Journal: Cell Death & Disease

Article Title: LINC01106 post-transcriptionally regulates ELK3 and HOXD8 to promote bladder cancer progression

doi: 10.1038/s41419-020-03236-9

Figure Lengend Snippet: a The relative expression of LINC01116 was analyzed in patients with different stages of BCa tissues using the GEPIA 2 bioinformatics website. b The overall survival rate was detected by GEPIA 2 bioinformatics website. c RT-qPCR was used to detect the expression of LINC01116 in human bladder epithelial immortalized cell line SV-HUC-1 and human BCa cell lines (RT-4, 5637, J82, and T24). d RT-qPCR analysis detected LINC01116 expression in J82 and T24 cells transfected with shRNAs targeting LINC01116. e , f Colony formation assay, and EdU assay tested whether LINC01116 knockdown affects the proliferation ability of BCa cell lines. g , h JC-1 assay, and flow cytometry analysis revealed the apoptosis rate of J28 and T24 cells transfected with shRNAs targeting LINC01116. i , j Tumor volume, and tumor weight were measured in response to LINC01116 knockdown. k IHC assay detected the staining of Ki67 and PCNA in tumors with or without LINC01116 knockdown. * P < 0.05, ** P < 0.01.

Article Snippet: The fixed J82 and T24 cells were rinsed in PBS, dehydrated, and air-dried for cultivating in hybridization buffer with LINC01116-specific RNA fluorescence in situ hybridization (FISH) probe (Ribobio).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Colony Assay, EdU Assay, Flow Cytometry, Staining

Journal: Cell Death & Disease

Article Title: LINC01106 post-transcriptionally regulates ELK3 and HOXD8 to promote bladder cancer progression

doi: 10.1038/s41419-020-03236-9

Figure Lengend Snippet: a , b Transwell assays were performed to explore the role of LINC01116 knockdown in BCa cells migration and invasion. c E-cadherin, Vimentin, and N-cadherin expression were measured by western blot in J82 and T24 cells transfected with sh-LINC01116#1 and sh-LINC01116#2. d IF assay determined the staining of E-cadherin and N-cadherin in J82 and T24 cells with or without LINC01116 silence. e The representative image of lungs from the two groups, and HE staining of metastasis nodules in these lungs. * P < 0.05, ** P < 0.01.

Article Snippet: The fixed J82 and T24 cells were rinsed in PBS, dehydrated, and air-dried for cultivating in hybridization buffer with LINC01116-specific RNA fluorescence in situ hybridization (FISH) probe (Ribobio).

Techniques: Migration, Expressing, Western Blot, Transfection, Staining

Journal: Cell Death & Disease

Article Title: LINC01106 post-transcriptionally regulates ELK3 and HOXD8 to promote bladder cancer progression

doi: 10.1038/s41419-020-03236-9

Figure Lengend Snippet: a , b . Nuclear separation followed by RT-qPCR and AGE, as well as FISH assay, monitored the subcellular location of LINC01116 in BCa cells. c , d GEPIA 2 analyzed the relationship between two protein-coding genes (ELK3 and HOXD8) and LINC01116 in BLCA tumors. e , f RT-qPCR and western blot analyzed the expression of ELK3 in BCa cells under LINC01116 inhibition. g , h RT-qPCR and western blot analyzed the expression of HOXD8 in BCa cells. i , j Dual-luciferase reporter assay further validated the combination between ELK3/HOXD8 and LINC01116. * P < 0.05, ** P < 0.01.

Article Snippet: The fixed J82 and T24 cells were rinsed in PBS, dehydrated, and air-dried for cultivating in hybridization buffer with LINC01116-specific RNA fluorescence in situ hybridization (FISH) probe (Ribobio).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Inhibition, Luciferase, Reporter Assay

Journal: Cell Death & Disease

Article Title: LINC01106 post-transcriptionally regulates ELK3 and HOXD8 to promote bladder cancer progression

doi: 10.1038/s41419-020-03236-9

Figure Lengend Snippet: a Ago2-RIP assay was performed to prove the existence of LINC01116 and ELK3 in RISCs. b , c By means of searching on starBase, miR-3612 was predicted to have the potentials to bind with LINC01116 andELK3. d , e Luciferase reporter gene assays predicted the relationship between miR-3612 and LINC01116/ELK3. f RNA pull-down assay further examined the combination between miR-3612 and LINC01116/ELK3. g Ago2-RIP assay confirmed the interaction among LINC01116, miR-3612, and ELK3 in RISCs. h RNA pull-down assay detected the impact of LINC01116 on the interaction of miR-3612 with ELK3. * P < 0.05, ** P < 0.01.

Article Snippet: The fixed J82 and T24 cells were rinsed in PBS, dehydrated, and air-dried for cultivating in hybridization buffer with LINC01116-specific RNA fluorescence in situ hybridization (FISH) probe (Ribobio).

Techniques: Luciferase, Pull Down Assay

Journal: Cell Death & Disease

Article Title: LINC01106 post-transcriptionally regulates ELK3 and HOXD8 to promote bladder cancer progression

doi: 10.1038/s41419-020-03236-9

Figure Lengend Snippet: a StarBase software predicted thirteen RBPs shared between HOXD8 and LINC01116. b RT-qPCR detected the relative expression of HOXD8 in J28 cells transfected with shRNAs targeting indicated RBPs. c RNA pull-down assay tested the interaction of LINC01116 with DKC1 or ELACL1. d RNA pull-down assay detected the interaction between HOXD8 and DKC1. e RIP assay verified whether LINC01116 or HOXD8 could be enriched in the anti-DKC1 pallet. f , g RT-qPCR analysis, and western blot analyzed that the expression of DKC1/HOXD8 in J82 and T24 cells transfected with shRNAs against DKC1. h RT-qPCR examined the stability of HOXD8 under ActD treatment in J82 and T24 cells transfected sh-LINC01116#1 or sh-DKC1#1. * P < 0.05, ** P < 0.01.

Article Snippet: The fixed J82 and T24 cells were rinsed in PBS, dehydrated, and air-dried for cultivating in hybridization buffer with LINC01116-specific RNA fluorescence in situ hybridization (FISH) probe (Ribobio).

Techniques: Software, Quantitative RT-PCR, Expressing, Transfection, Pull Down Assay, Western Blot

Journal: Cell Death & Disease

Article Title: LINC01106 post-transcriptionally regulates ELK3 and HOXD8 to promote bladder cancer progression

doi: 10.1038/s41419-020-03236-9

Figure Lengend Snippet: Four groups of J82 and T24 cells were involved, including sh-NC, sh-LINC01116#1, sh-LINC01116#1 + pcDNA3.1/ELK3, sh-LINC01116#1 + pcDNA3.1/ELK3 + pcDNA3.1/HOXD8. a , b Colony formation assay and EdU assay detected the proliferation ability of cells in these four groups. c Flow cytometry analysis detected the apoptosis rate of cells in these four groups. d , e Transwell assays detected the migration and invasion capacity of indicated J28 and T24 cells. f Western blot examined the expression of E-cadherin, N-cadherin, and Vimentin in indicated J28 and T24 cells. * P < 0.05, ** P < 0.01.

Article Snippet: The fixed J82 and T24 cells were rinsed in PBS, dehydrated, and air-dried for cultivating in hybridization buffer with LINC01116-specific RNA fluorescence in situ hybridization (FISH) probe (Ribobio).

Techniques: Colony Assay, EdU Assay, Flow Cytometry, Migration, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: LINC01106 post-transcriptionally regulates ELK3 and HOXD8 to promote bladder cancer progression

doi: 10.1038/s41419-020-03236-9

Figure Lengend Snippet: a The expression of LINC01116 was examined in J82 and T24 cells transfected with shRNAs targeting HOXD8 or ELK3 by RT-qPCR. b ChIP assay was further conducted to explore the functional relationship between LINC01116 and HOXD8. c Five binding sites between LINC01116 promoter and HOXD8 were predicted using JASPAR software. d Luciferase reporter gene assays were performed to verify which site in LINC01116 promoter was recognized by HOXD8. e Luciferase reporter assays also detected the influence of HOXD8 on the activity of P1. * P < 0.05, ** P < 0.01.

Article Snippet: The fixed J82 and T24 cells were rinsed in PBS, dehydrated, and air-dried for cultivating in hybridization buffer with LINC01116-specific RNA fluorescence in situ hybridization (FISH) probe (Ribobio).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Functional Assay, Binding Assay, Software, Luciferase, Activity Assay

Journal: Cell Death & Disease

Article Title: LINC01106 post-transcriptionally regulates ELK3 and HOXD8 to promote bladder cancer progression

doi: 10.1038/s41419-020-03236-9

Figure Lengend Snippet: The drawing analyzed the two functional pathways of LINC01116-miR-3612-ELK3 and LINC01116-DKC1-HOXD8 in BCa cells. * P < 0.05, ** P < 0.01.

Article Snippet: The fixed J82 and T24 cells were rinsed in PBS, dehydrated, and air-dried for cultivating in hybridization buffer with LINC01116-specific RNA fluorescence in situ hybridization (FISH) probe (Ribobio).

Techniques: Functional Assay

Journal: Journal of Cancer

Article Title: Linc01513 inhibits the malignant potential of Nasopharyngeal carcinoma by binding to PTBP1

doi: 10.7150/jca.62112

Figure Lengend Snippet: Linc01513 binded to PTBP1. (A) Localization of linc01513 was detected by FISH. Bar=50 uM. (B) RNA protein interaction Prediction of linc01513 with PTBP1 using the catRAPID graphic. (C) The interaction profile between PTBP1 protein and linc01513 predicted by catRAPID fragment. (D) Western blot analysis of RNA pull-down assay in 6-10B and SUNE-1 cell. (E) RIP-PCR analysis of interaction between PTBP1 and linc01513. n =3; ** p <0.01 vs IgG group.

Article Snippet: The probes of linc01513 were synthesized by RiboBio (Guangzhou, China), and FISH in situ hybridization kit (RiboBio, Guangzhou, China) was used for fluorescence staining.

Techniques: Western Blot, Pull Down Assay

Journal: BMC Developmental Biology

Article Title: Msx1 and Msx2 are required for endothelial-mesenchymal transformation of the atrioventricular cushions and patterning of the atrioventricular myocardium

doi: 10.1186/1471-213X-8-75

Figure Lengend Snippet: Expression of Msx1 and Msx2 in the developing AV canal . Section in situ hybridization showed that the domains of Msx1 and Msx2 expression overlap in a subpopulation of endocardial and cushion mesenchymal cells during AV cushion morphogenesis between E9.5 and E11.5 (A-F). Msx2 also displayed strong expression in the myocardium of the AV canal and right ventricle at all developmental stages examined (B, D and F). Red asterisks indicate AV cushions. endo, endocardium; FG, foregut; IAS and IVS, interatrial and interventricular septum; LA and LV, left atrium and ventricle; myo, myocardium; RA and RV, right atrium and ventricle. Scale bars: 0.1 mm in A (for A, B), 0.2 mm in C (for C, D) and E (for E, F).

Article Snippet: Stored embryos were rehydrated through graded methanol, washed in PBS, and cryopreserved in sucrose solutions with increasing concentrations, then frozen in O.C.T Sections were cut at 10-μm thickness and RNA section in situ hybridization was performed using the In Situ Hybridization kit (BioChain) with the protocol modified according to Dijkman et al. [ ].

Techniques: Expressing, In Situ Hybridization

Journal: BMC Developmental Biology

Article Title: Msx1 and Msx2 are required for endothelial-mesenchymal transformation of the atrioventricular cushions and patterning of the atrioventricular myocardium

doi: 10.1186/1471-213X-8-75

Figure Lengend Snippet: Impaired expression of endocardial and cushion mesenchymal genes in Msx1-/- ; Msx2-/- mutant AV cushions . At E10.5, the immunostaining intensity of NFATc1 was reduced in Msx1-/- ; Msx2-/- mutant AV endocardium compared with the littermate control (red arrows in A, B), while the intensity was comparable between control and Msx1/2 double mutant OFT endocardium (red asterisks in A, B). At the same developmental stage, RNA section in situ hybridization revealed greatly diminished expression of Notch1 (arrows in C, D) in the Msx1/2 double mutant AV cushion mesenchyme and endocardium compared with the control. In contrast, Notch1 expression was normal in the Msx1/2 mutant OFT cushions (compare red asterisks in C and D). Note also a decreased number of Has2 -expressing cells in the Msx1/2 null mutant AV cushion mesenchyme (red arrows in E, F). endo, endocardium; LA and LV, left atrium and ventricle; mes, mesenchyme; RA and RV, right atrium and ventricle. Scale bars: 0.1 mm in B (for A-D), 0.2 mm in F (for E, F).

Article Snippet: Stored embryos were rehydrated through graded methanol, washed in PBS, and cryopreserved in sucrose solutions with increasing concentrations, then frozen in O.C.T Sections were cut at 10-μm thickness and RNA section in situ hybridization was performed using the In Situ Hybridization kit (BioChain) with the protocol modified according to Dijkman et al. [ ].

Techniques: Expressing, Mutagenesis, Immunostaining, Control, In Situ Hybridization

Journal: BMC Developmental Biology

Article Title: Msx1 and Msx2 are required for endothelial-mesenchymal transformation of the atrioventricular cushions and patterning of the atrioventricular myocardium

doi: 10.1186/1471-213X-8-75

Figure Lengend Snippet: Perturbed expression of chamber-specific genes in Msx1-/- ; Msx2-/- mutant myocardium . RNA section in situ hybridization at E10.5 demonstrated that Tbx2 expression was dramatically reduced in the Msx1/2 null mutant AV myocardium compared with the littermate control (compare the staining intensity in the regions pointed by red notched arrowheads in A and B). In contrast, the level of Tbx expression in the pharyngeal mesoderm was comparable between the control and the Msx1/2 double mutant (indicated by black arrows in A and B). Concomitant with decreased Tbx2 expression in the Msx1-/-; Msx2-/- mutant AV myocardium, Anf expression exhibited increased expression in the Msx1/2 double mutant AV myocardium (compare the staining intensity in the regions pointed by red notched arrowheads in C and D). In addition, we detected ectopic Anf expression in the double mutant right ventricle (red asterisk in D), which was likely a hemodynamic effect secondary to the changes of gene expression in the AV canal. Msx1/2 null mutants also exhibited decreased expression of Hand1 (F) and Hand2 (H) in the AV myocardium (red notched arrowheads). In the Msx1/2 mutant AV myocardium, Hand1 expression was almost undetectable and Hand2 expression was significantly reduced compared with the control (red notched arrowheads in E-H). LA and LV, left atrium and ventricle; RA and RV, right atrium and ventricle. Scale bars: 0.1 mm in A (for A-D) and E (for E-H).

Article Snippet: Stored embryos were rehydrated through graded methanol, washed in PBS, and cryopreserved in sucrose solutions with increasing concentrations, then frozen in O.C.T Sections were cut at 10-μm thickness and RNA section in situ hybridization was performed using the In Situ Hybridization kit (BioChain) with the protocol modified according to Dijkman et al. [ ].

Techniques: Expressing, Mutagenesis, In Situ Hybridization, Control, Staining, Gene Expression

Journal: International Journal of Biological Sciences

Article Title: ALKBH5/MAP3K8 axis regulates PD-L1+ macrophage infiltration and promotes hepatocellular carcinoma progression

doi: 10.7150/ijbs.70149

Figure Lengend Snippet: ALKBH5 regulates MAP3K8 in an m6A -dependent manner. (a) The overall level of m6A changed after intervention of ALKBH5 expression by m6A colorimetry. (b) RNA-seq for HUH-7 cells with or without ALKBH5 knockdown n=2. (c) Intersection of the mRNA-seq and RIP-seq datasets (ALKBH5 binding target genes) (GSE144963). (d) Intersection of the mRNA-seq and m6A-seq datasets (Genes with increased m6A modification after silencing ALKBH5) (GSE87515). (e) RT-qPCR analysis of HUH-7 cells with or without silencing ALKBH5. (f) RIP-qPCR analysis of HUH-7 with or without silencing ALKBH5 in order to detect the combination of ALKBH5 with three genes (MAP3K8, MROH1, and UAP1L1). (g) M6a-IP-qPCR analysis of HUH-7 with or without silencing ALKBH5. (h) RT-qPCR analysis of Sk-hep1 with or without silencing ALKBH5. (i) Western blot analysis of the effect of cycloleucine on MAP3K8 expression. (j) Western blot analysis of the effect of mut-ALKBH5 H204A on MAP3K8 expression. (k) Western blot analysis of the effect of silencing YTHDF2 on MAP3K8 expression. (l) RT-qPCR analysis of the effect of silencing YTHDF2 on the half-life of MAP3K8 mRNA. (m) Colocalization of the YTHDF2 protein and MAP3K8 mRNA was observed by IF-FISH. *P < 0.05; **P < 0.01; ***P < 0.001. All data are presented as the means ± SEM. Student's t-test for independent samples and unequal variances was used to assess statistical significance. Comparisons among multiple groups were analyzed by one-way ANOVA. Comparisons at different time points were analyzed by repeated-measures ANOVA. Cell experiments were independently repeated three times.

Article Snippet: FISH was performed using an mRNA in situ hybridization kit (Shanghai GenePharma Co., Ltd., China).

Techniques: Expressing, Colorimetric Assay, RNA Sequencing Assay, Binding Assay, Modification, Quantitative RT-PCR, Western Blot