Fig 1: ETV4 interacts with COP1 to regulate nuclear β-catenin stability(A) GIST T1 cells were transfected with either ETV4 siRNA or control siRNA for 72h, and then cells were treated with MG132 10 μM for 6h prior to harvest. Nuclear protein extracts were analyzed by western blot as indicated. (B) GIST T1 cells were transfected with control or human ETV4 plasmids for 48h and treated with MG132 10 μM overnight before harvesting. Nuclear protein extracts were analyzed by western blot as indicated. (C) Representative CHX-chase assays (of 2 performed) to determine the stability (half-life) of nuclear β-catenin in GIST T1 cells 48h after transfection with control siRNA or ETV4 siRNA. Cells were collected after the addition of 200 mg/ml CHX at the indicated time points. Relative nuclear β-catenin levels were determined by normalizing to the loading control (lamin B1) and then normalizing to the t = 0h control siRNA. Immunoblots of nuclear extracts are shown. (D) GIST T1 cells were transfected with the indicated siRNA and harvested 48h later and nuclear extracts were analyzed by western blot. (E) GIST T1 cells were transfected with control or ETV4 siRNA for 72h and then treated with MG132 10 μM for 6h prior to harvest. Nuclear extracts were immunoprecipitated by either anti-β-catenin or anti-COP1 and western blot was performed as indicated. (F) GIST T1 cells were transfected with the indicated constructs and harvested 48h later and nuclear extracts were analyzed by western blot.
Fig 2: COP1 was required for KMT5A silencing-induced CD73 ubiquitination. a By analyzing ChIP-seq data of DLBCL, 864 selected genes were revealed to interact with H4K20 me1. b Expression levels of 20 E3 ligases selected by combining CHIP-seq analysis and GO annotations of E3 ligase. c COP1 downregulation restored CD73 expression. d COP1 knockdown DLBCL cells were treated with MG132 and subjected to coimmunoprecipitation and western blot analyses. e DLBCL cells were transfected with various plasmids, as indicated, and subjected to ubiquitination analysis. f DLBCL cells were transfected as indicated and subjected to coimmunoprecipitation and western blot analyses. g DLBCL cells were transfected as indicated and subjected to ubiquitination analysis. h-i Survival analysis of DLBCL patients classified according to KMT5A (h) or COP1 (i) expression
Fig 3: MiR-340-5p suppressed tumor growth by enhancing CD8+ T-TILs in vivo. a KMT5A significantly abrogated the lower tumor growth rate caused by miR-340-5p. Statistical tests were performed using tumor volume data on Day 24. b-d IHC analysis of Ki-67 (b), COP1 (c) and CD8 (d) expression in tumors, as indicated. The CD8+ T-TIL percentage was increased under miR-340-5p treatment and significantly reversed by overexpressing KMT5A, as shown by IHC analysis. e Representative flow cytometry data and cumulative data for CD8+ T-TILs isolated from murine model tumors. In all experiments, cells were first gated on the CD3+ population and then on the CD8+ population. f Representative flow cytometry data and cumulative data for CD69-positive CD8+ T-TILs. g IHC analysis of granzyme B and perforin. Asterisk (*) indicates a significant difference. *, P < 0.05
Fig 4: βTrCP-mediated ubiquitination of TFRC is essential for TRIB2 to decline labile iron.A, B TRIB2 knockout increased while TRIB2 overexpression decreased TFRC expression. TFRC was measured by qPCR and IB in Bel-7404 cells with or without TRIB2 knocked out (A) and in SK-Hep1 cells with or without TRIB2 overexpressed (B). C TRIB2 knockout prolonged while TRIB2 overexpression shortened the half-life of TFRC protein. CHX chase experiments were performed in Bel-7404 cells with or without TRIB2 knocked out and in SK-Hep1 cells with or without TRIB2 overexpressed. D TRIB2 regulated TFRC expression in a PSMB5-independent manner. TFRC was measured by IB in Bel-7404 cells with single or double knockout (DKO) of TRIB2 and PSMB5 or in SK-Hep1 cells with or without TRIB2 overexpression, in the presence or absence of PSMB5 knockout. E βTrCP but not Smurf1 and COP1 was required for TRIB2 to reduce TFRC in SK-Hep1 cells. TFRC was measured by IB before and after overexpressing TRIB2 in SK-Hep1 cells with or without knockout of Smurf1, COP1, and βTrCP. F βTrCP increased the ubiquitination of TFRC. Exogenous Ub-HA, βTrCP-FLAG, and TFRC-Myc were ectopically expressed, as indicated in HEK-293T treating with MG132 (8 µM). The conjugation of TFRC-Myc by Ub-HA was measured by IB using anti-HA antibodies in the immunoprecipitates that immunoprecipitated by anti-Myc antibodies. G TFRC could be ubiquitinated by βTrCP, as measured by an in vitro ubiquitination assay using anti-TFRC antibodies after incubation with purified proteins as indicated. H TRIB2 deletion-reduced ubiquitination of TFRC was blocked after βTrCP was simultaneously knocked out. Ubiquitination of TFRC was measured by anti-Ub antibodies in the immunoprecipitates that immunoprecipitated by the anti-TFRC antibodies in Bel-7404 cells with single or double knockout of TRIB2 and βTrCP. The immunoprecipitates that pulled down by IgG antibodies were parallel examined by anti-TFRC antibodies. I TRIB2 overexpressing-induced ubiquitination of TFRC was diminished after βTrCP was simultaneously knocked out. The measurement for the ubiquitination of TFRC was similarly performed as that in H. J, K TRIB2 altered labile iron was βTrCP-dependent. Expression of TRIB2 and βTrCP and labile iron were measured in Bel-7404 (J) and SK-Hep1 cells (K) under the treatments as indicated. Data were analyzed by Student’s t test and expressed as mean ± SD from three independent experiments. NS non-significance; ***P < 0.001. Images of all the immunoblots are representative of three independent experiments.
Fig 5: COP1 and APCP exhibited in vivo activity on B-cell lymphoma (a-b) APCP treatment significantly decreased the tumor growth rate in COP1 knockdown cells (a), consistent with Ki-67 expression in IHC analysis b. Statistical tests were performed using tumor volume data on Day 15. c IHC analysis showed that fewer CD8+ T-TILs caused by COP1 knockdown were significantly rescued by APCP treatment. d Representative flow cytometry data and cumulative data for CD8+ T-TILs isolated from murine model tumors. In all experiments, cells were first gated on the CD3+ population and then on the CD8+ population. e Representative flow cytometry data and cumulative data for CD69-positive CD8+ T-TILs. f IHC analysis of granzyme B and perforin Asterisk (*) indicates a significant difference. *, P < 0.05
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