Journal: Theranostics

Article Title: MicroRNA Biogenesis is Enhanced by Liposome-Encapsulated Pin1 Inhibitor in Hepatocellular Carcinoma

doi: 10.7150/thno.34588

Figure Lengend Snippet: API-LP increases the nucleus-to-cytoplasm export of XPO5 and pre-miRNAs and downregulates the expression of oncogenes. (A-B) Confocal imaging (A) and Western blotting assay (B) of XPO5 subcellular distribution in SK-Hep1 cells treated with DMSO, API-1 or API-LP (1 μM). (C) Relative expression of mature miRNAs, detected by quantitative RT-PCR, in SK-Hep1 cells incubated with DMSO, API-1 or API-LP (1 μM). Data were shown as the means ± SD, n=3. *p < 0.05. (D-E) Immunoblotting (D) and quantitative analysis (E) of DNMT1, STAT3, c-Myc, and GAPDH expression in SK-Hep1 cells treated with DMSO, API-1 or API-LP (1 μM).

Article Snippet: DMPC, DMPG, and DSPE-PEG 2000 were purchased from the Lipoid Company, Germany; CHOL and Sephadex G-25 were obtained from Sigma; anti-DNMT1 antibody was purchased from New England Biolabs; anti-XPO5, anti-ERK, anti-p-ERK, anti-STAT3, anti-c-Myc and anti-GAPDH antibodies were purchased from Cell Signaling Technology.

Techniques: Expressing, Imaging, Western Blot, Quantitative RT-PCR, Incubation

Journal: MedComm

Article Title: Regulation of XPO5 phosphorylation by PP2A in hepatocellular carcinoma

doi: 10.1002/mco2.125

Figure Lengend Snippet: PP2A catalyzes the dephosphorylation of XPO5. (A) HEK‐293T cells were transfected with plasmids expressing MEKDD and different catalytic subunits of serine/threonine phosphatases (PP1, PP2B, and PP2A). The phosphorylated and total XPO5/ERK proteins were detected via Western blot. (B) HEK‐293T cells were transfected with plasmids expressing MEKDD, myc‐XPO5 and C subunits of PP2A and myc‐XPO5 was immunoprecipitated from cell lysates. The immunoprecipitates were immunoblotted with the indicated antibodies. (C) The lysates from HEK‐293T cells co‐transfected with the indicated plasmids were subjected to immunoprecipitation via anti‐myc antibody. The enriched complexes were immunoblotted with the indicated antibodies. MEKDD: constitutively active MEK; p‐XPO5 (416): phosphorylated XPO5 at serine 416.

Article Snippet: Anti‐XPO5, anti‐actin, anti‐PP2A catalytic subunit, anti‐p‐ERK, anti‐ERK, and anti‐myc tag antibodies for Western blot were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: De-Phosphorylation Assay, Transfection, Expressing, Western Blot, Immunoprecipitation

Journal: MedComm

Article Title: Regulation of XPO5 phosphorylation by PP2A in hepatocellular carcinoma

doi: 10.1002/mco2.125

Figure Lengend Snippet: B55β is specifically engaged in XPO5 dephosphorylation. (A) HEK‐293T cells were transfected with plasmids expressing MEKDD and different regulatory subunits. The phosphorylated and total XPO5/ERK proteins were detected via Western blot. (B) SK‐Hep1 cells were transfected with plasmids expressing different regulatory subunits. The phosphorylated and total XPO5 proteins were detected via Western blot and the protein bands were quantified by ImageJ software. Comparison of the intensity of p‐XPO5 bands (using total XPO5 as loading control) was indicated as fold change relative to ctrl set. (C) SK‐Hep1 cells were co‐transfected with the indicated plasmids and myc‐XPO5 was immunoprecipitated from cell lysates. The immunoprecipitates were immunoblotted with the indicated antibodies.

Article Snippet: Anti‐XPO5, anti‐actin, anti‐PP2A catalytic subunit, anti‐p‐ERK, anti‐ERK, and anti‐myc tag antibodies for Western blot were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: De-Phosphorylation Assay, Transfection, Expressing, Western Blot, Software, Immunoprecipitation

Journal: MedComm

Article Title: Regulation of XPO5 phosphorylation by PP2A in hepatocellular carcinoma

doi: 10.1002/mco2.125

Figure Lengend Snippet: B55β regulates XPO5 distribution and miRNA expression. (A) Immunofluorescence of subcellular localization of XPO5 (Red) in Huh‐7 cells transfected with the indicated plasmids. (B) Immunofluorescence of subcellular localization XPO5 (Red) in SK‐Hep1 Ctrl and B55β overexpression cells. (C) The miR‐122 expression in Huh‐7 cells transfected with the indicated plasmids was determined by qRT‐PCR. (D) The expression of miR‐122 and miR‐200b in SK‐Hep1 ctrl and B55β overexpression cells was examined by qRT‐PCR. Data are shown as the means ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: Anti‐XPO5, anti‐actin, anti‐PP2A catalytic subunit, anti‐p‐ERK, anti‐ERK, and anti‐myc tag antibodies for Western blot were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Immunofluorescence, Transfection, Over Expression, Quantitative RT-PCR

Journal: MedComm

Article Title: Regulation of XPO5 phosphorylation by PP2A in hepatocellular carcinoma

doi: 10.1002/mco2.125

Figure Lengend Snippet: B55β represses HCC progression via tuning miRNA expression in vivo. (A) Photograph and (B) tumor growth curve of SK‐Hep1 ctrl and B55β overexpression xenografts in nude mice. (C) Phosphorylated and total XPO5 as well as B55β in xenografts was detected by Western blot. (D) miRNA expression in xenografts was measured by qRT‐PCR. Data are shown as the means ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: Anti‐XPO5, anti‐actin, anti‐PP2A catalytic subunit, anti‐p‐ERK, anti‐ERK, and anti‐myc tag antibodies for Western blot were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, In Vivo, Over Expression, Western Blot, Quantitative RT-PCR

Journal: MedComm

Article Title: Regulation of XPO5 phosphorylation by PP2A in hepatocellular carcinoma

doi: 10.1002/mco2.125

Figure Lengend Snippet: Schematic diagram of how PP2A modulates miRNA expression. In normal conditions, phosphatase PP2A dephosphorylates XPO5 and modulating the transport ability of XPO5, thus promoting miRNA expression. In HCC, PP2A downregulation coupled with ERK activation favors XPO5 phosphorylation, leading to the compromised miRNA expression.

Article Snippet: Anti‐XPO5, anti‐actin, anti‐PP2A catalytic subunit, anti‐p‐ERK, anti‐ERK, and anti‐myc tag antibodies for Western blot were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Activation Assay

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a Immunoblotting of Dicer, Drosha, DGCR8, Exportin-5, and β-actin in LM2 cells transduced with GFP, Drosha, DGCR8, Exportin-5, or Dicer. b Clonogenic survival assays of LM2 cells transduced with GFP, Drosha, DGCR8, Exportin-5, or Dicer after X-ray ionizing radiation (IR) treatment. n = 3 wells per group. c Immunoblotting of DGCR8, Drosha, Exportin-5, Dicer, γH2AX, H2AX, and β-actin in LM2 cells collected at the indicated times after X-ray IR treatment. Quantification results were normalized to β-actin. d Immunoblotting of Drosha, DGCR8, and β-actin (left panel) and clonogenic survival assays (right panel) of LM2 stable cell lines overexpressing GFP, wild-type (WT) DGCR8, or Δ692-DGCR8 (the Drosha binding-deficient mutant). n = 3 wells per group. e Schematic representation of the generation of radioresistant sublines (LM2-R and MCF-7-R) from parental LM2 and MCF-7 breast cancer cell lines by three rounds of X-ray irradiation. f Clonogenic survival assays of parental LM2 and LM2-R cells after X-ray IR treatment. n = 3 wells per group. g Immunoblotting of γH2AX, H2AX, and β-actin in parental LM2 and LM2-R cells collected at the indicated times after IR. h Immunoblotting of DGCR8, Drosha, Dicer, Exportin-5, and β-actin in LM2 and LM2-R cells collected at the indicated times after IR. LE long exposure, SE short exposure. Quantification results were normalized to β-actin. i , j Immunoblotting of DGCR8 and β-actin ( i ) and clonogenic survival assays ( j ) of control or DGCR8-knockdown LM2-R cells transduced with GFP, WT DGCR8, or Δ692-DGCR8. n = 3 wells per group. k Tumor size of mice bearing xenograft tumors formed by control or DGCR8-knockdown LM2-R cells transduced with WT DGCR8 or Δ692-DGCR8, with or without fractionated doses of localized X-ray IR treatment (XRT) using an X-RAD 320 irradiator. n = 5 mice per group. l Kaplan–Meier curves of relapse-free survival of breast cancer patients (dataset: GSE2034; n = 286 patients, 87% of whom received radiotherapy), stratified by DGCR8 expression levels. Data were generated from the KM Plotter (probes: 64474_g_at in the left panel and 91617_at in the right panel). The auto-select best cutoff was used in the analysis. Statistical significance was determined by a log-rank test. HR hazard ratio. Statistical significance in b , d , f , j , and k was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. Source data are provided as a file.

Article Snippet: The following antibodies were used: antibodies against DGCR8 (1:2000, Abcam, #ab191875), Dicer (1:1000, Cell Signaling Technology, #5362S), Drosha (1:1000, Cell Signaling Technology, #3364S), Exportin-5 (1:1000, Cell Signaling Technology, #12565), γH2AX (1:1000, Cell Signaling Technology, #9718S), H2AX (1:1000, Cell Signaling Technology, #2595S), H2A (1:1000, Cell Signaling Technology, #2578S), p-CHK1 (1:1000, Cell Signaling Technology, #12302S), CHK1 (1:1000, Cell Signaling Technology, #2360S), p-CHK2 (1:1000, Cell Signaling Technology, #2661S), CHK2 (1:1000, Cell Signaling Technology, #6334S), p-ATM (1:1000, Cell Signaling Technology, #5883S), ATM (1:1000, Cell Signaling Technology, #2873S), p-ATR (1:1000, Cell Signaling Technology, #2853S), ATR (1:1000, Cell Signaling Technology, #2790S), p-S/TQ (1:1000, Cell Signaling Technology, 2851S), MBP (1:1000, Cell Signaling Technology, 2396S), GST (1:1000, Cell Signaling Technology, #2622S), MDC1 (1:1000, R&D Systems, #MAB6497), RNF8 (1:1000, Millipore, #09-813), RNF168 (1:1000, Millipore, #ABE367), USP36 (1:1000, a gift from Dr Masayuki Komada at Tokyo Institute of Technology), USP51 (1:3000, a gift from Dr Sharon Dent at MD Anderson Cancer Center), β-actin (1:1000, Santa Cruz Biotechnology, #sc-47778), FLAG (1:5000, Sigma, #F3165, clone M2), HA (1:2000, Santa Cruz Biotechnology, #sc-7392), and MYC (1:2000, Santa Cruz Biotechnology, #sc-40, clone 9E10).

Techniques: Western Blot, Transduction, Stable Transfection, Binding Assay, Mutagenesis, Irradiation, Expressing, Generated, Two Tailed Test