Journal: PLoS ONE

Article Title: Retigeric Acid B Exhibits Antitumor Activity through Suppression of Nuclear Factor-κB Signaling in Prostate Cancer Cells in Vitro and in Vivo

doi: 10.1371/journal.pone.0038000

Figure Lengend Snippet: (A) and (B) RB slightly inhibited expression of p65 in PC3 and DU145 cells, while significantly for the Ser-536 phosphorylation of p65 ( p–p65 ), in both dosage-dependent and time-dependent manner. Lysates from whole cells treated with RB of different concentrations for 24 h (A) or with 10 µM RB for indicated times were used for western blot (B). GAPDH served as the loading control. Protein amount was normalized to the amount of GAPDH, and was quantified by densitometry of X-ray films. Results of one of at least three independent experiments are shown. (C) and (D) RB moderately down-regulated p65 mRNA level in PC3 and DU145 cells as detected by QRT-PCR assay. The procedure was performed as described in Materials and Methods . Results shown are representatives of three independent experiments, p<0.05 (*), versus RB-untreated control group respectively.

Article Snippet: QRT-PCR was performed using the Eppendorf QRT-PCR System.

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Retigeric Acid B Exhibits Antitumor Activity through Suppression of Nuclear Factor-κB Signaling in Prostate Cancer Cells in Vitro and in Vivo

doi: 10.1371/journal.pone.0038000

Figure Lengend Snippet: (A) RB dosage-dependently inhibited the mRNA expression of Bcl-x L , Bcl-2, survivin, and cyclin D1 as analyzed by QRT-PCR. GAPDH was used for normalization. Results are the mean ± SD of three independent experiments, each performed in triplicate. p<0.05 (*), p<0.01 (**), p<0.001 (***) versus RB-untreated control group respectively. (B) RB dosage-dependently inhibited expression of Bcl-x L , Bcl-2, survivin, and cyclin D. The results of western blot analysis of whole cell lysates from PC3 cells treated with different doses of RB were shown; GAPDH was included as a loading control. (C) RB inhibited invasion of PCa cells in a concentration-dependently manner as detected by transwell assay, (scale bar, 100 μm). The procedure was described in Materials and Methods . (D) The number of cells that invade through matrigel.

Article Snippet: QRT-PCR was performed using the Eppendorf QRT-PCR System.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Concentration Assay, Transwell Assay

Journal: Journal of Translational Medicine

Article Title: Circular RNA circLOC101928570 suppresses systemic lupus erythematosus progression by targeting the miR-150-5p/c-myb axis

doi: 10.1186/s12967-022-03748-2

Figure Lengend Snippet: Identification and characterization of circLOC101928570, a circRNA that is significantly downregulated in SLE. A Volcano plot describing the profile of differentially expressed circRNAs between the control and SLE groups. The selected 4 significantly changed circRNAs are marked with green and red dots (FDR < 0.05, |fold change|> 2.0). B CircLOC101928570 expression was significantly lower in SLE patients. The expression of circLOC101928570 in CD4 + T cells and CD8 + T cells from HC and SLE patients. The results are represented as the mean ± SD (n = 3), *P < 0.05, ***P < 0.001. C , D Liner regression analysis for the expression of circLOC101928570 and the SLEDAI score and complement C3 level, respectively. E ROC curve of relative circLOC101928570 expression for differentiating the 62 patients with SLE from the 62 healthy controls. F Structure of the circLOC101928570 genome and transcript. CircLOC101928570 is produced by exon 3, and the junction point of circLOC101928570 was identified by qRT–PCR followed by Sanger sequencing. G Two CPG islands existed in the transcriptional core region of circLOC101928570. H Relative expression of circLOC101928570, linear LOC101928570 and actin were tested by PCR upon RNase R treatment. I FISH showed circLOC101928570 localization. circLOC101928570 probes were labeled with Cy3, and nuclei were stained with DAPI; scale bar: 10 µm

Article Snippet: After reverse transcription with a Mir-X™ miRNA First Strand Synthesis Kit (Takara, Japan), complementary DNA (cDNA) was amplified using a Mir-X™ miRNA qRT–PCR TB Green® Kit (Takara, Japan), the expression of miRNAs was normalized to the expression of U6, and the probe sequences used are listed in Additional file : Table S2.

Techniques: Expressing, Produced, Quantitative RT-PCR, Sequencing, Labeling, Staining

Journal: Journal of Translational Medicine

Article Title: Circular RNA circLOC101928570 suppresses systemic lupus erythematosus progression by targeting the miR-150-5p/c-myb axis

doi: 10.1186/s12967-022-03748-2

Figure Lengend Snippet: CircLOC101928570 acted as a sponge for miR-150-5p. A ceRNAs that could competitively bind to this exonic circRNA. MiR-150 has two targets marked with red lines. B The relative levels of 10 miRNA candidates in the PBMC lysates were detected by real-time PCR. miR-150-3p/5p was pulled down by circLOC101928570 in PBMCs. C The interaction of circLOC101928570 with miR-150 was tested by RNA immunoprecipitation (RIP) of AGO2 from HEK293T cells. Relative circLOC101928570 enrichment levels were quantified by RIP-qPCR (AGO2 RIP/IgG RIP). D Schematic illustration of wild-type or mutant transcripts of the circLOC101928570 luciferase reporter. E , F Luciferase reporter and effects of miR-150-3p/5p on the expression of wild-type or mutant transcripts of the circLOC101928570 luciferase reporter. Luciferase activity was normalized to the value obtained in cells transfected with NC oligonucleotides. G The expression of miR-150-5p was analyzed by qRT–PCR in cells transfected with circLOC101928570, mock vector, shRNA-circLOC101928570 or shRNA-NC. H The expression levels of circLOC101928570 were determined with qRT–PCR in cells transfected with miR-150-3p/5p mimics or inhibitor. I The colocalization of circLOC101928570 and miR-150-5p in PBMCs was detected using a FISH assay. CircLOC101928570 was captured with a Cy3-labeled probe (red), while miR-150-5p was captured with a digoxin-labeled probe followed by further visualization using an anti-digoxin rhodamine-conjugated antibody (green). Blue: stained with DAPI. Scale bar: 10 µm. IgG: immunoglobulin G. All data are presented as the means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: After reverse transcription with a Mir-X™ miRNA First Strand Synthesis Kit (Takara, Japan), complementary DNA (cDNA) was amplified using a Mir-X™ miRNA qRT–PCR TB Green® Kit (Takara, Japan), the expression of miRNAs was normalized to the expression of U6, and the probe sequences used are listed in Additional file : Table S2.

Techniques: Real-time Polymerase Chain Reaction, Immunoprecipitation, Mutagenesis, Luciferase, Expressing, Activity Assay, Transfection, Quantitative RT-PCR, Plasmid Preparation, shRNA, Labeling, Staining

Journal: Molecular Cell

Article Title: Single-Cell Analyses Reveal Megakaryocyte-Biased Hematopoiesis in Myelofibrosis and Identify Mutant Clone-Specific Targets

doi: 10.1016/j.molcel.2020.04.008

Figure Lengend Snippet: Expression of Cell Surface G6B, a Cell Surface Protein, Identifies Mutant Clone-Derived HSPCs in Myelofibrosis (A) Left: expression of 6 megakaryocyte markers from a panel of 20 HSPC and megakaryocyte cell surface antigens assayed by mass spectrometry time of flight (CyTOF) shows expression of G6B on CD34 + HSPCs from patients with primary myelofibrosis (PMF), post-essential thrombocythemia myelofibrosis (PET-MF), and post-polycythaemia vera myelofibrosis (PPV-MF) with either JAK2V617F (JAK2+) or calreticulin (mut CALR ) driver mutations. Histograms show cell count (y axis) by expression level (x axis). Right: viSNE dimensionality reduction plots on a representative control and myelofibrosis sample for CD9 and G6B, illustrating more substantial differential expression of G6B than CD9 in myelofibrosis versus control cells (B) Flow cytometric analysis of G6B expression on CD34 + Lin − HSPCs showing significant increase in G6B+ cells in myelofibrosis (% GFP+ cells, 28.8% ± 5.5% versus 2.4% ± 1.0%); chart shows mean + SEM (left) and example plot (right) shown, illustrating expression in both CD41+ and negative cells. ∗∗ p ≤ 0.01 (t test). Controls (N = 8); myelofibrosis (N = 11). (C) Immunohistochemical staining for G6B (diaminobenzidine, DAB brown) of bone marrow biopsy sections from controls and myelofibrosis patients with JAK2V617F and mut CALR -positive myelofibrosis showing marked expansion of G6B+ megakaryocytes and progenitors in myelofibrosis. (D) Mononuclear cells from healthy donors and patients with JAK2V617F + myelofibrosis were combined and 50 cell “mini-bulk” replicates were sorted from the G6B+ and G6B− fractions for Taqman qRT-PCR to quantify expression of JAK2V617F mutated and wild-type JAK2 . Chart shows JAK2V617F relative to wild-type JAK2 expression for all mini-bulks from 3 replicate experiments. (E) Internalization of a CD34 × G6B bi-specific antibody and isotype control antibody conjugated to a pH-sensitive cyanine CypHer5E dye that fluoresces at an acidic pH following internalization. Left: representative images show clear intracellular fluorescence for CD34 × G6B bi-specific but not isotype control. Right: mean fluorescence intensity of cells measured by flow cytometry 30 min after addition of antibody with/without two endocytosis inhibitors, Dynasore and Pitstop 2. Data shown using SET-2 cells, chart shows mean + SEM, ** - P < 0.01 n= 3. See also Figure S7 .

Article Snippet: 50 G6B+ and G6B- cells were sorted into each well of a 96-well PCR plate (10 replicates per population for each experiment), containing CellsDirect One-Step qRT-PCR kit 2X Reaction Buffer and SuperScript III RT/Platinum Taq Mix (Thermo Fisher Cat#11753100), Ambion SUPERase-In RNase inhibitor (Thermo Fisher Cat#AM2694), TE buffer, JAK2 forward and reverse primers and wild-type and JAK2V617F-specific probe mix (see ).

Techniques: Expressing, Mutagenesis, Derivative Assay, Mass Spectrometry, Cell Counting, Immunohistochemical staining, Staining, Quantitative RT-PCR, Fluorescence, Flow Cytometry