fluorescence in situ hybridisation (fish) kit  (GenScript corporation)

Journal: Clinical and Translational Medicine

Article Title: Circular RNA circARPC1B functions as a stabilisation enhancer of Vimentin to prevent high cholesterol‐induced articular cartilage degeneration

doi: 10.1002/ctm2.1415

Figure Lengend Snippet: High cholesterol inhibits the circARPC1B expression in chondrocytes. (A) The flowchart illustrating the selecting processes of circARPC1B based on the sequencing data. (B) Volcano plot of 3089 circRNA were identified from sequencing data. (C) Heatmap of 15 circRNA (pval<0.05 and log2FC>5). (D) 15 circRNA expression levels in C28/I2 cells ( n = 3). (E) hsa_circ_0007004, hsa_circ_ARPC1B and hsa_circ_0008796 expression levels in C28/I2 cells treated with 10 μg/mL cholesterol ( n = 3). (F) hsa_circ_0007004, hsa_circ_ARPC1B and hsa_circ_0008796 expression levels in C28/I2 cells treated with IL‐1β or TNFα ( n = 3). (G) Aggrecan and COL2A1 protein level in C28/I2 cells infected with hsa_circ_0007004, hsa_circ_ARPC1B or hsa_circ_0008796 siRNA ( n = 3). (H) Representative images of RNA fluorescence in situ hybridisation (FISH), Safranin‐O/Fast green staining and Alcian blue staining in human cartilage tissues from stage 0 (S0) to stage 4 (S4). Scale bar, 100 mm. (I) OARSI grade used for evaluation of the cartilage degradation of patients with mild, middle, and severe OA ( n = 74). (J) The expression of circARPC1B in human cartilage specimens of patients with mild, middle, and severe OA ( n = 74). (K) Schematic illustration demonstrating the circularisation of ARPC1B exon 6−7 to form circARPC1B. The presence of circARPC1B was validated by RT‐PCR followed by Sanger sequencing. The red arrow represents “head‐to‐tail” circARPC1B splicing sites. (L) The presence of circARPC1B in C28/I2 cells was validated by RT‐qPCR. Divergent primers amplified circARPC1B from cDNA but not from genomic DNA; β‐actin served as the negative control. (M) The levels of circARPC1B and ARPC1B in C28/I2 cells treated with actinomycin D at the indicated time points were detected by RT‐qPCR ( n = 3). (N) The expression of circARPC1B and linear ARPC1B mRNA in C28/I2 cells treated with or without RNase R was detected by qRT‐PCR ( n = 3). (O) Representative images of FISH staining for circARPC1B localisation in C28/I2 cells. Scale bars, 50 μm. (P) Expression of circARPC1B assessed by RT‐qPCR in the nuclear and cytoplasmic fractions ( n = 3). The results were presented as mean ± SEM. * P <.05, ** P <.01, *** P <.005 and **** P <.001 as compared with the control group.

Article Snippet: The probe signal was detected using fluorescent in situ hybridisation (FISH) Kit (RiboBio, Guangzhou, China), and the nucleus was stained with DAPI.

Techniques: Expressing, Sequencing, Infection, Fluorescence, In Situ, Hybridization, Staining, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Amplification, Negative Control, Control

Journal: Cell Death and Differentiation

Article Title: Circular RNA cESRP1 sensitises small cell lung cancer cells to chemotherapy by sponging miR-93-5p to inhibit TGF-β signalling

doi: 10.1038/s41418-019-0455-x

Figure Lengend Snippet: Deregulated circRNAs in chemoresistant SCLC and cESRP1 characterisation. a The heatmap shows the top ten circRNAs with the most increased and decreased expression in H69AR cells compared with H69 cells, as determined using a circRNA Arraystar Chip. b We validated the differential expression of 14 circRNAs in H69AR cells and H69 cells using qRT-PCR. An independent-sample t -test was used; qRT-PCR, quantitative reverse transcription PCR. Data are mean ± SD, n = 3. c The relative expression of the ten indicated circRNAs listed in a from the chemoresistant cells and matched chemosensitive cells was measured by qRT-PCR; PDC1-S, patient-derived cells that are relatively sensitive to chemotherapy; PDC1-R, patient-derived cells that are relatively resistant to chemotherapy. Data are mean ± SD, n = 3. d A schematic diagram of the genomic location and splicing pattern of cESRP1 is shown. e Random hexamer or oligo (dT)18 primers were used in reverse transcription experiments. The relative RNA levels were analysed by qRT-PCR and normalised to the level measured using the random hexamer primers. Data are mean ± SD, n = 3. f The relative RNA levels of cESRP1 and mESRP1 in H69 and H446 cells were analysed by qRT-PCR after treatment with actinomycin D at the indicated time points. Data are mean ± SD, n = 3. g cESRP1 and mESRP1 were abundant in the cytoplasm of H69 cells. β-actin and U6 were used as positive controls in the cytoplasm and nucleus, respectively. Data are mean ± SD, n = 3. h RNA fluorescence in situ hybridisation for cESRP1 was performed in H446 cells. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm

Article Snippet: A fluorescence in situ hybridisation (FISH) kit (RiboBio, Guangzhou, China) was used to detect probe signals according to the manufacturer’s instructions after culturing cells for 24 h. To determine the cESRP1 status of PDX tumours, 4-μm-thick sections were cut from paraffin-embedded blocks and then processed, hybridised, and analysed.

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Random Hexamer Labeling, Fluorescence, In Situ, Hybridization, Staining

Journal: Cell Death and Differentiation

Article Title: Circular RNA cESRP1 sensitises small cell lung cancer cells to chemotherapy by sponging miR-93-5p to inhibit TGF-β signalling

doi: 10.1038/s41418-019-0455-x

Figure Lengend Snippet: cESRP1 functions as a sponge of miR-93-5p in SCLC. a RIP experiments were performed using an antibody against AGO2 with extracts from SCLC cells. Data are mean ± SD, n = 3. b The Venn diagram shows the intersection of miRNA lists. The heatmap shows the expression of the overlapping miRNAs in chemoresistant and chemosensitive SCLC cell lines. c The bar graph shows the effect of miR-93-5p, miR-182-5p, and miR-125a-5p inhibition on the drug sensitivity of H69AR cells. Data were pooled from four biological replicates ± SD, n = 4. d qRT-PCR was used to analyse the cESRP1 levels in streptavidin-captured fractions from H69 cell lysates after transfection with 3′-end biotinylated miR-93-5p or a negative control. Data are mean ± SD, n = 3. e H69 cells were co-transfected with LUC-cESRP1-WT or LUC-cESRP1-MUT vectors and an miR-93-5p mimic or a negative control (miR-NC). Luciferase activity was detected with luciferase reporter assays. Data are mean ± SD, n = 3. f The colocalization of cESRP1 and miR-93-5p was observed by RNA in situ hybridisation in H446 and H69AR cells. Nuclei were stained with a DAPI solution. g – h Cell cycle and cell apoptosis rate analyses of H69 cells that received the indicated treatments are shown. Data are mean ± SD, n = 3. i The IC50 values of H69 cells transfected with the indicated transcripts and treated with drugs were measured using CCK-8 assays. Data are mean ± SD, n = 4. j Images of subcutaneous tumours comprising H69AR or H446DDP cells after cisplatin/etoposide (C/E) treatment or combination treatment (Comb) with the miR-93-5p antagomir (Anta) are shown ( n = 4). NS normal saline. k The growth curves of xenografted tumours derived from SCLC cells with or without miR-93-5p antagomir or cisplatin/etoposide treatment are shown. Data are mean ± SD, n = 4. l Tumour weight (means) was measured at the endpoint

Article Snippet: A fluorescence in situ hybridisation (FISH) kit (RiboBio, Guangzhou, China) was used to detect probe signals according to the manufacturer’s instructions after culturing cells for 24 h. To determine the cESRP1 status of PDX tumours, 4-μm-thick sections were cut from paraffin-embedded blocks and then processed, hybridised, and analysed.

Techniques: Expressing, Inhibition, Quantitative RT-PCR, Transfection, Negative Control, Luciferase, Activity Assay, In Situ, Hybridization, Staining, CCK-8 Assay, Derivative Assay