for ccRCC PDO. Scale bar: 50 μm. O Immunoblotting of ACOX2 in the indicated ccRCC PDO. P Relative PDO viability at the end of olaparib treatment detected by CellTiter-Glo 3D Cell Viability Assay kit. Scale bar: 50 μm. Q Schematic illustration for generation of ccRCC patient-derived xenograft (PDX). R Representative IHC staining of ACOX2 in the indicated ccRCC PDX. Scale bar: 100 μm. S Immunoblotting of ACOX2 in the indicated ccRCC PDX. T , U Growth curves ( T ) and tumor weight ( U ) of the indicated ccRCC PDX groups. Statistical significance was determined by pearson correlation analysis ( A ), two-tailed unpaired t-test ( D , G , K , P , U ), and two-way ANOVA ( J , T ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns P ≥ 0.05. Experiments were independently repeated three times with similar results; data of one representative experiment are shown ( B , D , E , G , I , M - O , R , S )

Article Snippet: Human embryonic kidney cell HEK293T, human renal cortex proximal tubule epithelial cell HK-2, and human RCC cell lines containing 786-O, 769-P, A-498, Caki-1, Caki-2, ACHN, and SW839 were obtained from ATCC, authenticated by STR profiling, and tested negative for mycoplasma contamination.

Techniques: Derivative Assay, Expressing, Concentration Assay, Western Blot, Colony Assay, Immunohistochemistry, Staining, Viability Assay, Two Tailed Test