insect cells  (BPS Bioscience)

Journal: eLife

Article Title: Phosphodiesterase 1A physically interacts with YTHDF2 and reinforces the progression of non-small cell lung cancer

doi: 10.7554/eLife.98903

Figure Lengend Snippet: ( A ) The expression of PDE1A was detected in non-small cell lung cancer (NSCLC) and normal lung tissue. It was obtained from The Human Protein Atlas ( https://www.proteinatlas.org/ ). IOD/area means integral optical density/area. t-Test was used to compare the difference between lung cancer and normal lung groups, ** P < 0.01. ( B ) The expression of PDE1A in human lung fibroblasts (HELF), A549, NCI-H1299, and NCI-H460 cells was detected by western blot. ( C and D ) Box plot analysis of the PDE1A messenger RNA (mRNA) levels in clinical lung cancer tissue samples. It was collected, and statistical analyses were performed from SurvExpress ( http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp ). Gene: PDE1A; Access database numbers: Chitale lung (n=185); Censored: recurrence months or survival months. ( E ) It was collected from PROGgene V2 Prognostic Database ( http://www.progtools.net/gene/index.php ). Survival analysis is done using backend R script which employs R library ‘survival’ to perform Cox proportional hazards analysis (function ‘coxph’) and to plot prognostic plots (function ‘survfit’). Single-user input genes: PDE1A; Cancer type: LUNG; Survival measure: death; Bifurcate gene expression at: median; GSE30219 -Off-context gene expression in lung cancer identifies a group of metastatic-prone tumors. Figure 1—source data 1. Raw images for western blot shown in (labelled). Figure 1—source data 2. Raw images for western blot shown in .

Article Snippet: Antibodies against PDE1A (12442-2-AP), YTHDF2 (247441-AP), lamin B (12595-1-AP), and E-cadherin (20874-1-AP) were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Expressing, Western Blot, Gene Expression

Journal: eLife

Article Title: Phosphodiesterase 1A physically interacts with YTHDF2 and reinforces the progression of non-small cell lung cancer

doi: 10.7554/eLife.98903

Figure Lengend Snippet: Data collected from LinkedOmics ( https://www.linkedomics.org/admin.php ) are shown. Overrepresentation enrichment analysis (ORA) ( A ) and gene set enrichment analysis (GSEA) ( B ) were performed to analyze the biological processes related to PDE1A in NSCLC. Sample cohort: TCGA_NSCLC; Institute: UNC; Data type: RNAseq; Platform: HiSeq RNA; Attribute: PDE1A; Statistical methods: Spearman’s correlation test; Patients: 515; Tools: ORA and GSEA; Gene Ontology analysis: biological process. ( C ) GSEA demonstrated that PDE1A participated in mesenchyme development and the cellular response to VEGF stimulus.

Article Snippet: Antibodies against PDE1A (12442-2-AP), YTHDF2 (247441-AP), lamin B (12595-1-AP), and E-cadherin (20874-1-AP) were obtained from Proteintech (Rosemont, IL, USA).

Techniques:

Journal: eLife

Article Title: Phosphodiesterase 1A physically interacts with YTHDF2 and reinforces the progression of non-small cell lung cancer

doi: 10.7554/eLife.98903

Figure Lengend Snippet: ( A–B ) NSCLC cells were transfected with PDE1A siRNA or control siRNA ( A ) and empty vector or PDE1A plasmid ( B ) for 24 hr in six-well plates, then transferred to 96-well plates for the indicated times, and finally subjected to sulforhodamine B (SRB) assay (n=3).

Article Snippet: Antibodies against PDE1A (12442-2-AP), YTHDF2 (247441-AP), lamin B (12595-1-AP), and E-cadherin (20874-1-AP) were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Transfection, Control, Plasmid Preparation, Sulforhodamine B Assay

Journal: eLife

Article Title: Phosphodiesterase 1A physically interacts with YTHDF2 and reinforces the progression of non-small cell lung cancer

doi: 10.7554/eLife.98903

Figure Lengend Snippet: ( A–B ) NSCLC cells were transfected with control siRNA and PDE1A siRNA for 24 hr. Cells were transferred to Transwell chambers without or with a Matrigel coating on the insert membrane, and the cell migrative and invasive abilities were determined, respectively (n=3). ( C–D ) NSCLC cells were transfected with control siRNA and PDE1A siRNA for 24 hr, and the wound healing assay was established in NSCLC cells (n=3). ( E ) NSCLC cells were transfected with control siRNA and PDE1A siRNA for 48 hr, and the expression of indicated proteins was detected. ( F–G ) NSCLC cells were treated with DMSO or vinpocetine (5, 10, 20 µM) for 24 hr, and the migrative ability of treated NSCLC cells was determined using the Transwell assay for 24 hr (n=3). ( H ) NSCLC cells were treated with DMSO or vinpocetine (5, 10, 20 µM) for 24 hr, and the expression of indicated proteins was determined. ( I ) The pulmonary metastatic nodules were stained using H&E staining and counted in nude mice harboring NCI-H1299 cells transfected with PDE1A shRNA and control shRNA (n=6). ** P < 0.01, *** P < 0.001. Figure 2—source data 1. Raw images for western blots shown in (labelled). Figure 2—source data 2. Raw images for western blots shown in .

Article Snippet: Antibodies against PDE1A (12442-2-AP), YTHDF2 (247441-AP), lamin B (12595-1-AP), and E-cadherin (20874-1-AP) were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Transfection, Control, Membrane, Wound Healing Assay, Expressing, Transwell Assay, Staining, shRNA, Western Blot

Journal: eLife

Article Title: Phosphodiesterase 1A physically interacts with YTHDF2 and reinforces the progression of non-small cell lung cancer

doi: 10.7554/eLife.98903

Figure Lengend Snippet: ( A–B ) NSCLC cells were transfected with PDE1A plasmid and empty vector for 24 hr. Cells were transferred to Transwell chambers without ( A ) or with ( B ) a Matrigel coating on the insert membrane, and the cell migrative and invasive abilities were determined, respectively, (n=3). ( C ) NSCLC cells were transfected with PDE1A plasmid and empty vector for 24 hr, and the wound healing assay was established in NSCLC cells, (n=3). ( D ) NSCLC cells were transfected with PDE1A plasmid and empty vector for 48 hr, and the expression of indicated proteins was detected. ( E ) The highly invasive NSCLC cells were separated using the Transwell chamber assay, and P3 cells were obtained from P0 cells after three generations. ( F–G ) The messenger RNA (mRNA) ( F ) and protein ( G ) levels of indicated genes were determined in P3 and P0 NSCLC cells. ( H ) The pulmonary metastatic nodules were stained using H&E and Bouin’s solution and counted in nude mice harboring NCI-H1299 cells transfected with PDE1A plasmid and empty vector, (n=6). ** P < 0.01, *** P < 0.001. Figure 3—source data 1. Raw images for western blots shown in (labelled). Figure 3—source data 2. Raw images for western blots shown in .

Article Snippet: Antibodies against PDE1A (12442-2-AP), YTHDF2 (247441-AP), lamin B (12595-1-AP), and E-cadherin (20874-1-AP) were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Transfection, Plasmid Preparation, Membrane, Wound Healing Assay, Expressing, Transwell Chamber Assay, Staining, Western Blot

Journal: eLife

Article Title: Phosphodiesterase 1A physically interacts with YTHDF2 and reinforces the progression of non-small cell lung cancer

doi: 10.7554/eLife.98903

Figure Lengend Snippet: ( A ) It was collected from LinkedOmics. Statistical tests in LinkFinder include Pearson’s correlation coefficient, Spearman’s rank correlation, Student’s t-test, Wilcoxon test, analysis of variance, Kruskal-Wallis analysis, Fisher’s exact test, Chi-squared test, Jonckheere’s trend test, and Cox’s regression analysis. Multiple-test correction is performed using the Benjamini and Hochberg method to generate the false discovery rate. ( B–D ) NSCLC cells were transfected with empty vector/PDE1A overexpressing plasmid or control siRNA/siPDE1A for 48 hr, and then NSCLC cells were placed on the upper panel of Transwell with 0.4 μm insert, human umbilical vein endothelial cells (HUVECs) were placed on the lower panel of Transwell, and wound healing assay was performed to determine the migrative abilities of HUVECs, (n=3). ( E ) NSCLC cells with PDE1A overexpression were treated with 10 µM GW4869, and a wound healing assay was performed to determine the migrative abilities of HUVECs, (n=3). ( F ) NSCLC cells were transfected with empty vector or shPDE1A, then cells were transplanted into nude mice via subcutaneous injection, and the blood vessels were counted after 60 days, (n=6). ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against PDE1A (12442-2-AP), YTHDF2 (247441-AP), lamin B (12595-1-AP), and E-cadherin (20874-1-AP) were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Transfection, Plasmid Preparation, Control, Wound Healing Assay, Over Expression, Injection

Journal: eLife

Article Title: Phosphodiesterase 1A physically interacts with YTHDF2 and reinforces the progression of non-small cell lung cancer

doi: 10.7554/eLife.98903

Figure Lengend Snippet: Non-small cell lung cancer (NSCLC) cells were transfected with control siRNA and PDE1A siRNA. After 24 hr, cells were collected for Transwell assay. Cells were treated with DMSO or 5 µM SQ22536 for 24 hr, and the migrative abilities of NSCLC cells were determined, (n=3). ns, not significant, ** P < 0.01. Figure 5—figure supplement 1—source data 1. Raw images for western blots related to (labelled). Figure 5—figure supplement 1—source data 2. Raw images for western blots related to .

Article Snippet: Antibodies against PDE1A (12442-2-AP), YTHDF2 (247441-AP), lamin B (12595-1-AP), and E-cadherin (20874-1-AP) were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Transfection, Control, Transwell Assay, Western Blot

Journal: eLife

Article Title: Phosphodiesterase 1A physically interacts with YTHDF2 and reinforces the progression of non-small cell lung cancer

doi: 10.7554/eLife.98903

Figure Lengend Snippet: ( A ) A Venn diagram was generated using LinkedOmics, and overrepresentation enrichment analysis (ORA) was performed to analyze the molecular pathway regulated by PDE1A in NSCLC. Sample cohort: TCGA_NSCLC; Institute: UNC; Data type: RNAseq; Platform: HiSeq RNA; Attribute: PDE1A; Statistical methods: Spearman’s correlation test; Patients: 515; Tools: ORA; Gene Ontology analysis: WikiPathways and PANTHER Pathway; Select rank criteria: FDR; Select sign: Positively correlated; Significance level: 0.05; TOP40 was selected to generate the Venn diagram. ( B ) Gene set enrichment analysis (GSEA) was performed to analyze the biological process of PDE1A in NSCLC. ( C ) NSCLC cells were transfected with PDE1A plasmid and empty vector for 48 hr, and the expression of indicated proteins was detected. ( D ) NSCLC cells were transfected with control siRNA and PDE1A siRNA for 48 hr, and the expression of indicated proteins was detected. ( E ) NSCLC cells were treated with DMSO or vinpocetine (5, 10, 20 µM) for 24 hr, and the expression of indicated proteins was determined. ( F ) NSCLC cells overexpressing PDE1A were transfected with control siRNA and STAT3 siRNA for 48 hr, and the migrative abilities of NSCLC cells were determined by Transwell assay, (n=3). ( G ) NSCLC cells overexpressing PDE1A were treated with STAT3 inhibitor SH-4–54 (5 µM) for 24 hr. The migrative abilities of NSCLC cells were determined by Transwell assay, (n=3). ( H ) NSCLC cells overexpressing PDE1A were transfected with control siRNA and STAT3 siRNA for 48 hr, and the expression of indicated protein was detected by western blot. ( I ) NSCLC cells overexpressing PDE1A were treated with STAT3 inhibitor SH-4–54 (5 µM) for 24 hr, and the expression of indicated protein was detected by western blot. ( J ) The interaction between PDE1A and STAT3 was determined by immunoprecipitation. ( K ) NCI-H1299 cells were transfected with empty vector and PDE1A overexpressing plasmid for 48 hr, and the contribution of PDE1A in the cytoplasm and nucleus was determined. ** P < 0.01, *** P < 0.001. Figure 5—source data 1. Raw images for western blots shown in (labelled). Figure 5—source data 2. Raw images for western blots shown in .

Article Snippet: Antibodies against PDE1A (12442-2-AP), YTHDF2 (247441-AP), lamin B (12595-1-AP), and E-cadherin (20874-1-AP) were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Generated, Transfection, Plasmid Preparation, Expressing, Control, Transwell Assay, Western Blot, Immunoprecipitation

Journal: eLife

Article Title: Phosphodiesterase 1A physically interacts with YTHDF2 and reinforces the progression of non-small cell lung cancer

doi: 10.7554/eLife.98903

Figure Lengend Snippet: ( A ) Venn diagram showing the overlap among PDE1A-interacting proteins (it was collected from mass spectrometry analysis in NSCLC cells), STAT3-coexpressed genes (collected from gene correlation using UALCAN), upregulated proteins in NSCLC compared with normal tissues (analyzed by UALCAN based on CPTAC database), and upregulated genes in NSCLC compared with normal tissues (analyzed by UALCAN based on TCGA database). Pearson’s correlation analysis of UALCAN was used to evaluate gene correlation analyses, and Welch’s t-test was estimated to detect the significance of differences in expression levels between two groups. ( B ) GO enrichment analysis of PDE1A-interacting genes. ( C ) Immunoprecipitation followed by silver staining was performed to identify protein and protein interaction using A549 cell lysate with the anti-PDE1A antibody. ( D ) Immunoprecipitation was used to confirm protein and protein interaction in NCI-H1299 cells. ( E ) NSCLC cells overexpressing PDE1A were transfected with control siRNA and YTHDF2 siRNA for 48 hr, and the migrative abilities of NSCLC cells were determined by Transwell assay, (n=3). ( F ) NSCLC cells overexpressing PDE1A were transfected with control siRNA and YTHDF2 siRNA for 48 hr, and the expression of indicated protein was detected by western blot. *** P < 0.001. Figure 6—source data 1. Raw images for western blots shown in (labelled). Figure 6—source data 2. Raw images for western blots shown in .

Article Snippet: Antibodies against PDE1A (12442-2-AP), YTHDF2 (247441-AP), lamin B (12595-1-AP), and E-cadherin (20874-1-AP) were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Mass Spectrometry, Expressing, Immunoprecipitation, Silver Staining, Transfection, Control, Transwell Assay, Western Blot

Journal: eLife

Article Title: Phosphodiesterase 1A physically interacts with YTHDF2 and reinforces the progression of non-small cell lung cancer

doi: 10.7554/eLife.98903

Figure Lengend Snippet: ( A–B ) Non-small cell lung cancer (NSCLC) cells were transfected with control siRNA and YTHDF2 siRNA for 48 hr, and the knockdown efficiency of YTHDF2 was confirmed by western blot ( A ) and quantitative real‑time PCR (qRT-PCR) ( B ), (n=3). ( C–D ) The messenger RNA (mRNA) and protein levels of phosphodiesterase 1A (PDE1A) in NSCLC and normal lung tissues are shown. It was collected from UALCAN and shown. Gene: YTHDF2; TCGA dataset: Lung adenocarcinoma ( C ); CPTAC dataset: Lung adenocarcinoma ( D ). ( E ) The prognostic value of YTHDF2 in NSCLC patients was identified using the PROGgeneV2 online tool ( https://www.progtools.net/gene ). *** P < 0.001. Figure 6—figure supplement 1—source data 1. Raw images for western blots shown in (labelled). Figure 6—figure supplement 1—source data 2. Raw images for western blots shown in .

Article Snippet: Antibodies against PDE1A (12442-2-AP), YTHDF2 (247441-AP), lamin B (12595-1-AP), and E-cadherin (20874-1-AP) were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Transfection, Control, Knockdown, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Frontiers in Genetics

Article Title: Transcriptome profiling of intact bowel wall reveals that PDE1A and SEMA3D are possible markers with roles in enteric smooth muscle apoptosis, proliferative disorders, and dysautonomia in Crohn’s disease

doi: 10.3389/fgene.2023.1194882

Figure Lengend Snippet: Primer sequences.

Article Snippet: The staining antibodies were as follows: SEMA3D (dilution 1/50; Cat.# NBP1-85517, NOVUS, Centennial, United States of America) and PDE1A (dilution 1/200; Cat.# 12442-2-AP, Proteintech, Wuhan, China) ( ).

Techniques:

Journal: Frontiers in Genetics

Article Title: Transcriptome profiling of intact bowel wall reveals that PDE1A and SEMA3D are possible markers with roles in enteric smooth muscle apoptosis, proliferative disorders, and dysautonomia in Crohn’s disease

doi: 10.3389/fgene.2023.1194882

Figure Lengend Snippet: Abundant expression of SEMA3D and PDE1A proteins in layers of muscularis propria and mucosa in inflamed CD specimens. (A) Immunohistochemistry (IHC) images showing representative colon specimens from patients with CD and UC, including non-inflammatory and inflammatory samples. Staining demonstrates expression of SEMA3D and PDE1A at a magnification of ×20. The scale bar corresponds to 40 μm. (B,C) Graphs presenting the quantification of staining (percentage of DAB-positive tissue) in niBD, iUC, and iCD cohorts (niBD, n = 4; iUC, n = 7; iCD, n = 15). Statistical analysis was performed using Fisher’s LSD test. Nonsignificant p -values (>0.05) are denoted. Asterisks (*) indicate statistical significance levels: * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: The staining antibodies were as follows: SEMA3D (dilution 1/50; Cat.# NBP1-85517, NOVUS, Centennial, United States of America) and PDE1A (dilution 1/200; Cat.# 12442-2-AP, Proteintech, Wuhan, China) ( ).

Techniques: Expressing, Immunohistochemistry, Staining