Figures and data
WSB2 interacts with multiple members of BCL-2 family proteins in cells.
(A) The top 20 co-dependent genes of WSB2 in Broad’s 21Q2 DepMap dataset. Red, anti-apoptotic BCL-2 family proteins. Blue, pro-apoptotic BCL-2 family proteins.
(B) A co-essential module containing WSB2 and other BCL-2 family proteins using a dataset of CRISPR screens in 485 cell lines from the Achilles DepMap project. Cellular component: Bcl-2 family protein complex. Molecular function: BH domain binding.
(C-E) WB analyses of the indicated proteins in the whole cell lysates (WCL) and co-IP samples of anti-FLAG antibody obtained from 293 T cells transfected with the indicated plasmids.
(F-J) WB analyses of the indicated proteins in WCL and co-IP samples of IgG or anti-NOXA
(F), anti-MCL-1 (G), anti-BCL-2 (H), anti-BCL-XL (I), or anti-BAD (J) antibodies obtained from 293T cells.
(K) Representative IF images from HeLa cells transfected with FLAG-WSB2, stained with FLAG (WSB2), HSP60, and DAPI. Scale bar, 20 µm.
(L) The cytoplasmic (Cyto), mitochondrial (Mito), and nuclear fractions (Nuc) from HeLa cells were prepared as described in Methods section. Histone H3 (nucleus), GAPDH (cytoplasm), and HSP60 (mitochondria) were used as subcellular fraction markers. WB analyses were performed to detect the indicated proteins in three fractions from HeLa cells.
(M) Mitochondria from HeLa cells were purified as intact mitochondria or treated with hypotonic swelling buffer or lysed with Triton X-100 buffer. Different mitochondrial preparations were then digested with or without Proteinase K. WB analyses of the indicated proteins in three fractions were then performed. OM: outer membrane; IMS: intermembrane space.
CRL5WSB2 E3 ubiquitin ligase complex control NOXA protein turnover.
(A) WB analyses of the indicated proteins in the WCL from C4-2B cells infected with lentivirus expressing WSB2-specific shRNA or negative control (NC).
(B, C) WB analyses of the indicated proteins in the WCL from parental or WSB KO C4-2B or Huh-7 cells.
(D) WB analyses of the indicated proteins in the WCL from 293 T cells transfected with the indicated plasmids for 24 h and treated with DMSO or MG132 (20 µM) for 8 h.
(E) WB analyses of the indicated proteins in the WCLs from 293 T cells transfected with the indicated plasmids.
(F) WB analyses of the indicated proteins in the WCL from parental or WSB2 KO C4-2B cells stably overexpressing empty vector (EV), WSB2-WT or its mutants.
(G) WB analyses of the indicated proteins in the WCL from C4-2B cells transfected with the indicated siRNAs.
(H, I) WB analyses of indicated proteins in WCLs of parental and WSB2 KO C4-2B cells treated with cycloheximide (CHX, 50 µg/ml) and harvested at different time points. (G) At each time point, the intensity of NOXA was normalized to the intensity of GAPDH and then to the value at 0h. Data are shown as means ± SD (n=3).
(K-M) WB analyses of the products of in vivo ubiquitination assays performed using WCL from 293T cells transfected with the indicated plasmids and treated with MG132(20 µM).
(N) WB analyses of the products of in vivo ubiquitination assays. Co-IP using anti-IgG or anti-NOXA antibody in the WCL prepared from parental and WSB2 KO C4-2B cells transfected with HA-Ub for 24 h and treated with MG132(20 µM) for 8 h.
P values are calculated by the Two-way ANOVA test in (G) and One-way ANOVA test in (H).
Identification of the mutual-binding regions of WSB2 and NOXA.
(A) Schematic representation of WSB2 deletion mutants.
(B) WB analyses of the indicated proteins in WCLs and co-IP samples of anti-FLAG antibody obtained from 293T cells transfected with the indicated plasmids.
(C) WB analyses of the indicated proteins in the WCL from 293 T cells transfected with the indicated plasmids.
(D) WB analyses of the products of in vivo ubiquitination assays performed using WCL from 293T cells transfected with the indicated plasmids for 24 h and treated with MG132(20 µM).
(E) Schematic representation of NOXA deletion or point mutants.
(F-H) WB analyses of the indicated proteins in the WCLs and co-IP samples of anti-FLAG antibody obtained from 293T cells transfected with indicated plasmids.
(I) WB analyses of the indicated proteins in the WCL from 293 T cells transfected with the indicated plasmids.
(J) WB analyses of the products of in vivo ubiquitination assays performed using WCL from 293T cells transfected with the indicated plasmids and treated with MG132(20 µM).
(K, L) WB analyses of the indicated proteins in the WCL from 293 T cells transfected with the indicated plasmids treated with cycloheximide (CHX, 50 µg/ml) and harvested at different time points. (L) At each time point, the intensity of NOXA was normalized to the intensity of GAPDH and then to the value at 0 h. Data are shown as means ± SD (n=3). P values are calculated by the Two-way ANOVA test.
(M) 293T cells were transfected with the indicated plasmids. WSB2-NOXA complex was co-immunoprecipitated by anti-FLAG antibody, and then the bound complex was incubated with the increasing amounts of C-terminal NOXA peptide (200, 400 µg/ml) or the corresponding scramble peptide for 12 h. Bound material was subjected to WB analyses. C-ter: C-terminal NOXA peptide.
(N) WB analyses of the indicated proteins in WCLs from C4-2B and Huh-7 cells treated with the increasing concentration of C-terminal cell-penetrating peptide of NOXA or the corresponding R8 peptide for 12 h.
Combined inhibition of anti-apoptotic BCL-2 family members and WSB2 induces synthetic lethality.
(A, B) WB analyses of the indicated proteins in the WCL from C4-2B cells infected with lentivirus expressing BCL-XL-specific shRNA or NC and then transfected with the indicated siRNAs for 36 h. (B) Annexin-V-FITC/PI assays were used to stain the harvested cells in (A), of which later flow cytometry analysis was performed. Data are shown as means ± SD (n=3). (C, D) Similar to (A, B), but MCL-1 was knocked down in C4-2B cells.
(E, F) Similar to (A, B), but MARCH5 was knocked down in C4-2B cells.
(G, H) WB analyses of the indicated proteins in the WCL from parental and WSB2 KO C4-2B cells treated with ABT-737 (20 µM) for 6 h. (H) Annexin-V-FITC/PI assays were used to stain the harvested cells in (H), of which later flow cytometry analysis was performed. Data are shown as means ± SD (n = 3).
(I, J) Similar to (A, B), but AZD5991 (10 µM) was used for treatment in C4-2B cells.
(K, L) Similar to (A, B), but BAY-1143572 was used for treatment in C4-2B cells.
P values are calculated by the One-way ANOVA test in (B, D, F) and Two-way ANOVA test in (H, J, L), ns no significant.
WSB2 primarily exerts its anti-apoptotic function largely via destabilizing NOXA.
(A, B) WB analyses of indicated proteins in WCLs from parental or WSB2 KO C4-2B cells infected with lentivirus expressing NOXA-specific shRNA or NC, treated with DMSO or ABT-737 (20 µM) for 6 h. (B) Annexin-V-FITC/PI assays were used to stain the harvested cells, of which later flow cytometry analysis was performed. Data are shown as means ± SD (n = 3). (C, D) Similar to (A, B), but AZD5991 (10 µM) was used for treatment in C4-2B cells.
(E, F) Similar to (A, B), but Huh-7 cells were treated.
(G, H) WB analyses of the indicated proteins in the WCL from Huh-7 cells treated with the cell-penetrating C-terminal NOXA peptide (5 µM) or the corresponding scramble peptide (5 µM) for 12 h and then the cells were treated with increasing doses of ABT-737(10, 20 µM). Annexin-V-FITC/PI assays were used to stain the harvested cells, of which later flow cytometry analysis was performed. Data are shown as means ± SD (n = 3).
(I, J) Huh-7 cells were injected subcutaneously (s.c.) into the right flank of BALB/c mice and treated with ABT-737(30 mg/kg), R8-C-ter (20 mg/kg) or R8 (20 mg/kg) as control at different day points. 6 mice per experimental group. Tumors in each group at day 20 were harvested and photographed (I) and tumor weight (J) was documented. Data are shown as means ± SD (n = 6).
P values are calculated by the Two-way ANOVA test in (B, D, F, H).
Validation of the anti-apoptotic function of WSB2 using Wsb2 KO mouse models.
(A, B) WB analyses of the indicated proteins in the WCL from heart (A) or liver (B) tissues obtained from Wsb2+/+ and Wsb2-/- mice after gavage administration of vehicle or ABT-199 (100 mg/kg/day) for 7 days.
(C) Representative IF images from the heart or liver tissues of Wsb2+/+ and Wsb2-/- mice after oral administration of vehicle or ABT-199 (100 mg/kg/day) for 7 days, and stained with cl-CASP3 and DAPI. Scale bar, 20 µm.
(D, E) The mean fluorescence intensity of cl-CASP3 from the heart (D) or liver tissues (E) obtained from Wsb2+/+ and Wsb2-/- mice. Data were shown as means ± SD (n = 5).
(F) WB analyses of the indicated proteins in the WCL from the primary hepatocytes of Wsb2+/+ and Wsb2-/- mice treated with ABT-199 (10, 20 µM) for 6 h.
(G) The levels of myocardial zymogram in serum from Wsb2+/+ and Wsb2-/- mice after gavage administration of vehicle or ABT-199 (100 mg/kg/day) for 7 days.
(H, I) WB analyses of indicated proteins in WCLs from Wsb2+/+ and Wsb2-/- MEFs infected with lentivirus expressing NOXA-specific shRNA or NC, treated with DMSO or ABT-199 (20 µM) for 6 h. (B) Annexin-V-FITC/PI assays were used to stain the harvested cells, of which later flow cytometry analysis was performed. Data are shown as means ± SD (n = 3).
P values are calculated by the One-way ANOVA test in (G) and Two-way ANOVA test in (D, E).
WSB2 expression is upregulated in multiple human cancers.
(A) WSB2 mRNA expression in normal and tumor tissues from the TCGA cohort.
(B–E) Relationship between WSB2 mRNA expression and Gleason score (B), nodal metastasis status (C), clinical T stage (D), and pathological T stage (E) in PRAD patients from the TCGA cohort.
(F–H) Relationship between WSB2 mRNA expression and clinical stage (F), pathological grade (G), and nodal metastasis status (H) in LIHC patients from the TCGA cohort.
(I, J) Kaplan–Meier survival plots of OS according to WSB2 mRNA expression in PRAD (I) and LIHC (J) patients from the TCGA cohorts.
(K) Representative IHC staining results for WSB2 in PRAD TMA, scale bar, 50 µm.
(L) Quantification analysis of WSB2 IHC staining in PRAD patients by Gleason score categories. n=84.
(M) Representative IHC staining results for WSB2 in LIHC TMA, scale bar, 50 µm.
(N) Quantification analysis of WSB2 IHC staining in LIHC patients by tumor grade categories. n=29.
(O) The WSB2 mRNA levels in 81 sorafenib-response and 66 sorafenib-non-response LIHC patients from GSE104580 dataset.
(P) The WSB2 mRNA levels in 42 sorafenib-response and 98 sorafenib-non-response LIHC patients from GSE109211 dataset.
P values are calculated by the unpaired t test in (A, B-H, L, N, O, P) and log-rank test in (I, J). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
The schematic diagram illustrates that the disruption of the CRL5WSB2 E3 Ubiquitin ligase complex results in NOXA stabilization and increased sensitivity to BCL-2 family protein inhibitors.
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