Journal: Thoracic Cancer

Article Title: Gentiana macrophylla flavonoids from Tibetan medicine decreases circ_0059665 to alleviate the progression of non‐small cell lung cancer under hypoxia

doi: 10.1111/1759-7714.15165

Figure Lengend Snippet: Primer sequences used for qRT‐PCR.

Article Snippet: The primary antibodies used were anti‐c‐Myc (ab32072), anti‐MMP2 (ab86607) (Abcam), NOVA2 (Cat# PA5‐83784) (Invitrogen).

Techniques:

Journal: Thoracic Cancer

Article Title: Gentiana macrophylla flavonoids from Tibetan medicine decreases circ_0059665 to alleviate the progression of non‐small cell lung cancer under hypoxia

doi: 10.1111/1759-7714.15165

Figure Lengend Snippet: Circ_0059665 targets miR‐512‐5p, and NOVA2 is a target of miR‐512‐5p. (a) Two predicted miRNAs (miR‐197‐3p and miR‐512‐3p) were identified in the overlapping of Circinteractome and CircBank databases. (b) Quantitative real‐time‐polymerase chain reaction (qRT‐PCR) analysis for miR‐197‐3p and miR‐512‐3p expression after circ_0059665 or pCD5‐ciR transfection. (c) The binding sites of miR‐512‐3p on circ_0059665. (d) The transfection efficiency of miR‐512‐3p or miR‐NC was validated by qRT‐PCR. (e, f) The interaction analysis between circ_0059665 and miR‐512‐3p by dual‐luciferase reporter assay. (g) qRT‐PCR analysis for miR‐512‐3p expression in non‐small cell lung cancer (NSCLC) and normal tissues. (h) Correlation analysis between circ_0059665 and miR‐512‐3p in NSCLC tissues. (i) qRT‐PCR analysis for miR‐512‐3p expression in NSCLC cell lines (H1299 and A549) and normal cell lines (16HBE). (j) qRT‐PCR for miR‐512‐3p level in H1299 and A549 cells after hypoxia stimulation alone or GF treatment and hypoxia stimulation. (k) The binding sites of miR‐512‐3p on NOVA2. (l, m) The interaction analysis between NOVA2 and miR‐512‐3p by dual‐luciferase reporter assay. (n) qRT‐PCR analysis for NOVA2 expression in NSCLC and normal tissues. (o) Correlation analysis between NOVA2 and miR‐512‐3p in NSCLC tissues. (p) Western blotting analysis for NOVA2 expression in NSCLC cell lines (H1299 and A549) and normal cell lines (16HBE). (q) Western blotting for NOVA2 level in H1299 and A549 cells after hypoxia stimulation alone or GF treatment and hypoxia stimulation. *** p < 0.001; **** p < 0.0001.

Article Snippet: The primary antibodies used were anti‐c‐Myc (ab32072), anti‐MMP2 (ab86607) (Abcam), NOVA2 (Cat# PA5‐83784) (Invitrogen).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Transfection, Binding Assay, Luciferase, Reporter Assay, Western Blot

Journal: Thoracic Cancer

Article Title: Gentiana macrophylla flavonoids from Tibetan medicine decreases circ_0059665 to alleviate the progression of non‐small cell lung cancer under hypoxia

doi: 10.1111/1759-7714.15165

Figure Lengend Snippet: NOVA2 reverses the suppressing effects of circ_0059665 knockdown on NSCLC cell proliferation, invasion and M2 polarization under hypoxic conditions. (a) The transfection efficiency of si‐NC or si‐circ_0059665 was validated using quantitative real‐time‐polymerase chain reaction (qRT‐PCR). (b–j) H1299 and A549 cells were transfected with si‐circ_0059665 alone or si‐circ_0059665 and NOVA2, followed by hypoxia stimulation. (b) Western blotting for NOVA2 level. (c–e) Cell counting kit‐8 (CCK‐8), 5‐ethynyl‐2′‐deoxyuridine (EdU) and colony formation assays were used for cell proliferation analysis. (f, g) Transwell assay for cell invasion. (h, i) Western blotting analysis for C‐Myc and MMP2 protein levels. (j) Flow cytometry for M2‐polarized macrophages. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The primary antibodies used were anti‐c‐Myc (ab32072), anti‐MMP2 (ab86607) (Abcam), NOVA2 (Cat# PA5‐83784) (Invitrogen).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Cell Counting, CCK-8 Assay, Transwell Assay, Flow Cytometry

Journal: Thoracic Cancer

Article Title: Gentiana macrophylla flavonoids from Tibetan medicine decreases circ_0059665 to alleviate the progression of non‐small cell lung cancer under hypoxia

doi: 10.1111/1759-7714.15165

Figure Lengend Snippet: NOVA2 weakens the suppressing effects of Gentiana macrophylla flavonoids (GF) on non‐small cell lung cancer (NSCLC) cell proliferation, invasion and M2 polarization under hypoxic conditions. (a–h) H1299 and A549 cells were transfected with pcDNA and NOVA2, and treated with GF, followed by hypoxia stimulation. (a) Western blotting for NOVA2 level. (b–d) Cell counting kit‐8 (CCK‐8), 5‐ethynyl‐2′‐deoxyuridine (EdU) and colony formation assay for cell proliferation analysis. (e) Transwell assay for cell invasion. (f, g) Western blotting analysis for C‐Myc and MMP2 protein levels. (h) Flow cytometry for M2‐polarized macrophages. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The primary antibodies used were anti‐c‐Myc (ab32072), anti‐MMP2 (ab86607) (Abcam), NOVA2 (Cat# PA5‐83784) (Invitrogen).

Techniques: Transfection, Western Blot, Cell Counting, CCK-8 Assay, Colony Assay, Transwell Assay, Flow Cytometry

Journal: Thoracic Cancer

Article Title: Gentiana macrophylla flavonoids from Tibetan medicine decreases circ_0059665 to alleviate the progression of non‐small cell lung cancer under hypoxia

doi: 10.1111/1759-7714.15165

Figure Lengend Snippet: Gentiana macrophylla flavonoids (GF) treatment suppresses tumor growth of non‐small cell lung cancer (NSCLC) in vivo via circ_0059665. The in vivo growth curve (a), representative images and the weight at the end points (b) of xenografts. (c, d) Quantitative real‐time‐polymerase chain reaction (qRT‐PCR) analysis for the levels of circ_0059665 and miR‐512‐3p in xenografts. (e) Western blotting analysis for NOVA2 protein level in xenografts. (f) Immunohistochemistry (IHC) analysis for the protein of NOVA2, c‐Myc, and MMP2 in xenografts. * p < 0.05.

Article Snippet: The primary antibodies used were anti‐c‐Myc (ab32072), anti‐MMP2 (ab86607) (Abcam), NOVA2 (Cat# PA5‐83784) (Invitrogen).

Techniques: In Vivo, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: medRxiv

Article Title: Spatial proteo-transcriptomic profiling reveals the molecular landscape of borderline ovarian tumors and their invasive progression

doi: 10.1101/2023.11.13.23298409

Figure Lengend Snippet: a) Principal Component Analysis (PCA) for the epithelium clearly separates serous borderline tumors, serous borderline tumors with a micropapillary pattern, invasive low grade serous cancer, and corresponding metastasis. This transition is evident in the diagonal of PC1 and PC2 from lower right to upper left. AI-based recognition of epithelial cells using immunofluorescence (EpCAM-purple, decorin-green) below the white diagonal line, followed by AI segmentation (yellow). b) Volcano plot of the differential epithelial protein expression between SBT and LGSC-PT in the epithelial compartment. A fold change cutoff of 1.5 and a q-value cutoff of 0.05 are indicated by vertical and horizontal lines, respectively. Proteins matching the significance for differential regulation (DR) criteria are highlighted in black, markers of secretory cells in orange and ciliated cells in red. c) Proteins of the mitogen-activated protein kinase (MAPK)-signaling pathway show a gradual increase towards LGSC and corresponding metastasis (Heatmap). Commonly altered Ras and Ras-regulating proteins (box plots). d) Boxplots of significantly changed membrane-associated progesterone receptor component 2 (PGRMC2) between the four groups (Student’s T-test). e) Heatmap. Proteins involved in apoptosis show reduced abundance from SBT to LGSC-Met. f) Upset plot. Comparison of MS-detected peptides/proteins detected in specific groups but completely absent in others and therefore not included in and (methods). The set size is the number of identified proteins, while the intersection size shows the number of overlapping proteins. g) Bar plot of the protein subset highlighted in (f). Bars present the percentage of samples in which the four most frequent proteins were identified per group using mass spectrometry. NOVA2 was solely detectable in more than 75% of LGSC-PT and LGSC-Met, but not in SBT and SBT-MP. h) Immunohistochemistry for NOVA2.

Article Snippet: The membrane was blocked with 5% non-fat dry milk (NFDM) (Lab Scientific) in Tris-buffered saline with Tween 20 (TBST) for 1 hour at room temperature The membranes were incubated with primary antibodies against NOVA2 (1:1000) and GAPDH (1:1000) (Proteintech) in 2% bovine serum albumin (Sigma-Aldrich) in TBST at 4 °C overnight.

Techniques: Immunofluorescence, Expressing, Membrane, Comparison, Mass Spectrometry, Immunohistochemistry

Journal: International journal of molecular sciences

Article Title: Alternative Splicing Changes Promoted by NOVA2 Upregulation in Endothelial Cells and Relevance for Gastric Cancer.

doi: 10.3390/ijms24098102

Figure Lengend Snippet: Figure 1. Novel AS events identified upon NOVA2 upregulation in mouse ECs. (A) Left panel: HA- tagged NOVA2 mRNA levels in empty vector control (HA) or NOVA2 (HA-NOVA2)-overexpressing mouse ECs (moEC). Data represent the mean ± SEM (n = 3 independent experiments) *** p ≤0.001. Unpaired Student’s t-test. Right panel: Immunoblotting using anti-HA antibody in HA and HA- NOVA2 moEC. Vinculin is used as loading control. (B) RT-PCR analysis of selected NOVA2 splicing targets in moEC-overexpressing HA-tagged NOVA2. Transcripts generated from skipping/inclusion of the AS exon are represented near the corresponding RT-PCR bands. The percentages of exon inclusion (PSI) are also indicated. For each AS event, the genomic region containing the AS exon and the flanking sequences are represented; grey boxes: AS exons; black boxes: constitutive exons; blue/red dots: YCAY clusters predicted to function as NOVA silencer/enhancer; blue/red bars: NOVA-silenced/enhanced exon inclusion events.

Article Snippet: NOVA2 IHC detection was obtained by using anti-NOVA2 (polyclonal rabbit, 1:100; Atlas Antibodies, Cat# HPA045607, RRID: AB_10962628) antibody.

Techniques: Plasmid Preparation, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Generated

Journal: International journal of molecular sciences

Article Title: Alternative Splicing Changes Promoted by NOVA2 Upregulation in Endothelial Cells and Relevance for Gastric Cancer.

doi: 10.3390/ijms24098102

Figure Lengend Snippet: Figure 2. Validation of NOVA2-mediated AS events in human ECs knockdown for NOVA2. (A) NOVA2 mRNA levels in HUVEC hTERT transfected with control (siCTR) or two different NOVA2 siRNAs (siNOVA2 #1, siNOVA2 #2). Data represent the mean ± SEM (n = 3 independent exper- iments) **** p ≤0.0001. One-way ANOVA with multiple Tukey’s comparisons test. (B) NOVA2 immunoblotting in siCTR, siNOVA2 #1 and siNOVA2 #2 HUVEC hTERT. (C) RT-PCR analysis of selected NOVA2 targets in siCTR, siNOVA2 #1 and siNOVA2 #2 HUVEC hTERT. Transcripts generated from skipping/inclusion of the AS exon are represented near the corresponding RT-PCR bands. The percentages of exon inclusion (PSI) are also indicated. For each AS event, the genomic region containing the AS exon and the flanking regions are represented; grey boxes: AS exons; black boxes: constitutive exons; blue/red dots: YCAY clusters predicted to function as NOVA silencer/enhancer; blue/red bars: NOVA silenced/enhanced exon inclusion events.

Article Snippet: NOVA2 IHC detection was obtained by using anti-NOVA2 (polyclonal rabbit, 1:100; Atlas Antibodies, Cat# HPA045607, RRID: AB_10962628) antibody.

Techniques: Biomarker Discovery, Knockdown, Transfection, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Generated

Journal: International journal of molecular sciences

Article Title: Alternative Splicing Changes Promoted by NOVA2 Upregulation in Endothelial Cells and Relevance for Gastric Cancer.

doi: 10.3390/ijms24098102

Figure Lengend Snippet: Figure 3. NOVA2 expression levels are increased in GC and associated with poor overall patients’ survival. (A) Expression of NOVA2 mRNA levels (mean ± SEM) in normal and GC samples from TCGA-STAD project (from TSVdb); Unpaired Student t-test with Welch’s correction (p = 0.0099). ** p ≤0.01. (B) Fold change of NOVA2 expression in normal and GC samples from Wang dataset (GSE19826) and DErrico dataset (GSE13911) (probe: 206477_s_t, p = 0.0145 (* p ≤0.05) and p = 0.0003 (*** p < 0.001, respectively) with Unpaired Student’s t-test. (C) Kaplan–Meier plot of overall survival in GC patients from TCGA-STAD project classified according to NOVA2 expression (cutoff: median) (red curve, high expression; black curve, low expression). Log-rank (Mantel–Cox) test (p = 0.0415). (D) Kaplan–Meier plot of overall survival in GC patients from GSE14210, GSE15459, GSE22377, GSE29272; GSE38749, GSE51105, GSE62254 datasets classified according to NOVA2 expression (cutoff: median; probe: 206477_s_t; Log-rank (Mantel–Cox) test p = 1.2 × 10−9) (red curve, high expression; black curve, low expression).

Article Snippet: NOVA2 IHC detection was obtained by using anti-NOVA2 (polyclonal rabbit, 1:100; Atlas Antibodies, Cat# HPA045607, RRID: AB_10962628) antibody.

Techniques: Expressing

Journal: International journal of molecular sciences

Article Title: Alternative Splicing Changes Promoted by NOVA2 Upregulation in Endothelial Cells and Relevance for Gastric Cancer.

doi: 10.3390/ijms24098102

Figure Lengend Snippet: Figure 4. Expression of NOVA2 in gastric cancer vessels. (A) Correlation analysis showing a significant association between COL4A1 and NOVA2 expression levels from TGCA-STAD project (Pearson r = 0.6372, p < 0.0001). (B) Serial sections of GC samples (n = 27) stained for NOVA2 (left panel) or ERG (right panel). Arrows indicate NOVA2-positive nuclei of ECs. No immunoreactivity is present in the nuclei of tumor cells. Arrowheads indicate ERG-positive nuclei of ECs in the same area on a consecutive section of tissue. Scale bar: 20 µm. (C) Kaplan–Meier plot of overall survival in our cohort of GC patients classified according to NOVA2 expression (red curve, high expression; black curve, low expression).

Article Snippet: NOVA2 IHC detection was obtained by using anti-NOVA2 (polyclonal rabbit, 1:100; Atlas Antibodies, Cat# HPA045607, RRID: AB_10962628) antibody.

Techniques: Expressing, Staining

Journal: International journal of molecular sciences

Article Title: Alternative Splicing Changes Promoted by NOVA2 Upregulation in Endothelial Cells and Relevance for Gastric Cancer.

doi: 10.3390/ijms24098102

Figure Lengend Snippet: Figure 5. Expression of RapGEF6 exon 21A in the TCGA-STAD dataset. (A) Coding transcript and protein domains of RapGEF6 adapted from DoChaP web tool. Black lines with numbers show the position in the transcript (nucleotides) and protein (amino acids). Different exons are represented in different colors; exon 21A is highlighted. RapGEF6 protein (NP_001157858) is depicted as a black line, with elliptical shapes representing functional domains: cyclic-nucleotide-binding domain (green); N-terminal domain for RasGEF-like protein domain (orange); post-synaptic density protein, disc large tumor suppressor, zonula occludens-1 protein (PDZ) domain (yellow); ras-associating domain (blue); ras-like guanine nucleotide exchange factor domain (purple). DR stands for disordered region encoded by exon 21A. (B) RapGEF6 exon 21A in healthy donor (blue) and tumor patient (red) samples of TCGA-STAD dataset. p value was calculated with unpaired student t-test with Welch’s correlation. (C) Correlation between NOVA2 and RapGEF6 exon21A expression in TCGA-STAD samples. Linear regression (red line) and Pearson r coefficient with two-tailed p value are reported. (D) RapGEF6 exon 21A expression in RSEM stratified as upper and lower quartiles according to tumor size (T1 and >T1). Unpaired student t-test. (E) Histotype distribution of STAD-GC tumors for low and high RapGEF6 exon 21A expression (comparing lower and upper quartile). Fisher’s exact test. (F) Kaplan–Meier analysis of 10-year overall survival for STAD patients stratified for RapGEF6 exon 21A expression in high (upper quartile, red line), medium (medium quartile, black line) and low (lower quartile, green line) levels. Log-rank (Mantel–Cox) test. p value legend: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (G,H) NOVA2 and RapGEF6 exon 21A correlation with a gastric angiogenesis-related gene (ARG) signature.

Article Snippet: NOVA2 IHC detection was obtained by using anti-NOVA2 (polyclonal rabbit, 1:100; Atlas Antibodies, Cat# HPA045607, RRID: AB_10962628) antibody.

Techniques: Expressing, Functional Assay, Binding Assay, Two Tailed Test