1. Volume 23, Issue 3, Pages 838-851 (April 2018)
Mutual Stabilization between TRIM9 Short Isoform and MKK6 Potentiates p38 Signaling to Synergistically Suppress Glioblastoma Progression 
Kunpeng Liu, Chuanxia Zhang, Bowen Li, Weihong Xie, Jindong Zhang, Xichen Nie, Peng Tan, Limin Zheng, Song Wu, Yunfei Qin, Jun Cui, Feng Zhi 
Cell Reports 
Volume 23, Issue 3, Pages (April 2018)
DOI: /j.celrep
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2. Cell Reports 2018 23, 838-851DOI: (10.1016/j.celrep.2018.03.096)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
TRIM9s Inhibits Glioblastoma Progression by Promoting p38 Activation
(A) U251 and LN229 cells were transfected with control (ctrl) siRNA or TRIM9-specifc siRNAs. Cell extracts were collected for immunoblot (IB) analysis with the indicated antibodies.
(B) Schematic diagram of human TRIM9 short and long isoform-containing domains. BB, B-box domain; CC, coiled-coil domain; FN3, fibronectin type 3 domain; SPRY_PRY, splA and the ryanodine receptor domain and associated PRY domain.
(C) Lysates from control or TRIM9s-overepxressing LN229 cells were used for immunoblotting with the indicated antibodies. EV, empty vector.
(D) Immunoblot analysis of TRIM9 expression in each clone of TRIM9s and TRIM9l-knockout (KO) LN229 cells.
(E) Lysates of control (ctrl) or TRIM9s-knockout (KO) LN229 or U251 cells were used for immunoblotting with the indicated antibodies.
(F) Lysates of control (ctrl) or TRIM9s-knockdown (KD) primary glioblastoma (GBM) cells were used for immunoblotting with the indicated antibodies.
(G) Proliferation assay of TRIM9s- and TRIM9l-overexpressing LN229 (left) and U251 (right) cells compared with control LN229 and U251 cells containing empty vector (EV).
(H) Anchorage-independent soft agar assay of TRIM9s- and TRIM9l-overexpressing U251 and LN229 cells. Scale bar, 200 μm. Representative cell clones in soft agar are shown. Quantitative analysis of colony numbers is shown in the right panel.
(I) Proliferation assay of each clone of TRIM9s and TRIM9l-KO LN229 cells.
(J) Anchorage-independent soft agar assay of TRIM9s and TRIM9l-KO LN229 cells. Representative cell clones in soft agar are shown. Quantitative analysis of the colony numbers is shown in the right panel.
(K) Xenograft tumors derived from TRIM9s-overepxressing LN229 cells or control LN229 cells containing empty vector (EV) were harvested at 2.5 months post-injection. Representative images of tumor growth are shown; the measured tumor weights (n = 5) are presented in the right panel.
(L) Lysates from control or TRIM9s-overepxressing LN229 xenograft tumors were used for immunoblotting with the indicated antibodies. EV, empty vector.
(M) Xenograft tumors derived from control (ctrl) or TRIM9s-KO LN229 cells were harvested at 1 month post-injection. Representative images of tumor growth are shown; the measured tumor weights (n = 5) are presented in the right panel.
(N) Lysates from control or TRIM9s-KO LN229 xenograft tumors were used for immunoblotting with the indicated antibodies.
The data in (F)–(I) are presented as means ± SD of at least three independent experiments. ∗∗∗p < (Student’s t test or one-way ANOVA test).
See also Figure S1.
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4. Figure 2 TRIM9s Targets MKK6 to Inhibit Glioblastoma Cell Growth
(A) Lysates of LN229 cells transfected with HA-TRIM9s and other indicated plasmids were immunoprecipitated with anti-FLAG beads, followed by immunoblot analysis with the indicated antibodies.
(B) Lysates of U251 cells transfected with FLAG-TRIM9s, TRIM9l, and TRIM9l mutants were immunoprecipitated with anti-FLAG beads, followed by immunoblot analysis with the indicated antibodies. FL, full-length; SD, shared domain with TRIM9s; UD, the unique domain of TRIM9l.
(C) Lysates of control (ctrl) or TRIM9s/MKK6-knockout LN229 cells were immunoprecipitated with anti-TRIM9 antibody, followed by immunoblot analysis with the indicated antibodies.
(D) MKK6-knockout (KO) or control (ctrl) LN229 cells overexpressing GFP-TRIM9s or empty vector (EV) were harvested for immunoblot analysis with the indicated antibodies.
(E) Proliferation assay of LN229 control or MKK6-KO cells with the ectopic expression of empty vector (EV) or TRIM9s.
(F) Colony formation assay of LN229 control or MKK6-KO cells with the ectopic expression of empty vector (EV) or TRIM9s. Quantitative analysis of colony numbers is shown in the right panel.
(G) qPCR analysis was performed to measure the mRNA levels of BIRC5, CCNE2, and CCNA1 in the indicated cells. The data in (E)–(G) are presented as means ± SD of at least three independent experiments. ∗∗∗p < (Student’s t test or one-way ANOVA test).
See also Figure S2.
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5. Figure 3
TRIM9s Stabilizes MKK6 by Mediating the K48-K63 Ubiquitination Transition of MKK6
(A) Single clones of TRIM9s or TRIM9l-knockout (KO) LN229 cells were harvested for immunoblotting with the indicated antibodies. Bottom: RT-PCR analysis of MKK6 mRNA is shown; GAPDH mRNA (encoding glyceraldehyde phosphate dehydrogenase) serves as a loading control. The data from three independent experiments are presented as means ± SD. ∗∗∗p < (Student’s t test).
(B) Immunoassay of extracts of TRIM9s-KO or control (ctrl) LN229 cells treated with CHX (100 μg/mL) at the indicated time points. Densitometric analysis of MKK6/Actin in control or TRIM9s-KO LN229 cells is shown in the right panel.
(C) Immunoassay of extracts of U251 cells transfected with various combinations of plasmids for FLAG-MKK6, Myc-TRIM9s, HA-ubiquitin, or its mutants and treated with MG132 (10 μM, 8 hr), followed by immunoprecipitation with anti-FLAG beads and immunoblot analysis with anti-HA.
(D) Co-immunoprecipitation of extracts of LN229 cells with or without TRIM9s/9l deficiency with anti-MKK6 antibody, followed by immunoblot analysis of K63 and K48 ubiquitination of MKK6 with the indicated antibodies.
(E) U251 cells were transfected with Myc-MKK6, FLAG-TRIM9s, FLAG-TRIM9s mutant without Ring domain (ΔRing), and HA-tagged K48 or K63 ubiquitin mutants, in the presence of MG132 (10 μM). Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblot analysis.
(F) TRIM9s- or TRIM9l-overexpressing U251 cells were transfected with the indicated plasmids in the presence of MG132 (10 μM). Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblot analysis.
(G) U251 cells were transfected with Myc-MKK6 or Myc-MKK6-K82R mutant, together with HA-K48/63 ubiquitin mutants in the presence of MG132 (10 μM). Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblot analysis.
(H) U251 cells were transfected with various combinations of plasmids for wild-type Myc-MKK6 (WT), Myc-MKK6-K82R, FLAG-TRIM9s, and HA-K48 or K63 ubiquitin mutants in the presence of MG132 (10 μM). Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblot analysis.
See also Figure S3.
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6. Figure 4 MKK6 Is Essential for Glioblastoma Suppression
(A) Cell counting of MKK6-overexpressing U251 cells and LN229 cells. Bottom: immunoblot analysis of extracts of MKK6-overexpressing (FLAG-MKK6) U251 and LN229 cells.
(B) Colony formation assay of MKK6-overexpressing U251 cells and LN229 cells.
(C) Cell counting and colony formation assay were used to measure the proliferation of MKK6-KO or control LN229 cells.
(D) Cell counting of MKK6-knockout (KO) LN229 cells with or without MKK6-EE/AA overexpression. Right: immunoblot analysis of extracts of MKK6-EE/AA-overexpressing LN229 cells is shown.
(E) Control (ctrl), TRIM9s-overexpressing, and MKK6-overexpressing LN229 cells were intracranially injected into athymic nude mice. After 6 weeks, the mice were sacrificed and tumor growth was examined. Top: H&E-stained coronal brain sections show representative tumor xenografts. Bottom, left panel: the TRIM9/MKK6 levels were detected by immunoblot analyses in these established stable cells. Bottom, right panel: the tumor areas were measured using the length (a) and width (b) and were calculated using the equation V = ab/2. Data represent the results of three mice per group of two independent experiments. Data are presented as the mean ± SD of at least three independent experiments. ∗∗p < 0.01 (one-way ANOVA test).
(F) Control (ctrl), TRIM9s-overexpressing, and MKK6-overexpressing LN229 cells were intracranially injected into athymic nude mice. After 10 weeks, the mice were sacrificed. Survival curves of mice bearing intracranial GBM were determined in TRIM9s-overexpressed tumors (n = 6) or MKK6-overexpressed tumors (n = 6) compared with the control group (n = 6). p = between TRIM9s overexpression and EV group comparison, log rank test; p = between MKK6 overexpression and EV group comparison, log rank test.
(G) MKK6 mRNA levels were detected in normal brain tissue (n = 20) or glioblastoma tissue (n = 78) samples. RPL13A was used as an internal control.
(H and I) Representative images of the histological analysis of MKK6 expression in glioblastoma tissues compared with that in normal brain tissues (H). Scale bar, 100 μm. Quantitative scoring of the staining of MKK6 is shown in (I).
(J) IHC assays using anti-MKK6 antibody were performed for human glioblastoma specimens compared with adjacent tissue of the tumor; representative images of the tumors are shown. Scale bar, 200 μm.
(K) Kaplan-Meier survival curves of overall survival duration based on the scores of MKK6 IHC staining.
The data in panels (A)–(D), (G), and (I) are presented as means ± SD of at least three independent experiments. ∗∗∗p < (Student’s t test or one-way ANOVA).
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7. Figure 5
MKK6 Stabilizes TRIM9s by Blocking the Ubiquitin-Proteasome Pathway
(A) A single clone of LN229 cells with MKK6-knockout (KO) or control (ctrl) LN229 cells was harvested for immunoblot analysis with the indicated antibodies (top), as well as RT-PCR analysis of TRIM9 mRNA (bottom). GAPDH mRNA (encoding glyceraldehyde phosphate dehydrogenase) served as a loading control.
(B) U251 cells transfected with the indicated plasmids were treated with cycloheximide (CHX, 100 μg/mL) for the indicated time points, and then they were harvested for immunoblot analysis. Densitometric analysis of TRIM9s/Actin in U251 cells expressing empty vector (EV) or FLAG-MKK6 is shown in the bottom panel.
(C) U251 cells transfected with Myc-TRIM9s, FLAG-MKK6, or HA-ubiquitin (Ub) were treated with MG132 (10 μM) for 8 hr, and then they were harvested for immunoprecipitation by anti-Myc beads, followed by immunoblotting with the indicated antibodies.
(D) U251 cells transfected with FLAG-MKK6 or empty vector were treated with MG132 for 8 hr. After immunoprecipitation with the anti-TRIM9 antibody, immunoblot analysis was performed to detect the K48-linked ubiquitination of endogenous TRIM9.
(E) Lysates of U251 cells transfected with Myc-MKK6-EE, Myc-MKK6-AA, and FLAG-TRIM9s, together with HA-K48-Ub in the presence of MG132 (10 μM), were immunoprecipitated with anti-FLAG antibody, followed by immunoblot analysis to detect TRIM9s ubiquitination.
(F) U251 cells transfected with the indicated plasmids were treated with CHX for the indicated time points and were harvested for immunoblot analysis. Densitometric analysis of HA-TRIM9s/Actin in U251 cells expressing FLAG-MKK6-EE or FLAG-MKK6-AA is shown in the right panel. The data in (B) and (F) are presented as means ± SD of at least three independent experiments. ∗∗∗p < (one-way ANOVA).
See also Figure S4.
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8. Figure 6
The MKK6-p38 Axis Enhances TRIM9s Phosphorylation to Inhibit the Ubiquitination of TRIM9s
(A) Control or TRIM9-knockout (KO) U251 cells were transfected with the indicated plasmids for 24 hr before harvesting. Cell lysates were immunoprecipitated with anti-TRIM9 antibody, followed by immunoblot analysis.
(B) TRIM9 antibody was used to pull down endogenous TRIM9 proteins in control or MKK6/TRIM9-KO LN229 cells, followed by immunoblot analysis.
(C) U251 cells transfected with Myc-MKK6, HA-ubiquitin, FLAG-TRIM9s, or FLAG-TRIM9s-SA mutant plasmids were treated with MG132 (10 μM) for 8 hr, and then they were harvested for immunoprecipitation with anti-FLAG beads, followed by immunoblotting with the indicated antibodies.
(D) Co-immunoprecipitation and immunoblot analysis of U251 cells transfected with Myc-TRIM9s and FLAG-p38-EE or p38-AA mutants.
(E) Immunoassay of extracts of U251 cells transfected with HA-TRIM9s and EV or FLAG-p38-EE, followed by treatment with CHX for the indicated time points. Densitometric analysis of HA-TRIM9s/Actin in U251 cells expressing control vector or FLAG-p38-EE is shown in the bottom panel. The data from three independent experiments are presented as means ± SD ∗∗∗p < (Student’s t test).
(F) Co-immunoprecipitation and immunoblot analysis of the extracts of U251 cells transfected with various combinations of plasmids for FLAG-p38-EE and HA-Ub together with Myc-TRIM9s or its SA mutant, followed by treatment with MG132 (10 μM) for 8 hr.
(G) Co-immunoprecipitation and immunoblot analysis of the extracts of control or p38-KO LN229 cells transfected with or without FLAG-MKK6, followed by treatment with MG132 (10 μM) for 8 hr.
See also Figure S5.
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9. Figure 7
Correlated Reduction of MKK6 and TRIM9s Predicts Poor Survival Outcomes
(A) The TRIM9 mRNA expression level in the normal brain (normal) (n = 20) and glioblastoma tissue (tumor) (n = 78) samples was determined by qPCR assay. The data are presented as means ± SD. ∗∗p < 0.01 (Student’s t test).
(B) Immunohistochemical staining of 20 normal human brain tissue specimens and 78 glioblastoma specimens with the anti-TRIM9 antibody. Scale bar, 100 μm. Quantitative analysis of TRIM9 staining is shown as scores. The data are presented as means ± SD. ∗∗∗p < (Student’s t test).
(C) Immunoblot analysis of the TRIM9, phosphor-MKK6 (p-MKK6), MKK6, phosphor-p38 (p-p38), and p38 protein levels in glioblastoma tissues compared with those in adjacent tissues. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as a loading control.
(D) Human glioblastoma specimens were immunohistochemically stained with anti-MKK6 or anti-TRIM9 antibodies. Representative images are shown. Scale bar, 100 μm. The correlation of MKK6 and TRIM9 was statistically significant among different specimens (n = 78, r = , p < ).
(E) Paired samples of adjacent tissues (n = 10) and glioblastoma tissues (n = 10) were double-stained with anti-MKK6 (red) and TRIM9 (green) antibodies. Representative images are shown. Scale bars, 200 μm. In three randomly selected microscope fields of each specimen, the percentages of TRIM9/MKK6 double-stained cells and TRIM9-negative but MKK6-positive cells were analyzed and compared (mean ± SEM; ∗∗∗p < 0.001, unpaired Student’s t test).
(F) Kaplan-Meier survival curves of overall survival duration based on the TRIM9 and MKK6 protein levels in the glioblastoma tissues of patients (n = 78).
(G) Work model of the mutual stabilization of MKK6 and TRIM9s in human glioblastoma.
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