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: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Diagram showing the Spearman’s correlation of top co-dependent proteins with ANKRD52 or PPP6C in CRISPR (Avana) Public 20Q3 database. Solid lines depict significant positive correlations (Correlation > 0.25, P < 0.001) and dashed lines depict weak correlation (Correlation > 0.1, P < 0.01). b , c Volcano plot showing the Spearman’s correlation and estimated significance of DICER1 ( b ) or XPO5 ( c ) with SOCS1 mRNA levels from RNA-seq data across all TCGA cancer types. Each dot represents a cancer type in TCGA; blue dots indicate significant negative correlations ( P < 0.05, TIMER). d SOCS1 mRNA level in MC38 cells with targeted sgRNAs ( n = 3 per group). Data are representative of three independent experiments and represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. e Killing of OVA-treated MC38 cells with targeted sgRNAs by OT-I T cells ( n = 3 per group per condition). Data are representative of two independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. f Tumor growth curves of Ago2 -null or control MC38 tumors in WT mice treated with PD-1 antibody or not ( n = 5 for NT tumor, n = 6 for NT with anti-PD-1 and n = 7 for Ago2 -null with or without anti-PD-1). Data are represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. g Heatmap showing the Spearman’s correlation of ANKRD52 , AGO2 , DICER1 , XPO5 , DROSHA , PPP6C , or SOCS1 mRNA levels with CD4 + and CD8 + T cell abundance in tumors across all TCGA cancer types. h Model of miRNA machinery in regulation of cancer-intrinsic evasion from T cell attack. See also Supplementary Figs. – . Source data are provided as a source data file.

Article Snippet: For western blot, primary antibodies against mouse ANKRD52 (Santa Cruz, sc-398544) and CK1 a (Santa Cruz, sc-6477) were used with dilution as 1:100; human ANKRD52 (Bethyl, A302-372A), OVA (Sigma, SAB5300165), β-Actin (CST, 4967), JAK1 (CST, 3332), p-STAT1 (Tyr701) (CST, 9167), p-STAT3 (Tyr705) (CST, 9145), SOCS3 (CST, 52113), TAP1 (CST, 12341), FLAG (Sigma, F3165), Vinculin (Sigma, v9131), PPP6C (Abcam, EPR8764), AGO2 (CST, 2897 S), XPO5 (CST, 12565 S), and DGCR8 (Abcam, ab191875; Proteintech, 10996-1-AP) were used with dilution as 1:1000.

Techniques: CRISPR, RNA Sequencing Assay, Two Tailed Test

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