Journal: The Journal of Clinical Investigation

Article Title: Disruption of the KLHL37–N-Myc complex restores N-Myc degradation and arrests neuroblastoma growth in mouse models

doi: 10.1172/JCI176655

Figure Lengend Snippet: ( A ) MYCN -amplified (MYCN-Amp) or non- MYCN -amplified (MYCN-Non) cells were transfected for 72 hours with a siRNA library specifically targeting KLHL proteins. The effect of the siRNAs on cell proliferation was examined and visually presented as a heatmap. SRB, sulforhodamine B. ( B ) KLHL37 expression in tumor ( n = 59) or adjacent ( n = 8) tissue was analyzed. FPKM, fragments per kilobase of transcript per million mapped reads. ( C ) The correlation between expression levels of KLHL12 , KLHL30 , and KLHL37 and clinical progression staging of patients with neuroblastoma was analyzed in comparison with the GEO GSE120572 cohort. The numbers of patients with stage 1, n = 54; stage 2, n = 67; stage 3, n = 58; stage 4, n = 168; stage 4S, n = 46, respectively. INSS, International Neuroblastoma Staging System. ( D ) Kaplan-Meier survival curves for OS of neuroblastoma patients with high ( n =138) or low ( n = 138) KLHL12 , KLHL30 , and KLHL37 expression levels. ( E ) Analysis of KLHL37 expression in patients with MYCN -amplified ( n = 55) or non- MYCN -amplified ( n = 222) neuroblastoma. Data were derived from the GEO GSE85047 cohort. ( F ) Effect of KLHL37-Flag overexpression on the clonogenic capacity of tumor cells with high N-Myc expression (SH-SY5Y and RH30) or low N-Myc expression (RD and NCI-H1299). ( G ) GSEA of the correlation of MYCN target gene signature enrichment with high KLHL37 expression in the GEO GSE85047 cohort. NES, normalized enrichment score; WEI, gene set. ( H ) Analysis of the impact of high KLHL37 expression and MYCN amplification on the progression-free survival (PFS) or OS of the patients with neuroblastoma based on the GEO GSE85047 cohort. The numbers of patients in the 4 groups are 211, 11, 44, and 11, respectively. ( I ) Effect of KLHL37 overexpression on the colony-forming ability of NIH-3T3 cells exogenously overexpressing N-Myc or C-Myc. * P < 0.05, ** P < 0.01 and *** P < 0.001, by unpaired, 2-tailed Student’s t test ( B , E , and F ) and 1-way ANOVA ( C and I ). Data represent the mean ± SEM, except in F and I where data represent the mean ± SD.

Article Snippet: Antibodies against KLHL37 (7a, sc-517590) and Ub (P4D1, sc-8017) were purchased from Santa Cruz Biotechnology.

Techniques: Amplification, Transfection, Expressing, Comparison, Derivative Assay, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: Disruption of the KLHL37–N-Myc complex restores N-Myc degradation and arrests neuroblastoma growth in mouse models

doi: 10.1172/JCI176655

Figure Lengend Snippet: ( A ) Colony formation assay of neuroblastoma cells transduced with lentivirus-shKLHL37 (nos. 1 and 2). The colony-forming rate of shKLHL37 groups was calculated. ( B ) Overexpression of KLHL37-synonymous mutants, which were resistant to the knockdown effect of shKLHL37 no. 2, reversed the suppression of SK-N-DZ cell colony formation due to KLHL37 knockdown. ( C ) Effect of KLHL37 knockdown on apoptosis of SK-N-DZ, SK-N-BE(2), and SK-N-AS cells was determined by flow cytometry when cells were infected with shKLHL37 lentivirus for 5 days. ( D ) Analysis of histological staining for N-Myc and markers of proliferation and apoptosis in SK-N-DZ cells. ( E ) Images of SK-N-BE(2) xenograft tumors were captured at the end of the experiment. ( F ) Tumor volumes were measured every 2 days, and tumor growth curves are shown as the mean ± SEM. ( G ) Tumor weights of SK-N-BE(2) xenografts. ( H ) GSEA plots show the correlation between the enrichment of MYCN downstream genes and KLHL37 depletion. ( I ) Heatmap of changes in expression of survival-related genes in the KLHL37 depletion group. min, minimum; max, maximum. ( J ) Histological staining for N-Myc protein and markers of proliferation and apoptosis in SK-N-BE(2) xenograft tumors. Scale bar: 100 μm. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 1-way ANOVA ( A – D , G , and I ), unpaired, 2-tailed Student’s t test ( B ) and 2-way ANOVA ( F ) Data represent the mean ± SD in A , B , C , D , G , and J .

Article Snippet: Antibodies against KLHL37 (7a, sc-517590) and Ub (P4D1, sc-8017) were purchased from Santa Cruz Biotechnology.

Techniques: Colony Assay, Transduction, Over Expression, Knockdown, Flow Cytometry, Infection, Staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Disruption of the KLHL37–N-Myc complex restores N-Myc degradation and arrests neuroblastoma growth in mouse models

doi: 10.1172/JCI176655

Figure Lengend Snippet: ( A ) Effect of KLHL37 knockdown on endogenous N-Myc protein expression. ( B ) KLHL37 depletion efficiency and N-Myc and C-Myc protein expression changes in SK-N-BE(2)-derived xenograft tumors were detected using immunoblotting. ( C ) Effect of KLHL37 overexpression on endogenous N-Myc protein levels in neuroblastoma cells. ( D ) Effect of KLHL37 knockdown on exogenously expressed N-Myc and C-Myc in RD cells. ( E ) Effect of KLHL37 overexpression on exogenous N-Myc protein levels in NIH-3T3 cells. ( F ) Overexpression of KLHL37-synonymous mutants, which were resistant to the knockdown effect of shKLHL37 no. 2, rescued the downregulation of N-Myc protein levels due to KLHL37 knockdown. ( G ) Effect of the proteasome inhibitor MG132 on the decline in N-Myc protein expression induced by knockdown of KLHL37. Cells were infected with lentivirus-shKLHL37 or control shRNA for 3 day and then treated with MG132 (10 μM) for 8 hours. ( H ) Effect of KLHL37 overexpression on the degradation rate of N-Myc protein. HEK-293T cells were infected with lentivirus to express N-Myc protein and then transfected for 48 hours with plasmids to overexpress KLHL37. Before harvesting, the cells were treated with cycloheximide (CHX) (10 μg/mL) for the indicated durations. ( I ) Effect of KLHL37 overexpression on the ubiquitination of N-Myc protein in the cell system. HEK-293T cells with stable expression of N-Myc-HA were transfected with plasmids to overexpress KLHL37 and His-Ub for 48 hours. Then cells were treated with MG132 (10 μM) for 8 hours before being harvested. ( J ) Effect of KLHL37 on the ubiquitination of N-Myc in vitro with the RRL system. ( K ) Effect of KLHL37 overexpression on the endogenous ubiquitination of N-Myc. CHP-126 cells were transfected for 48 hours with plasmids to overexpress KLHL37. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 1-way ANOVA ( G ) and 2-way ANOVA ( H ). Data represent the mean ± SD in G and H .

Article Snippet: Antibodies against KLHL37 (7a, sc-517590) and Ub (P4D1, sc-8017) were purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Expressing, Derivative Assay, Western Blot, Over Expression, Infection, Control, shRNA, Transfection, Ubiquitin Proteomics, In Vitro

Journal: The Journal of Clinical Investigation

Article Title: Disruption of the KLHL37–N-Myc complex restores N-Myc degradation and arrests neuroblastoma growth in mouse models

doi: 10.1172/JCI176655

Figure Lengend Snippet: ( A ) The interaction between exogenous KLHL37 and N-Myc was detected using an IP assay. HEK-293T cells were transfected for 48 hours with plasmids overexpressing KLHL37-HA and N-Myc–Flag. ( B ) Cells were transfected for 48 hours with plasmids to overexpress KLHL37. KLHL37-Flag was enriched using anti-Flag resin, and then N-Myc signal was determined by immunoblotting. ( C ) A PLA was performed to detect the in situ interaction between KLHL37 and N-Myc proteins in SK-N-BE(2) cells. Scale bar: 10 μm. ( D ) Direct interaction between KLHL37-Flag and GST–N-Myc recombinant proteins in a cell-free system. KLHL37 bound to N-Myc was detected by immunoblotting, and other signals were visualized as Coomassie brilliant blue staining. ( E ) MST was performed to detect the interaction affinity of recombinant KLHL37 protein and recombinant GFP–N-Myc (rGFP–N-Myc) and GFP–C-Myc (rGFP–C-Myc) proteins. Mean ± SD.( F ) HEK293T cells were transfected with different plasmids to overexpress N-Myc, WT KLHL37, and the truncated form of KLHL37 (Kelch repeats) for 48 hours. ( G ) Competition between KLHL37 and FBXW7 in binding to N-Myc proteins. GST or GST–N-Myc recombinant proteins were preincubated with KLHL37-His recombinant protein at 25°C for 4 hours. They were then incubated with cell extracts from HEK-293T cells overexpressing FBXW7-HA at 4°C for 12 hours. ( H ) Cells were transfected with plasmids to overexpress KLHL37 for 48 hours, and the interaction between endogenous N-Myc and FBXW7 was determined by co-IP and immunoblotting. ( I ) Effect of KLHL37 on the in vitro ubiquitination of N-Myc in a RRL cell-free system when recombinant FBXW7 protein was added to the system to promote N-Myc ubiquitination. ( J ) Interaction between KLHL37 and the N-Myc 1–89 aa segment. HEK-293T cells were transfected for 48 hours with plasmids to overexpress the indicated proteins, and the interaction was determined by immunoblotting after a co-IP assay.

Article Snippet: Antibodies against KLHL37 (7a, sc-517590) and Ub (P4D1, sc-8017) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Western Blot, In Situ, Recombinant, Staining, Binding Assay, Incubation, Co-Immunoprecipitation Assay, In Vitro, Ubiquitin Proteomics

Journal: The Journal of Clinical Investigation

Article Title: Disruption of the KLHL37–N-Myc complex restores N-Myc degradation and arrests neuroblastoma growth in mouse models

doi: 10.1172/JCI176655

Figure Lengend Snippet: ( A ) Schematic view of the high-content screening assay for compounds that downregulate N-Myc expression on the basis pf N-Myc immunofluorescence (IF). SK-N-BE(2) cells were treated with a compound library (1 μM) for 9 hours. The library was composed of 306 inhibitors of posttranslational modification enzymes. The compounds are ranked in descending order according to their effectiveness in decreasing N-Myc IF signals. ( B ) Representative IF images of N-Myc and DAPI staining in the groups treated with the indicated compounds. Scale bar: 100 μm. ( C ) Effect of RTA-408 treatment at the indicated concentrations (9 hours) on downregulating N-Myc protein expression. ( D ) Effect of the MG132 (10 μM, 3 hours) on the reduction of N-Myc protein expression induced by RTA-408 treatment. ( E ) Proteome-wide mass spectrometry was performed after neuroblastoma cells [SK-N-DZ, SK-N-BE(2) and CHP-126] were acutely treated with RTA-408 (1 μM, 3 hours). The protein changes identified in the proteomics analysis of the 3 cell lines are presented in the form of a volcano plot. A fold change of 2 or greater and a P value of less than 0.05 indicated significantly altered abundance. ( F ) Kinetics profile of binding of RTA-408, SFN, and DMF to KLHL37-His recombinant protein from the SPR analysis. The binding equilibrium dissociation constant ( K D ) of each compound were presented. ( G ) Representative images following the PLA to determine the in situ effect of RTA-408 on N-Myc–KLHL37 interaction in cells. SK-N-BE(2) cells were treated with RTA-408 (1 μM) as well as MG132 (4 μM) before the PLA. Scale bar: 10 μm. ( H ) The effect of RTA-408 on the direct interaction of KLHL37 and N-Myc proteins in a recombinant protein system. ( I ) The effect of KLHL37 overexpression on the degradation of N-Myc protein induced by RTA-408 treatment in SK-N-BE(2) cells. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 1-way ANOVA ( C ) and 2-way ANOVA ( I ) Data represent the mean ± SD in C , F , and I .

Article Snippet: Antibodies against KLHL37 (7a, sc-517590) and Ub (P4D1, sc-8017) were purchased from Santa Cruz Biotechnology.

Techniques: High Content Screening, Expressing, Immunofluorescence, Drug discovery, Modification, Staining, Mass Spectrometry, Binding Assay, Recombinant, In Situ, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: Disruption of the KLHL37–N-Myc complex restores N-Myc degradation and arrests neuroblastoma growth in mouse models

doi: 10.1172/JCI176655

Figure Lengend Snippet: ( A ) Proliferation of SK-N-BE(2) cells treated with RTA-408 (0.0625–2 μM), SFN (0.3125–10 μM), DMF (0.625–20 μM), or cisplatin (0.625–20 μM) for 72 hours, as measured by SRB assay. The IC 50 values for each compound are shown as a scatter plot. ( B ) Proliferation of MYCN -amplified neuroblastoma PDCs treated with RTA-408 (0.03125–1 μM), SFN (0.3125–10 μM), DMF (0.625–20 μM), or cisplatin (1.5625–50 μM) for 72 hours, as measured by SRB assay. ( C ) PDCs from different patients with neuroblastoma were treated with RTA-408 (1 or 2 μM) for 72 hours, and then cell survival was determined by CellTiter-Glo Luminescent Cell Viability Assay. ( D ) Colony formation assay on SK-N-DZ cells treated with RTA-408 or cisplatin at the indicated concentrations. The colony formation rate was determined as the ratio of the number of clones in the RTA-408 (or cisplatin) treatment group to the number of clones in the control group. ( E ) Apoptosis of SK-N-DZ cells was determined by flow cytometry after cells were treated with RTA-408 or cisplatin (1 μM) for 48 hours. ( F ) Real-time qPCR analysis of representative MYCN downstream target genes in SK-N-DZ cells treated for 24 hours with RTA-408 (0.5 μM). YWHAZ gene is reported to be unaffected by N-Myc and was thus used as a reference control. ( G ) Effect of RTA-408 on the colony-forming ability of RD cells exogenously overexpressing N-Myc, C-Myc. ( H ) KLHL37, N-Myc, and C-Myc protein expression levels in different cancer cell lines were determined by immunoblotting. L.E., long exposure; S.E., short exposure. ( I ) Relationship of IC 50 of RTA-408 and DC 50 showing that RTA-408 promotes N-Myc, C-Myc degradation in different neuroblastoma cells. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed Student’s t test ( F ) and 1-way ANOVA ( A – E , and G ). ### P < 0.001, and NS, no significance, by 2-tailed Student’s t test (RTA-408 vs. cisplatin groups) ( D ). Data represent the mean ± SD.

Article Snippet: Antibodies against KLHL37 (7a, sc-517590) and Ub (P4D1, sc-8017) were purchased from Santa Cruz Biotechnology.

Techniques: Sulforhodamine B Assay, Amplification, Cell Viability Assay, Colony Assay, Clone Assay, Control, Flow Cytometry, Expressing, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Disruption of the KLHL37–N-Myc complex restores N-Myc degradation and arrests neuroblastoma growth in mouse models

doi: 10.1172/JCI176655

Figure Lengend Snippet: ( A ) Tumor growth of SK-N-DZ xenografts. Tumor volume was measured every day, and the growth curves are drawn to show the mean ± SEM ( n = 6). ( B ) Images of SK-N-DZ xenograft tumors were captured 14 days after RTA-408 administration. ( C ) Tumor weight of the SK-N-DZ xenografts 14 days after RTA-408 administration. ( D ) N-Myc and C-Myc protein levels in SK-N-DZ xenografts were detected by immunoblotting. ( E ) GSEA of the correlation of the MYCN target gene enrichment with KLHL37 inhibition via RTA-408 administration. ( F ) Heatmap of changes in expression of survival-related genes upon RTA-408 administration. ( G ) Histological staining for N-Myc protein and markers of proliferation and apoptosis in SK-N-DZ xenograft tumors. Scale bar: 100 μm. ( H ) Tumor growth of relapsed PDXs. Tumor volume was measured every day, and growth curves are drawn to show the mean ± SEM ( n = 5). ( I ) Images of PDX tumors were captured 16 days after RTA-408 administration. ( J ) Tumor weight of the PDX 16 days after RTA-408 or cisplatin administration. ** P < 0.01, and *** P < 0.001, by 2-way ANOVA ( A and G ), 1-way ANOVA ( C , E , and F ), and unpaired, 2-tailed Student’s t test ( D ). Data represent the mean ± SD in C , D , G , and J .

Article Snippet: Antibodies against KLHL37 (7a, sc-517590) and Ub (P4D1, sc-8017) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Inhibition, Expressing, Staining