Journal: International Journal of Molecular Sciences

Article Title: Sonic Hedgehog-Gli1 Signaling and Cellular Retinoic Acid Binding Protein 1 Gene Regulation in Motor Neuron Differentiation and Diseases

doi: 10.3390/ijms21114125

Figure Lengend Snippet: Crabp1 expression in motor neuron disease cell models. ( a ) CRABP1 mRNA levels in SOD1 WT and SOD1 G93A transfected MN1 cells (modeling ALS). ( b ) CRABP1 mRNA levels in AR-24Q and AR-65Q transfected MN1 cells (modeling SBMA). Abbreviations: ALS = amyotrophic lateral sclerosis, SBMA = spinal and bulbar muscular atrophy.

Article Snippet: ALS cell disease model: Plasmid plv-AcGFP-SOD1 WT (27138, Addgene, Cambridge, MA, USA) and G93A (27142, Addgene) were purchased from Addgene.

Techniques: Expressing, Transfection

Journal: Cell Death & Disease

Article Title: USP21 promotes self-renewal and tumorigenicity of mesenchymal glioblastoma stem cells by deubiquitinating and stabilizing FOXD1

doi: 10.1038/s41419-022-05163-3

Figure Lengend Snippet: a Co-IP showing that FOXD1 was physically conjugated with USP21 among the predicated proteins(COPS5, SENP3, USP22, USP21). Anti-Flag antibody was used to bind Flag-tagged USP21 WT or C221A. Anti-Myc antibody was used to bind Myc-tagged FOXD1. b Western blotting showing that transiently transfecting of USP21-overexpressing plasmid altered the expression of FOXD1 in a dose-dependent manner. c Western blotting showing that knockdown of USP21 attenuated the protein expression of FOXD1 in U87, U251, T98G and LN229 GBM cell lines. d Western blotting shows that knockdown of USP21 attenuated the expression of FOXD1 in MES 21 and 505 GSCs, whereas treatment with MG-132 (20 μM) abolished the effect of the knockdown of USP21 in MES 21 and 505 GSCs thus increasing the expression of FOXD1. e Western blotting showing that the overexpression of an shRNA-resistant WT, but not C221A mutant, USP21 altered the effect of the knockdown of USP21 in MES 21 and 505 GSCs thus increasing the expression of FOXD1. f Western blotting showing that the overexpression of wild-type USP21 (USP21 WT) but not the catalytically inactive USP21 mutant (USP21 C221A) stabilized FOXD1. *** P < 0.001. g , h Western blotting showing that the knockdown of USP21 in MES 21 ( g ) and 505 ( h ) GSCs resulted in accelerated degradation of FOXD1.*** P < 0.001.

Article Snippet: Anti-USP21 (catalog MA5-34953), anti-FOXD1 (catalog PA5-27142) were purchased from Invitrogen.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Expressing, Over Expression, shRNA, Mutagenesis

Journal: Cell Death & Disease

Article Title: USP21 promotes self-renewal and tumorigenicity of mesenchymal glioblastoma stem cells by deubiquitinating and stabilizing FOXD1

doi: 10.1038/s41419-022-05163-3

Figure Lengend Snippet: a Co-IP showing FOXD1 interaction with USP21 WT and C221A mutant. Anti-Flag antibody was used to bind Flag-tagged USP21 WT or C221A. Anti-Myc antibody was used to bind Myc-tagged FOXD1. b Co-IP validation of the interaction between USP21 and FOXD1 in MES 21 and 505 GSCs. c The GST-Pulldown assay showing the direct interaction between USP21 and FOXD1. d Schematic structures of USP21 and FOXD1, together with their truncated mutants. e Co-IP showing that the C terminal of USP21 is essential for the interaction with FOXD1. f Co-IP shows that the C terminal of FOXD1 is indispensable for the interaction with USP21.

Article Snippet: Anti-USP21 (catalog MA5-34953), anti-FOXD1 (catalog PA5-27142) were purchased from Invitrogen.

Techniques: Co-Immunoprecipitation Assay, Mutagenesis, GST Pulldown Assay

Journal: Cell Death & Disease

Article Title: USP21 promotes self-renewal and tumorigenicity of mesenchymal glioblastoma stem cells by deubiquitinating and stabilizing FOXD1

doi: 10.1038/s41419-022-05163-3

Figure Lengend Snippet: a Co-IP showing that USP21 WT, but not C221A, reduced polyubiquitinated FOXD1. Anti-HA antibody was used to bind HA-tagged Ub to indicate ubiquitination. b Co-IP showing that the knockdown of USP21 increased accumulation of polyubiquitinated FOXD1 in MES 21 and 505 GSCs. Anti-HA antibody was used to bind HA-tagged Ub to indicate ubiquitination. c GST-Pulldown assay showing that FOXD1 is a direct deubiquitinated substrate of USP21. d Co-IP showing that USP21 efficiently disassembled K48-linked polyubiquitylation of FOXD1 but had no significant effect on monoubiquitylation or the K11, K27, K63-linked polyubiquitylation of FOXD1. Anti-HA antibody was used to bind HA-tagged Ub to indicate ubiquitination. e Western blotting showing that enforced expression of Lys48R ubiquitin attenuated USP21 depletion–induced FOXD1 downregulation.

Article Snippet: Anti-USP21 (catalog MA5-34953), anti-FOXD1 (catalog PA5-27142) were purchased from Invitrogen.

Techniques: Co-Immunoprecipitation Assay, GST Pulldown Assay, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: USP21 promotes self-renewal and tumorigenicity of mesenchymal glioblastoma stem cells by deubiquitinating and stabilizing FOXD1

doi: 10.1038/s41419-022-05163-3

Figure Lengend Snippet: a Immunofluorescence assays showing that USP21 colocalizes with FOXD1 in the nuclei of MES 21 and 505 GSCs. Scale bar: 30 μm ( b , c ). Primary neurosphere formation showing that the silencing of USP21 considerably attenuated cell growth, and the effect of USP21-knockdown could be largely rescued by FOXD1. Right panel ( c ) showing the quantification of neurosphere formation efficiency (spheres/cells plated). Data are shown as mean ± S.D., n = 3, * P < 0.05, ** P < 0.01, Student’s t -test. d In vitro limiting dilution sphere-forming frequency showing that the knockdown of USP21 remarkably reduced the tumorsphere formation frequency of MES21 and 505 GSCs, and the effect of USP21-knockdown could be rescued by FOXD1. Stem cell frequencies were estimated as the ratio 1/x with the upper and lower 95% confidence intervals, where 1 = stem cell and x = all cells. * P < 0.05. e Western blotting showing that the USP21/FOXD1 axis has great importance in the maintenance of core MES-specific markers, including ALDH1A3, CD44, C/EBPβ, TAZ, phosphorylated STAT3 (p-STAT3), and c-MET. f Representative bioluminescent images showing that USP21-knockdown MES 21 and 505 GSCs had lower tumorigenicity abilities than control GSCs while the tumorigenicity abilities could be rescued by FOXD1. n = 10. g Kaplan–Meier survival curves showing that mice bearing xenograft tumors formed by USP21-knockdown GSCs had a longer lifespan than control GSCs, and the survival time in mice intracranially injected with USP21-depleted GSCs significantly decreased after FOXD1 overexpression. n = 10, *** P < 0.001, Log-rank test. h Representative H&E and IHC images showing that the xenografts carrying USP21 shRNA GSCs displayed restricted tumor growth, whereas this effect could be reversed by overexpression of FOXD1.

Article Snippet: Anti-USP21 (catalog MA5-34953), anti-FOXD1 (catalog PA5-27142) were purchased from Invitrogen.

Techniques: Immunofluorescence, In Vitro, Western Blot, Injection, Over Expression, shRNA

Journal: Cell Death & Disease

Article Title: USP21 promotes self-renewal and tumorigenicity of mesenchymal glioblastoma stem cells by deubiquitinating and stabilizing FOXD1

doi: 10.1038/s41419-022-05163-3

Figure Lengend Snippet: a Co-IP showing that the ability of USP21 to remove ubiquitin moieties from polyubiquitinated FOXD1 was almost completely abrogated by disulfiram. b Western blotting showing that disulfiram, like USP21 knockdown, promotes FOXD1 ubiquitination and degradation. c Western blotting showing that disulfiram induced a marked decrease in the stabilization of FOXD1 protein. *** P < 0.001, ** P < 0.01. d In vitro limiting dilution sphere-forming frequency showing that the disulfiram treatment reduced the tumorsphere formation frequency of MES21 and 505 GSCs. ** P < 0.01. e Western blotting showing that disulfiram treatment notably inhibited the core MES GSC markers including ALDH1A3, CD44, TAZ, p-STAT3, c-MET and c/EBPβ. f Representative bioluminescent images showing that tumor-bearing mice receiving disulfiram treatment showed retarded tumor growth compared with vehicle-treated mice. n = 10. g Kaplan–Meier survival curves showing that mice bearing xenograft tumors receiving disulfiram treatment had a shorter lifespan than vehicle-treated mice. n = 10, *** P < 0.001, Log-rank test. h Representative H&E and IHC images showing that disulfiram attenuated the tumor growth and the expression of FOXD1 and CD44 in tumor tissues.

Article Snippet: Anti-USP21 (catalog MA5-34953), anti-FOXD1 (catalog PA5-27142) were purchased from Invitrogen.

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Vitro, Expressing

Journal: Cell Death & Disease

Article Title: USP21 promotes self-renewal and tumorigenicity of mesenchymal glioblastoma stem cells by deubiquitinating and stabilizing FOXD1

doi: 10.1038/s41419-022-05163-3

Figure Lengend Snippet: a Representative IHC images of USP21 and FOXD1 expression in MES GBM specimens. b Correlation between USP21 and FOXD1 proteins in 91 MES GBM IHC results. c Kaplan–Meier plots of overall survival and progression-free survival of 91 MES GBM patients stratified by protein expression of USP21 showing that USP21 hi MES GBM patients displayed significantly shorter overall survival and progression-free survival than USP21 low MES GBM patients. ** P < 0.01. d Schematic illustration of USP21-mediated FOXD1 stabilization, promoting self-renewal and tumorigenicity of MES GSCs.

Article Snippet: Anti-USP21 (catalog MA5-34953), anti-FOXD1 (catalog PA5-27142) were purchased from Invitrogen.

Techniques: Expressing