Journal: Microbiome

Article Title: Tumor-colonized Streptococcus mutans metabolically reprograms tumor microenvironment and promotes oral squamous cell carcinoma.

doi: 10.1186/s40168-024-01907-9

Figure Lengend Snippet: Fig. 5 Single-cell transcriptomic landscape reveals the shift of the cellular types during oral carcinogenesis, and KYNA is a predominant driving force for expansions and infiltrations of neutrophils in the TME of OSCC. A Overview of the workflow for scRNA-seq analyses of OLK and OSCC rat tongue tissues. B UMAP plot of the clustering results for 13 major cell types from OLK and OSCC tissues. C Stacked histogram of the percentages of different cells from OLK and OSCC tissues. D Quantitative analysis of neutrophils, macrophages, and T cells in non-epithelial cells in OLK and OSCC tissues. E H&E and IHC staining for CD11b in OLK and OSCC tissues of experimental rats. Scale bar: 600 µm for 4 × magnification and 300 µm for 10 × magnification. F Representative IHC staining images and statistical analysis of CD16 in saline and KYNA-treated 4NQO rats. Scale bars: 200 µm for 10 × magnification and 50 µm for 40 × magnification. G Representative flow cytometry dot plots and statistical analysis of CD11b + CD16 + neutrophils from the peripheral blood of OSCC patients after KYNA treatment. H Relative SLC7A8 expression in neutrophils from OSCC patients after KYNA treatment detected by RT-qPCR. I Representative flow cytometry dot plots and statistical analysis of AHR + neutrophils from OSCC patients after treatments with KYNA in indicated concentrations. In D, unpaired Student’s t-test; E and F, Mann– Whitney U test; G, H, and I, one-way ANOVA

Article Snippet: At week 20, the rats were sacrificed, and the tongues were dissected, and a longitudinal mid-lingual incision was made. (4) IL-1β/PD-L1monoclonal antibody (mAb) intervention (8 rats/group): (A) 4NQO + IL-1β mAb (200 μg/rat, BE0246, cloneB122, Bioxcell, USA); (B) 4NQO + KYNA + IL-1β mAb; (C) 4NQO + PD-L1 mAb (200 μg/rat, BE0383, clone 368A.4H1, Bioxcell, USA); (D) 4NQO + KYNA + PD-L1 mAb; (E) 4NQO + control IgG (200 μg/rat, BE0091, Bioxcell, USA).

Techniques: Immunohistochemistry, Saline, Flow Cytometry, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cell Communication and Signaling : CCS

Article Title: Aurantio-obtusin modulates Wilms Tumour 1 within the breast tumour microenvironment reducing immunosuppression and tumour growth

doi: 10.1186/s12964-025-02292-y

Figure Lengend Snippet: WT1 regulates the STAT1/3 pathway in CAFs. A Protein levels of WT1, STAT1, STAT3, PD-L1 and the TGFβ receptor were determined in WT1 knockdown ( A ) and WT1 overexpressed ( B ) CAFs by Western blotting. Actin was used as a loading control. IDO release was determined by ELISA. RNA levels of PD-L1 was determined by qRT-PCR. TBP was used as a loading control. Representative experiment is shown of n = 3–5 biological replicates. Data represents mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: The blots were probed with primary antibodies against WT1 (Invitrogen, MA5-38,406; 1:500), STAT1 (Invitrogen, AHO0832; 1:500), STAT3 (Invitrogen, PA5-85,199; 1:500), PD-L1 (R&D systems, AF156; 1:100), TGF-β RI (R&D systems, AF3025; 1:500), p53 (R&D systems, AF1355; 1:1,000), β-actin (Sigma Aldrich, A5441; 1:20,000), and GAPDH (Cell Signaling Technology, 2118S; 1:1,000) overnight at 4 °C and detected by chemiluminescence using Goat anti-Rabbit IgG secondary antibody (Invitrogen, 31,460; 1:5,000), Rabbit anti-Goat IgG secondary antibody (Invitrogen, 31,402; 1:5,000), Goat anti-mouse IgG secondary antibody (Invitrogen, 31,430; 1:5,000).

Techniques: Knockdown, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Aurantio-obtusin modulates Wilms Tumour 1 within the breast tumour microenvironment reducing immunosuppression and tumour growth

doi: 10.1186/s12964-025-02292-y

Figure Lengend Snippet: Chemotherapeutic drugs DOX and PTX increase WT1 levels in CAFs and enhance their ability to inhibit T cell proliferation. A Schematic overview of drug treatment, co-culture of CAFs and PBMCs followed by T cell proliferation assay. B Protein expression of WT1 and p53 in CAFs following DOX and PTX treatment was determined by Western blotting. GAPDH was used as a loading control. C Protein levels of STAT1, STAT3, PD-L1 levels. IDO release from DOX and PTX treated CAFs was determined by ELISA. D DOX treated CAFs were co-cultured with PBMCs and CFSE-labelled CD4 + and CD8 + T cell proliferation was determined by flow cytometry ( E ) PTX treated CAFs were co-cultured with PBMCs and CFSE-labelled CD4 + and CD8. + T cell proliferation was determined by flow cytometry. Representative experiment is shown of n = 3 biological replicates. Data are mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: The blots were probed with primary antibodies against WT1 (Invitrogen, MA5-38,406; 1:500), STAT1 (Invitrogen, AHO0832; 1:500), STAT3 (Invitrogen, PA5-85,199; 1:500), PD-L1 (R&D systems, AF156; 1:100), TGF-β RI (R&D systems, AF3025; 1:500), p53 (R&D systems, AF1355; 1:1,000), β-actin (Sigma Aldrich, A5441; 1:20,000), and GAPDH (Cell Signaling Technology, 2118S; 1:1,000) overnight at 4 °C and detected by chemiluminescence using Goat anti-Rabbit IgG secondary antibody (Invitrogen, 31,460; 1:5,000), Rabbit anti-Goat IgG secondary antibody (Invitrogen, 31,402; 1:5,000), Goat anti-mouse IgG secondary antibody (Invitrogen, 31,430; 1:5,000).

Techniques: Co-Culture Assay, Proliferation Assay, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Aurantio-obtusin modulates Wilms Tumour 1 within the breast tumour microenvironment reducing immunosuppression and tumour growth

doi: 10.1186/s12964-025-02292-y

Figure Lengend Snippet: AO reduces WT1 levels in CAFs and reduces the ability of CAFs to suppress T cell proliferation. A Comparison of chemical structures of DOX and AO. B Protein levels of WT1, STAT1, STAT3, and PD-L1 after AO treatment of CAFs were determined by Western blotting. Actin was used as a loading control. C Dose–response curves of DOX, PTX and AO treatment of CAFs. P1 and P3 (Luminal A), P2 (TNBC). Curves were generated by non-linear regression analysis by GraphPad. D AO treated CAFs were co-cultured with PBMCs and CFSE-labelled CD4 + and CD8. + T cell proliferation was determined by flow cytometry. Representative experiment is shown of n = 3 biological replicates. Data represent mean ± SEM; * P < 0.05, and ** P < 0.01

Article Snippet: The blots were probed with primary antibodies against WT1 (Invitrogen, MA5-38,406; 1:500), STAT1 (Invitrogen, AHO0832; 1:500), STAT3 (Invitrogen, PA5-85,199; 1:500), PD-L1 (R&D systems, AF156; 1:100), TGF-β RI (R&D systems, AF3025; 1:500), p53 (R&D systems, AF1355; 1:1,000), β-actin (Sigma Aldrich, A5441; 1:20,000), and GAPDH (Cell Signaling Technology, 2118S; 1:1,000) overnight at 4 °C and detected by chemiluminescence using Goat anti-Rabbit IgG secondary antibody (Invitrogen, 31,460; 1:5,000), Rabbit anti-Goat IgG secondary antibody (Invitrogen, 31,402; 1:5,000), Goat anti-mouse IgG secondary antibody (Invitrogen, 31,430; 1:5,000).

Techniques: Comparison, Western Blot, Control, Generated, Cell Culture, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Aurantio-obtusin modulates Wilms Tumour 1 within the breast tumour microenvironment reducing immunosuppression and tumour growth

doi: 10.1186/s12964-025-02292-y

Figure Lengend Snippet: Proposed model highlighting how WT1 in CAFs modulates immune evasion and tumour growth in the breast TME. WT1 is upregulated in breast patient-derived CAFs. WT1 is a transcription factor, regulating expression of STAT1/STAT3 in CAFs. STAT1/STAT3 regulate PD-L1 expression and IDO release in CAFs. Both PD-L1 and IDO inhibit T cell activity and cytotoxic GZMB levels within the TME, contributing to increased tumour growth. Targeting WT1 in CAFs reduces the release of immunosuppressive factors into the TME. T cells are more active and are able to limit tumour growth

Article Snippet: The blots were probed with primary antibodies against WT1 (Invitrogen, MA5-38,406; 1:500), STAT1 (Invitrogen, AHO0832; 1:500), STAT3 (Invitrogen, PA5-85,199; 1:500), PD-L1 (R&D systems, AF156; 1:100), TGF-β RI (R&D systems, AF3025; 1:500), p53 (R&D systems, AF1355; 1:1,000), β-actin (Sigma Aldrich, A5441; 1:20,000), and GAPDH (Cell Signaling Technology, 2118S; 1:1,000) overnight at 4 °C and detected by chemiluminescence using Goat anti-Rabbit IgG secondary antibody (Invitrogen, 31,460; 1:5,000), Rabbit anti-Goat IgG secondary antibody (Invitrogen, 31,402; 1:5,000), Goat anti-mouse IgG secondary antibody (Invitrogen, 31,430; 1:5,000).

Techniques: Derivative Assay, Expressing, Activity Assay

Journal: Cell reports

Article Title: Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma

doi: 10.1016/j.celrep.2024.114497

Figure Lengend Snippet: (A) shRNA-mediated knockdown of EWS-FLI1 in A673 Ewing sarcoma cells. Tubulin serves as a loading control. (B) EWS-FLI1 knockdown resulted in 7.6-fold reduced protein levels of SMPD1 in the secretome. The quantification based on spectral counting by mass spectrometry is shown. (C) EWS-FLI1 knockdown reduces SMPD1 RNA and protein levels in A673 cells. (Left) The quantitative real-time RT-PCR data ( n = 3). (Right) The immunoblotting data. (D) EWS-FLI1 induces SMPD1 RNA and protein expression in human mesenchymal stem cells (hMSCs), putative cells of origin of Ewing sarcoma. (Left) The quantitative real-time RT-PCR data ( n = 3). (Right) The immunoblotting data. (E) EWS-FLI1 binds to the SMPD1 gene promoter. Chromatin immunoprecipitation analysis for EWS-FLI1 binding to the promoter of SMPD1 and known EWS-FLI1 target genes ( NR0B1, EZH2 , and CD133 ) as well as control ( GAPDH ), with and without EWS-FLI1 silencing ( n = 3). (F) SMPD1 is highly expressed in Ewing sarcoma tumors and cell lines. SMPD1 RNA expression was analyzed by RT-qPCR and was normalized to the levels in hMSCs ( n = 3). EWS, Ewing sarcoma; RMS, rhabdomyosarcoma; SS, synovial sarcoma. (G) SMPD1 knockdown inhibits Ewing sarcoma proliferation. SMPD1 was silenced by siRNAs, and cell proliferation was assessed by the IncuCyte live-cell imaging system. SMPD1 knockdown barely affected proliferation of 293T and HeLa cells. (Top) SMPD1 knockdown was verified by immunoblotting. (H) SMPD1 knockdown inhibits anchorage-independent growth of A673 and PDX1 cells. Scale bars: 100 μm (3 independent experiments; 9 fields each). (Top) shRNA-mediated silencing of SMPD1 was verified by immunoblotting. (I) SMPD1 knockdown inhibits xenograft tumorigenicity of A673 cells (4 mice per group; p = 0.0001). (J) Recombinant SMPD1 rescues proliferation arrest induced by SMPD1 knockdown. A673 and PDX1 cells were transfected with SMPD1 siRNAs or control siRNAs and treated with or without the indicated concentration of recombinant SMPD1. Cell proliferation was assessed by the IncuCyte.

Article Snippet: Importantly, growth arrest of Ewing sarcoma cells upon SMPD1 knockdown was rescued by adding recombinant purified SMPD1 protein (R&D Systems, #5348-PD) to the culture medium , indicating that Ewing sarcoma depends on extracellular SMPD1.

Techniques: shRNA, Knockdown, Control, Mass Spectrometry, Quantitative RT-PCR, Western Blot, Expressing, Chromatin Immunoprecipitation, Binding Assay, RNA Expression, Live Cell Imaging, Recombinant, Transfection, Concentration Assay

Journal: Cell reports

Article Title: Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma

doi: 10.1016/j.celrep.2024.114497

Figure Lengend Snippet: (A) Ceramide rescues proliferation arrest induced by SMPD1 knockdown. A673 cells were transfected with SMPD1 siRNAs or control siRNAs and left untreated or treated with 1 μM C16, C18, or C24 ceramide. Cell proliferation was assessed by the IncuCyte. (B) A mass spectrometry analysis identified GPR64 as an interactor of biotin-ceramide. (C) SMPD1 knockdown and GPR64 knockdown cause similar gene expression changes. The gene set enrichment analysis determined a positive normalized enrichment score of 1.84 ( p = 0.00) from 150 upregulated genes upon SMPD1 knockdown and a negative normalized enrichment score of 2.13 ( p = 0.00) from 101 downregulated genes upon SMPD1 knockdown. The list of genes used for the gene set enrichment analysis is shown in . (D) Biotin-ceramide pulls down endogenous GPR64 from A673 cells. A673 cells were transfected with control siRNA or GPR64 siRNA. Cells were lysed by Dounce homogenization, and cell lysate was incubated with biotin-ceramide or biotin. The interacting proteins were isolated by streptavidin agarose pull-down and analyzed by anti-GPR64 immunoblotting. (E) Biotin-ceramide pulls down transfected GPR64-HA from 293T cells. 293T cells were transfected with GPR64-HA. Cells were lysed by Dounce homogenization, and cell lysate was incubated with biotin-ceramide or biotin. The interacting proteins were isolated by streptavidin agarose pull-down and analyzed by anti-HA immunoblotting. (F) Endogenous GPR64 mediates cAMP signaling by ceramide treatment in Ewing sarcoma cells. A673 cells were transfected with control siRNA or GPR64 siRNA and treated with 1 μM C18 ceramide for 20 min. (Left) The levels of cAMP were assessed by cAMP ELISA. (Right) The levels of phospho-CREB, total CREB, and GPR64 were assessed by immunoblotting. (G) Transfected GPR64 mediates cAMP signaling by ceramide treatment in 293T cells. 293T cells were transfected with GPR64-HA or empty vector and treated with and without 1 μM C18 ceramide for 20 or 30 min. (Left) The levels of cAMP were assessed by cAMP ELISA. (Right) The levels of phospho-CREB, total CREB, and GPR64-HA were assessed by immunoblotting.

Article Snippet: Importantly, growth arrest of Ewing sarcoma cells upon SMPD1 knockdown was rescued by adding recombinant purified SMPD1 protein (R&D Systems, #5348-PD) to the culture medium , indicating that Ewing sarcoma depends on extracellular SMPD1.

Techniques: Knockdown, Transfection, Control, Mass Spectrometry, Gene Expression, Homogenization, Incubation, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Cell reports

Article Title: Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma

doi: 10.1016/j.celrep.2024.114497

Figure Lengend Snippet: (A) EWS-FLI1 knockdown reduces GPR64 RNA and protein levels in A673 cells. (Top) The quantitative real-time RT-PCR data ( n = 3). (Bottom) The immunoblotting data. (B) EWS-FLI1 induces GPR64 RNA and protein expression in hMSCs. (Top) The quantitative real-time RT-PCR data ( n = 3). (Bottom) The immunoblotting data. (C) EWS-FLI1 binds to the GPR64 gene promoter. Chromatin immunoprecipitation analysis for EWS-FLI1 binding to the promoter of GPR64 and known EWS-FLI1 target genes ( NR0B1 , EZH2 , and CD133 ), as well as control ( GAPDH ), with and without EWS-FLI1 silencing ( n = 3). Note the data for NR0B1, EZH2, CD133 , and GAPDH are the same as those in . (D) GPR64 is highly expressed in Ewing sarcoma tumors and cell lines. GPR64 RNA expression was analyzed by RT-qPCR and normalized to the levels in hMSCs ( n = 3). (E) GPR64 knockdown inhibits Ewing sarcoma proliferation. GPR64 was silenced by siRNAs, and cell proliferation was assessed by the IncuCyte system. GPR64 knockdown barely affected the proliferation of 293T and HeLa cells. (Top) GPR64 knockdown was verified by immunoblotting. (F) GPR64 knockdown inhibits anchorage-independent growth of A673 and PDX1 cells (3 independent experiments; 9 fields each). (Top) shRNA-mediated silencing of GPR64 was verified by immunoblotting. (G) GPR64 knockdown inhibits xenograft tumorigenicity of A673 cells (5 mice per group; p = 0.0002). (H) A model for the SMPD1-ceramide-GPR64 pathway in Ewing sarcoma. Created with BioRender.com .

Article Snippet: Importantly, growth arrest of Ewing sarcoma cells upon SMPD1 knockdown was rescued by adding recombinant purified SMPD1 protein (R&D Systems, #5348-PD) to the culture medium , indicating that Ewing sarcoma depends on extracellular SMPD1.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Chromatin Immunoprecipitation, Binding Assay, Control, RNA Expression, shRNA

Journal: Cell reports

Article Title: Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma

doi: 10.1016/j.celrep.2024.114497

Figure Lengend Snippet: (A) Anti-GPR64 C-terminal antibody detects GPR64 C-terminal fragments. A673 cells were transfected with control siRNA or GPR64 siRNA, and the protein levels of GPR64 were analyzed by immunoblotting. (Left) Anti-GPR64 N-terminal antibody immunoblotting. (Right) Anti-GPR64 C-terminal antibody immunoblotting, which detected approximately 33 and 17 kDa fragments, both silenced by GPR64 siRNA. (B) Ceramide induces GPR64 C-terminal fragments. A673 cells were left untreated or treated with 1 μM C18 ceramide for 16 h. The levels of GPR64 C-terminal fragments were assessed by anti-GPR64 C-terminal antibody immunoblotting. (C) Exogenously expressed GPR64 C-terminal intracellular domain (ICD) is located in the nucleus. A673 cells were infected with lentiviruses expressing the FLAG-tagged GPR64 ICD (879–1017) or empty vector, and the subcellular location of FLAG-ICD was examined by anti-FLAG immunofluorescence. The nuclei were stained with DAPI. Scale bars: 10 μm. (D) The GPR64 C-terminal antibody detects a nuclear signal in A673 cells, which is abolished by GPR64 siRNA knockdown. Scale bars: 10 μm. (E) GPR64 ICD rescues growth arrest induced by GPR64 knockdown. A673 cells were infected with lentiviruses expressing FLAG-ICD or empty vector, followed by transfection with control siRNA or GPR64 siRNA. (Left) The protein levels of FLAG-ICD, endogenous full-length GPR64, and tubulin were assessed by immunoblotting. Note that the GPR64 cDNA clone is codon optimized and harbors numerous silent nucleotide substitutions, making the ICD expressed from GPR64 ICD cDNA resistant to silencing by GPR64 siRNA. (Right) Proliferation of cells was assessed by the IncuCyte. (F) GPR64 ICD rescues growth arrest induced by SMPD1 knockdown. A673 cells were infected with lentiviruses expressing FLAG-ICD or empty vector, followed by transfection with control siRNA or SMPD1 siRNA. (Left) The protein levels of FLAG-ICD, SMPD1, and tubulin were assessed by immunoblotting. (Right) Proliferation of cells was assessed by the IncuCyte. (G) Forskolin and bromo-cAMP induce the GPR64 C-terminal fragments. A673 cells were treated with the indicated concentration of forskolin or bromo-cAMP for 16 h, and the levels of GPR64 C-terminal fragments were assessed by anti-GPR64 C-terminal antibody immunoblotting. (H) The suppression of cAMP-PKA signaling blocks the induction of the GPR64 C-terminal fragments by ceramide. A673 cells were treated with 1 μM C18 ceramide for 16 h, followed by treatment with the indicated concentration of NKY80 (adenylate cyclase inhibitor) or H-89 (PKA inhibitor) for 48 h. The levels of GPR64 C-terminal fragments were assessed by anti-GPR64 C-terminal antibody immunoblotting. (I) A γ-secretase inhibitor, DAPT, blocks the induction of the GPR64 C-terminal fragments by ceramide. A673 cells were treated with 1 μM C18 ceramide for 16 h, followed by treatment with the indicated concentration of DAPT for 48 h. The levels of GPR64 C-terminal fragments were assessed by anti-GPR64 C-terminal antibody immunoblotting.

Article Snippet: Importantly, growth arrest of Ewing sarcoma cells upon SMPD1 knockdown was rescued by adding recombinant purified SMPD1 protein (R&D Systems, #5348-PD) to the culture medium , indicating that Ewing sarcoma depends on extracellular SMPD1.

Techniques: Transfection, Control, Western Blot, Infection, Expressing, Plasmid Preparation, Immunofluorescence, Staining, Knockdown, Concentration Assay

Journal: Cell reports

Article Title: Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma

doi: 10.1016/j.celrep.2024.114497

Figure Lengend Snippet: (A) Exogenous GPR64 ICD reduces the RIF1 protein levels in A673 cells. A673 cells were infected with lentiviruses expressing the FLAG-GPR64 ICD or empty vector, and the levels of FLAG-ICD, RIF1, and tubulin were assessed by immunoblotting. (B) Knockdown of SMPD1 and GPR64 increases the RIF1 protein levels in A673 cells. A673 cells were transfected with SMPD1 siRNA, GPR64 siRNA, or control siRNA, and the protein levels of SMPD1, GPR64, RIF1, and tubulin were assessed by immunoblotting. (C) Ceramide treatment reduces the RIF1 protein levels in A673 cells. A673 cells were treated with and without 1 μM C18 ceramide for 16 h, and the protein levels of RIF1 and tubulin were assessed by immunoblotting. (D) RIF1 knockdown rescues growth arrest induced by GPR64 knockdown in A673 cells. A673 cells were infected with lentiviruses expressing RIF1 shRNA or control shRNA. The cells were subsequently transfected with GPR64 siRNA or control siRNA. (Left) The protein levels of GPR64, RIF1, and tubulin were assessed by immunoblotting. (Right) Cell proliferation was assessed by the IncuCyte. (E) RIF1 knockdown rescues growth arrest induced by SMPD1 knockdown in A673 cells. A673 cells were infected with lentiviruses expressing RIF1 shRNA or control shRNA. The cells were subsequently transfected with SMPD1 siRNA or control siRNA. (Left) The protein levels of SMPD1, RIF1, and tubulin were assessed by immunoblotting. (Right) Cell proliferation was assessed by the IncuCyte. (F) Ceramide-induced RIF1 degradation can be rescued by a proteasome inhibitor. A673 cells were treated with 1 μM C18 ceramide for 16 h, followed by the indicated concentration of MG-132 for 6 h. The protein levels of RIF1 and tubulin were assessed by immunoblotting. (G) FLAG-GPR64 ICD co-immunoprecipitates with endogenous RIF1 and SPOP in A673 cells. A673 cells were infected with lentiviruses expressing FLAG-GPR64 ICD or empty vector. Cells were treated with 25 μM MG-132 for 6 h and cell lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting for FLAG, RIF1, and SPOP. (H) FLAG-GPR64 ICD harboring mutations of the SPOP-binding motif does not co-immunoprecipitate with SPOP in A673 cells. A673 cells were infected with lentiviruses expressing the FLAG-GPR64 ICD, FLAG-GPR64 ICD with mutations of the SPOP-binding motif (3SA), or empty vector. Cells were treated with 25 μM MG-132 for 6 h, and cell lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting for FLAG and SPOP. (I) SPOP knockdown blocks RIF1 degradation by GPR64 ICD. A673 cells were infected with lentiviruses expressing the FLAG-GPR64 ICD or empty vector, followed by transfection with SPOP siRNA or control siRNA. The protein levels of SPOP, FLAG-ICD, RIF1, and tubulin were assessed by immunoblotting. (J) SPOP knockdown blocks RIF1 degradation by ceramide. A673 cells were transfected with SPOP siRNA or control siRNA and treated with 1 μM C18 ceramide for 16 h. The protein levels of SPOP, RIF1, and tubulin were assessed by immunoblotting. (K) GPR64 ICD harboring a deletion of residues 906–956 does not rescue growth arrest induced by GPR64 knockdown. A673 cells were infected with lentiviruses expressing FLAG-ICD del lacking residues 906–956 or empty vector, followed by transfection with control siRNA or GPR64 siRNA. (Left) The protein levels of endogenous full-length GPR64, FLAG-ICD del, RIF1, and tubulin were assessed by immunoblotting. (Right) Proliferation of cells was assessed by the IncuCyte. (L) GPR64 ICD harboring mutations of the SPOP-binding motif does not rescue growth arrest induced by GPR64 knockdown. A673 cells were infected with lentiviruses expressing FLAG-ICD 3SA with mutations of the SPOP-binding motif or empty vector, followed by transfection with control siRNA or GPR64 siRNA. (Left) The protein levels of endogenous full-length GPR64, FLAG-ICD 3SA, RIF1, and tubulin were assessed by immunoblotting. (Right) Proliferation of cells was assessed by the IncuCyte.

Article Snippet: Importantly, growth arrest of Ewing sarcoma cells upon SMPD1 knockdown was rescued by adding recombinant purified SMPD1 protein (R&D Systems, #5348-PD) to the culture medium , indicating that Ewing sarcoma depends on extracellular SMPD1.

Techniques: Infection, Expressing, Plasmid Preparation, Western Blot, Knockdown, Transfection, Control, shRNA, Concentration Assay, Immunoprecipitation, Binding Assay

Journal: Cell reports

Article Title: Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma

doi: 10.1016/j.celrep.2024.114497

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Importantly, growth arrest of Ewing sarcoma cells upon SMPD1 knockdown was rescued by adding recombinant purified SMPD1 protein (R&D Systems, #5348-PD) to the culture medium , indicating that Ewing sarcoma depends on extracellular SMPD1.

Techniques: Control, Virus, Recombinant, Transfection, SYBR Green Assay, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Plasmid Preparation