Targeting USP8 inhibits Cancer Progression and Prevents EV-TβRII-Induced Exhaustion of CD8+ T cells (2022)
Figure 3

Figure 3. USP8 directly deubiquitinates TβRII and stabilizes TβRII at the plasma membrane

a, Immunofluorescence and DAPI staining of HeLa cells transfected with TβRII-HA and Flag-USP8. Scale bar, 10 μm.

b, USP8 interacts with TβRII. Purified prokaryotically expressed USP8 and recombinant GST-TβRII-ICD were incubated in vitro and immunoprecipitated with anti-USP8 antibody; immunoprecipitates were then immunoblotted for TβRII-ICD and USP8 (top panel); Immunoblot analysis of total cell lysate derived from MDA-MB-231 cells treated with TGF-β as indicated and immunoprecipitated with control IgG (ns) or anti-TβRII antibody (bottom panel).

c, Immunoblot analysis of anti-TβRII immunoprecipitates from HEK293T cells stably expressing HA-Ub and in vitro incubated with purified USP8 wt/cs.

d, IB of Nickle-pull down precipitants derived from His-Ub stably expressed HEK293T cells incubated with purified USP8 protein as indicated time points.

e, IB of TCL and immunoprecipitants derived from HEK293T cells transfected with indicated plasmids and treat with or without MG132 (10 μM) for 4 h.

f, IB of TCL and immunoprecipitants derived from HA-Ub stably expressed HEK293T cells transfected with Myc-USP8 wt/cs (left panel) or infected with control (Co.sh) or USP8 shRNA lentivirus (#1 and #2) (right panel) and treated with TGF-β (5 ng/ml) as indicated.

g, IB of HeLa cells stably expressing TβRII-HA and transfected with control empty vector (Co.vector) and Flag-USP8 wt/cs plasmids and treated with CHX (20 μg/ml) for indicated time points.

h, Immunoblot analysis of MCF10A-RAS cells stably expressing USP8 wt/cs or USP8 shRNA (#1 and #2).

i, Immunoblot analysis of biotinylated TβRII in MDA-MB-231 cells ectopically expressing USP8 wt/cs and treated with TGF-β (5 ng/ml) as indicated (left panel). Quantification of the band intensities is shown in right panel. Band intensity was normalized to the t=0 controls (right panel). n = 2 technical replicates.

j, Immunoblot analysis of biotinylated TβRII in MDA-MB-231 cells infected with lentivirus encoding control shRNA (Co.sh), USP8 shRNA (# 1 and # 2) and treated with TGF-β (5 ng/ml) as indicated (left panel). Quantification of the band intensities is shown in right panel. Band intensity was normalized to the t=0 controls (right panel). n = 2 technical replicates.

k, Working model in which USP8 deubiquitylates TβRII thus maintains cell surface level of TβRII that sustains SMAD activation.

l, Expression of USP8 of normal tissues (n = 11) and breast cancer patients (n = 110). Data are means ± s.e.m. p values from Student's t tests were indicated.

m, Scatter-plot showing the positive correlation of USP8, TβRII and p-SMAD2 expression in patients (n = 110). H score of every tissue samples of USP8, TβRII and p-SMAD2 was calculated. Pearson's coefficient tests were performed to assess statistical significance.

n, Percentage of specimens displaying low or high USP8 expression compared to the expression levels of TβRII and P-SMAD2 in human breast cancer tissue (n = 110). p values from Student's t tests were indicated.

o, GEPIA analysis curves showing the metastasis-free survival of breast cancer patients was significantly correlated with USP8 expression by log-rank test in the TCGA and GTEx datasets (http://gepia.cancer-pku.cn/). Cutoff-high = 67%, cutoff-low = 25%.
Data information: *p < 0.05 (two-tailed Student's t test (l), two-way ANOVA (i, j), pearson correlation test (m), log-rank (o)). Data are shown as means ± SD (i, j, l).