Fig 1: SPOP-CHAF1A axis controls tumor autophagy of DLBCL in a TFEB-dependent manner. A Positive associations between CHAF1A and TFEB expressions based on TCGA-DLBCL samples. B The RT-qPCR assay showed that CHAF1A OE could promote TFEB expression (left), while CHAF1A KD reduced TFEB mRNA levels (right panel). C The ChIP-qPCR of H3K27ac markers and CHAF1A in promoter of TFEB gene in U2932 cells, as indicated (N = 4). D The RT-qPCR assay showed that CHAF1A KD could suppress TFEB transcriptional activity in U2932 cells (left panel) and FARAGE cells (middle panel). Relative luciferase activities were normalized versus control. The OCI-Ly7 cells were transfected with TFEB reporters together with TFEB vectors alone or TFEB + CHAF1A plasmids to show the regulation of TFEB reporter activity by CHAF1A (right panel). E Western blotting assay showed that CHAF1A-KD decreased the levels of autophagy-related markers (p62, and Beclin-1) in CHAF1A-silenced U2932 and FARAGE cells relative to control cells. F-G The luciferase activity (F) and mRNA levels (G) of lysosomal genes were measured in control and SPOP-OE OCI-Ly7 cells transfected with siCtrl or siTFEB, individually. H The tumor growth curves were obtained from OCI-Ly7-derived tumor models to show that targeting TFEB significantly suppressed the enhanced tumor growth induced by SPOP deficiency. I The tumor weight of mice from indicated groups was shown and compared. Experiments were performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001
Fig 2: CHAF1A knockdown reversed the promotive effects of SPOP deficiency on DLBCL cell autophagy. A, B The confocal microscopy showed the autophagy flux in the indicated groups of DLBCL cells. C The TEM analysis showed the number of autophagic vacuoles in the indicated groups of DLBCL cells
Fig 3: Aberrant SPOP-CHAF1A ubiquitination axis contributes to accumulated CHAF1A expressions in DLBCL. A Co-Immunoprecipitation (IP) analysis based on U2932 cells with IgG or anti-SPOP antibody indicated the endogeous interactions between SPOP and CHAF1A, as shown by western blotting assay. B The schematic representation of SPOP-WT and deletion mutants. Binding capacity of SPOP to CHAF1A is indicated with the representative symbol. C Western blotting assay showed the altered CHAF1A proteins in whole cell lysate (WCLs) from U2932 cells which were co-transfected with plasmids of FLAG-CHAF1A and WT SPOP or mutants. D Western blotting analysis showed the elevated CHAF1A proteins in CHAF1A KD cells (U2932, FARAGE) relative to control cells. Besides, the corresponding mRNA levels of CHAF1A in indicated groups were quantified and shown on the right, and no altereations were observed. E Quantification of CHAF1A proteins in WCLs of U2932 cells (parental, sgSPOP) for 12 h and then treated with 50 µg/ml cycloheximide (CHX) and harvested at different time points. At each time point, the intensity of CHAF1A was normalized to the intensity of actin and then to the data at 0 h. F Quantification of CHAF1A proteins in WCLs of FARAGE cells (EV, myc-SPOP) for 12 h and then treated with 50 µg/ml cycloheximide (CHX) and harvested at different time points. G After the 293 T cells were transfected with the indicated plasmids and treated with 20 µM MG132 for 8 h, the western blotting assay showed the in vivo CHAF1A ubiquitination levels by WT SPOP or mutants. H Illustration of tumor-associated mutations across SPOP gene in lymphoid malignancies. DLBCL: diffuse large B Cell lymphoma; PCM: plasma cell myeloma; ALL: acute lymphoid leukemia; NKTL: NK-T cell lymphoma; CLL: chronic lymphocytic leukemia; unclassified LNs unclassified lymphoid malignancies. I Western blotting assay showed the differential in vivo ubiquitination levels of CHAF1A in 293 T cells transfected with plasmids of FLAG-CHAF1A and Myc-SPOP or relevant mutations. J Comparison of putative SPOP binding sites in CHAF1A with the SPOP-binding consensus motif defined in the known SPOP substrates, where amino acid sequence alignment of the SBC motif (F-p-S-S/T-S/T; F: nonpolar residues, p: polar residues) was identified. K Western blotting assay showed the altered protein levels of WT-CHAF1A and mutants with deleted SBC motif when U2932 cells were transfected with Myc-SPOP, respectively. Experiments were performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001
Fig 4: Down-regulated or mutated SPOP promotes DLBCL maliganant aggressiveness depending on CHAF1A. A The violin plot exhibited the differential expression levels of SPOP between DLBCL and normal tissues based on the GSE83632 data set. B Kaplan–Meier (K–M) survival curves analysis also showed that low SPOP levels may appear to correlate with shorter OS months of DLBCL patients based on the GSE83632 data set. C The CCK8 assays in three DLBCL cell lines (DB, U2932, FARAGE) revealed that WT SPOP, but not the dBTB mutant, could suppress cell proliferation, but simultaneous overexpression of CHAF1A could partially rescue the impaired cell growth ability. D Proliferation ability of OCI-Ly7 cells was enhanced by SPOP depletion, which could be partailly impaired by CHAF1A KD. E Similarly, SPOP depletion could enhance migration of OCI-Ly7 cells, which could be partially suppressed by CHAF1A KD. F Tumor volumes curves of FARAGE-derived tumor models showed that WT SPOP, but not the dBTB mutant, could suppress in vivo tumor growth, whereas tumor-associated SPOP F102I mutant could enhance in vivo tumor growth. G The OCI-Ly7-derived tumor models showed that SPOP depletion could enhance tumor in vivo growth, which could be partially suppressed by CHAF1A KD. Tumor graph was shown on the left; Quantification of tumor volumes was shown on the middle panel; Tumor weight was compared and shown on the right panel. Experiments were performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001
Fig 5: The Cullin 3 SPOP E3 ubiquitin ligase negatively regulates the stability of HnRNPK. A Expression of HnRNPK and SPOP in PrCa cells transfected with miR-206 and miR-613 mimics was detected by Immunoblot analysis. B Relative SPOP mRNA expression levels in PrCa tissues and adjacent normal prostate tissues in a public data set (GSE60329). C Effect of the SPOP expression level on Biochemical recurrence in 36 prostate cancer patients who did not undergo androgen deprivation therapy, chemotherapy, radiotherapy or other anticancer treatment was analyzed, and Kaplan–Meier plots were generated using a Kaplan–Meier Plotter. D WB analysis of WCL and immunoprecipitates (IP) derived from 293 cells transfected with Flag-HnRNPK and various Myc-tagged Cullin constructs. 30 h post-transfection, cells were treated with 10 µM MG132 for 10 h before harvesting. E WB analysis of WCL derived from C4-2 and 22Rv1 (F) cells infected with the indicated lentiviral shRNAs. Infected cells were selected with 1 µg/ml puromycin for 72 h to eliminate non-infected cells before harvesting. G WB analysis of WCL and IP derived from 293 cells transfected with HA-HnRNPK and Flag-tagged BTB domain-containing protein constructs. 30 h post-transfection, cells were treated with 10 µM MG132 for 10 h before harvesting. EV, empty vector. H WB analysis of WCL derived from C4-2 or 293 I cells transfected with increasing doses (0.5–3 µg) of indicated plasmids. Where indicated, 10 µM MG132 was added for 10 h before harvesting. J WB analysis of WCL derived from C4-2 cells infected with the indicated lentiviral shRNAs. Infected cells were selected with 1 µg/ml puromycin for 72 h to eliminate non-infected cells before harvesting. K–N IB analysis of WCL derived from C4-2, 22Rv1, DU145 and mouse embryonic fibroblasts (MEFs) cells with SPOP knockout by the CRISPR technology. O WB analysis of WCLs and His pull-down products derived from 293 cells transfected with indicated constructs and treated with MG132 (10 µM) 10 h. P SPOP knockout cells (sg SPOP) as well as parental C4-2 cells (Con) were treated with 100 µg/ml cycloheximide (CHX), and cells were harvested at the indicated time points. Relative HnRNPK protein abundance was quantified by Image J and plotted in Q. Data was shown as mean ± SD for three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001
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