Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Circ-sh3rf3/GATA-4/miR-29a regulatory axis in fibroblast–myofibroblast differentiation and myocardial fibrosis

doi: 10.1007/s00018-023-04699-7

Figure Lengend Snippet: circ-sh3rf3 upregulates the expression of miR-29a by inhibiting GATA-4 expression. (A) Bioinformatic analysis of potential GATA-4 binding sites on the circ-sh3rf3 sequence using RBPmap. (B) Cell lysates from cardiac fibroblasts infected with recombinant circ-sh3rf3 adenovirus were mixed with biotinylated circ-sh3rf3 or random probe, incubated with streptavidin beads and finally subjected to Western blotting using anti-GATA-4 antibody. (C–D) Cell lysates from H9C2 cells (C) or cardiac fibroblasts (D) infected with recombinant circ-sh3rf3 adenovirus were subjected to immunoprecipitation with antibodies against rabbit IgG (control) or GATA-4, followed by qPCR. (E) The mRNA level of GATA-4 in cardiac fibroblasts transfected with circ-sh3rf3 was determined by q-PCR (Left), and the protein level of GATA-4 and GATA-6 were detected by Western blotting and quantitated by densitometry (right). (F) Expression of miR-29a (upper) and GATA-4 (lower) in cardiac fibroblasts transfected with RNAi GATA-4 by qPCR. (G) mRNA levels of CTGF, TGF-β1, and α-SMA in cardiac fibroblasts transfected with RNAi GATA-4 by qPCR. The empty vector served as the control (E–G). (H–I) qPCR for CTGF and α-SMA in cardiac fibroblasts treated with TGF-β1 and Ri-GATA4. (J) Cardiac fibroblasts were treated with TGF-β1 and/or transfected with Ri-GATA4, and F-actin (upper panel) and α-SMA (lower panel) staining was determined by immunofluorescence. PBS treatment served as the control (H–J). The data shown are the means ± SEMs from three independent experiments,**P < 0.01 compared with the control. Scale bars: 50 μm. rAd-circ-sh3rf3 Recombinant circ-sh3rf3 adenovirus, CFs cardiac fibroblasts, ctr control, c-sh3rf3 circular sh3rf3, Ri-G4 RNAi-GATA-4, Tβ TGF-β1

Article Snippet: Three micrograms of biotinylated DNA oligo probes against circ-sh3rf3 or random sequences were diluted in 500 μl washing/binding buffer (20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA) and incubated with 100 μl of Dynabeads M-280 Streptavidin (Invitrogen) at room temperature for 2 h. Then, the cell lysates were added to each bead-probe pellet and incubated at 4 °C overnight.

Techniques: Expressing, Binding Assay, Sequencing, Infection, Recombinant, Incubation, Western Blot, Immunoprecipitation, Control, Transfection, Plasmid Preparation, Staining, Immunofluorescence

Journal:

Article Title: JC Virus Induces Nonapoptotic Cell Death of Human Central Nervous System Progenitor Cell-Derived Astrocytes

doi: 10.1128/JVI.78.9.4884-4891.2004

Figure Lengend Snippet: Composite figure showing differences between uninfected and JCV-infected astrocyte morphologies. (A and B) Light microscopy of uninfected (A) and JCV-infected (B) human progenitor-derived astrocytes viewed with Hoffman interference optics. Infected astrocytes show a distinct morphology at 7 to 10 days p.i., including enlarged nuclei compared to those of uninfected controls. (C and D) In situ DNA hybridization of uninfected (C) and infected (D) astrocytes demonstrates active viral DNA replication in the majority of infected cells, detected by a JCV-specific biotinylated probe and DAB (3,3′-diaminobenzidine) visualization (brown). (E) Immunocytochemistry demonstrated that more than 95% of astrocytes in culture (infected and uninfected) were positive for the astrocyte marker GFAP (green). (F) Immunocytochemistry for the late viral protein VP-1 in infected astrocytes. An overlay of the nuclear stain bisbenzimide (blue) and VP-1 (red) shows that infected astrocytes are positive for the JCV capsid protein (purple). This figure represents results from three independent experiments.

Article Snippet: The presence of JCV in astrocyte cultures was detected by DNA-DNA in situ hybridization (ISH) using a biotinylated JCV DNA probe (ENZO Biochemical), as previously described ( 20 ), or by immunofluorescence against the structural protein VP-1 (see “Immunofluorescence” below).

Techniques: Infection, Light Microscopy, Derivative Assay, In Situ, DNA Hybridization, Immunocytochemistry, Marker, Staining

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: Interactome of MALAT1 lncRNA by RAT-seq. A. RAT-seq assay. MALAT1 lncRNA was in situ reverse transcribed using MALAT1-specific complementary primers at 60°C with biotin-dCTP. The biotin-MALAT1 cDNA chromatin complex was isolated by streptavidin beads and cDNAs were isolated for Illumina library sequencing. RAT-seq will generate a genome-wide target interaction network for MALAT1 lncRNA in breast cancer cells. B. Gene ontology enrichment pathway analysis of the MALAT1 RAT-Seq data. GO enrichment was analyzed with Cytoscape software. C. The MALAT1 RAT-seq interactome. The MALAT1 interactome was drawn based on the enrichment fold of the top RAT-Seq pathway target genes.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: In Situ, Reverse Transcription, Isolation, Sequencing, Genome Wide, Software

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 binds to the EEF1A1 promoter and epigenetically regulates its activity. A. The RAT-seq IGV binding of MALAT1 lncRNA at the EEF1A1 locus. MALAT1-RAT: the RAT-seq library created by the MALAT1-specific complementary primers; RC-RAT: the RAT-seq control library created by random oligonucleotide primers; pEEF1A1: EEF1A1 promoter; 3’-CT, 5’-CT: the 3’- and 5’-control sites; E1-E8: EEF1A1 exons. B. Quantitation of EEF1A1 binding in the MALAT1-specific RAT-seq products and the negative control RAT-seq products. C. EEF1A1 expression levels by Q-PCR in MALAT1-knockdown cells. β-Actin was used as an internal control. **P < 0.01 as compared with the control groups. D. Western blot of eEF1A1. Note the reduced expression of eEF1A1 in MALAT1-knockdown breast cancer cells. GAPDH was used as control.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Activity Assay, Binding Assay, Control, Quantitation Assay, Negative Control, Expressing, Knockdown, Western Blot

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 epigenetically regulates EF1A1. A. pEEF1A1-luciferase assay. The EEF1A1 promoter (pEEF1A1) sequence was cloned into the upstream of luciferase gene. Luciferase reporter assay was performed in CTL group, shNC group and shMALAT1 group by co-transfecting respectively with pGL3-Basic vector or luciferase reporter vector. Data were adjusted over the negative control (CTL) and were represented as means ± SD. **P < 0.01 as compared with the control groups. B. Quantitation of Histone 3-K4 (H3K4) trimethylation. All data are presented as the relative values after normalization over the input DNA. *P < 0.05 as compared with control.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Luciferase, Sequencing, Clone Assay, Reporter Assay, Plasmid Preparation, Negative Control, Control, Quantitation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 is dysregulated in breast cancer. A. Unsupervised hierarchical clustering analysis of the significantly differentially expressed genes in paracancer tissues and tumor tissues. Data from 64 mammary tissues was downloaded from TCGA database. In the heatmap, the normalized expression values are represented in shades of green and red, indicating the expression being above and below the median expression value across the samples. B. The normalized expression level of MALAT1 in 64 mammary tissues (32 paracancer tissues and 32 tumor tissues). ***P < 0.01 as compared with the paracancer group. C. Q-PCR quantitation of MALAT1 expression in breast cancer cell lines.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Expressing, Quantitation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: The role of MALAT1 in cell proliferation and cell cycle. A. MALAT1 shRNA knockdown in two breast cancer cell lines. MALAT1 expression was examined by Q-PCR in CTL (non-transfected control), shNC (random shRNA non-targeting control), shMALAT1 (MALAT1 shRNA-1 transfected cells), and shMALAT1-2 (MALAT1 shRNA-2 transfected cells). β-Actin was used as an internal control. **P < 0.01 as compared with CTL and shNC controls. B. Cell Proliferation. CCK-8 assay was used to determine cell growth viability at 0, 24, 48, 72 and 96 hour time points. C. Cell cycle. Flow cytometry was used to measure cell cycle profile with propidium iodide staining. Cell numbers were counted according to DNA content of G0/G1, S and G2/M phases. The statistical results are shown on the right panel. *P < 0.05 as compared with the control groups.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: shRNA, Knockdown, Expressing, Transfection, Control, CCK-8 Assay, Flow Cytometry, Staining

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 knockdown inhibits cell invasion in breast cancer cells. Representative images of invading MDA-MB231 cells (A) and SKBR3 cells (B) are showed on the left panel. shNC: random shRNA control; shMALAT1: Cells that were transfected with MALAT1 shRNA-1. Quantitation of invaded cells is shown in the right panel, mean ± SD, **P < 0.01 as compared with the control groups.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Knockdown, shRNA, Control, Transfection, Quantitation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: EEF1A1 rescues the effect induced by MALAT1 knockdown. A. Overexpression of EEF1A1 in breast cancer cells. The expression of EEF1A1 was quantitated by Q-PCR. shNC: random shRNA control; shMALAT1: Cells that were transfected with MALAT1 shRNA-1. β-Actin was used as an internal control. ***P < 0.001 as compared with the control groups. Overexpression of eEF1A1 in breast cancer cells was measured by Western blot. B. Cell growth viability as measured by CCK-8 assay. C. Cell invasion as examined by Transwell assay. Quantitation of invaded cells was shown as mean ± SD, **P < 0.01 as compared with the control groups.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Knockdown, Over Expression, Expressing, shRNA, Control, Transfection, Western Blot, CCK-8 Assay, Transwell Assay, Quantitation Assay

Journal: American Journal of Physiology-Heart and Circulatory Physiology

Article Title: Early sequence of cardiac adaptations and growth factor formation in pressure- and volume-overload hypertrophy

doi: 10.1152/ajpheart.2000.279.3.h976

Figure Lengend Snippet: Fig. 3. Top: expression of angiotensino- gen (AGTN), preproET-1 (ppET-1), IGF-I, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in heart ho- mogenates from pressure (left)- and vol- ume (right)-overloaded pigs. For repre- sentative RT-PCR experiments, L indicates DNA molecular size markers. Bottom: mean changes in the densito- metric ratio to GAPDH for AGTN (●), ppET-1 (‚), and IGF-I (Œ) in pressure (left)- and volume (right)-overloaded pigs. GF, growth factor. GF/GAPDH, GF-to-GAPDH ratio. *P , 0.05 vs. sham.

Article Snippet: The in situ hybridization procedure was performed as previously described (29) with the use of specific biotinylated cDNA probes for GAPDH [pHcGAP, American Type Culture Collection (ATCC) no. 57090], ppET-1 (ET1c, ATCC no. 65698), angiotensinogen (AGTN, ATCC no. 82996), and IGF-I (ATCC no. 59944).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction