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

Journal: Neurobiology of disease

Article Title: Identifying disulfidptosis-related biomarkers in epilepsy based on integrated bioinformatics and experimental analyses.

doi: 10.1016/j.nbd.2025.106789

Figure Lengend Snippet: Fig. 2. Identification of DE-DRMs in individuals with EP. (A) The heatmap of expression levels for nine DE-DRMs. (B) The expression levels of 11 DRMs were exhibited between Ctrl and EP groups in boxplots. (C) Correlation analysis of nine DE-DRMs. Red and green colors represent positive and negative correlations, respectively. (D) The PPI analysis of DE-DMRs showed that SLC3A2 interacted with SLC7A11, while NDUFS1 interacted with NDUFA11, NUBPL, and LRPPRC. Abbreviations: DE-DRMs, differentially expressed disulfidptosis-related molecules; EP, epilepsy; Ctrl, control; PPI, protein-protein interaction; SLC3A2, solute carrier family 3 member 2; SLC7A11, solute carrier family 7 member 11; NDUFS1, NADH:ubiquinone oxidoreductase core subunit S1; NDUFA11, NADH:ubiquinone oxidoreductase subunit A11; NUBPL, NUBP iron‑sulfur cluster assembly factor, mitochondrial; LRPPRC, leucine rich pentatricopeptide repeat containing. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The paraffin sections were incubated with the primary antibodies (overnight, 4 ◦C): anti-GYS1 antibody (Sangon Biotech, D122431, dilution: 1:100), anti-NDUFS1 antibody (Abcam, ab185733, dilution: 1:100), anti-OXSM antibody (Boster, A12866–1, dilution: 1:100), anti-LRPPRC antibody (Abcam, ab259927, dilution: 1:200), anti-NDUFA11 antibody (Abclonal, A16239, dilution: 1:100) (Yang et al., 2022), anti-NUBPL antibody (Boster, A10634–1, dilution: 1:100), anti-NCKAP1 antibody (Bioworld Technology, BS71703, dilution: 1:50), anti-SLC3A2 antibody (Santa, sc-390,154, dilution: 1:50), anti-SLC7A11 antibody (Abcam, ab307601. dilution: 1:500).

Techniques: Expressing, Control

Journal: Neurobiology of disease

Article Title: Identifying disulfidptosis-related biomarkers in epilepsy based on integrated bioinformatics and experimental analyses.

doi: 10.1016/j.nbd.2025.106789

Figure Lengend Snippet: Fig. 8. The expression of DE-DRMs in seizures models (in vitro and in vivo). (A) Constructing the in vitro seizures model. The amplitude and frequency of neuronal APs in the Mg2+-free group were significantly increased (n = 6 in each group; Independent sample t-test; #, P < 0.01). (B) Protein expression of nine DE-DRMs in the in vitro seizures model. The expression of GYS1, NDUFS1, OXSM, LRPPRC, NDUFA11, NUBPL, NCKAP1, and SLC3A2 were not significantly changed in the Mg2+-free group, while the expression of SLC7A11 was significantly increased in Mg2+-free group (n = 6 in each group; Independent sample t-test; #, P < 0.01). (C) Con structing the in vivo seizures model. No epileptoid discharges were observed in six rats of the Ctrl group, while significant epileptoid discharges were observed in six rats of the PTZ group (scale: Y-axis,50uV; X-axis, 0.5 s). (D) Protein expression of nine DE-DRMs in vivo models. The expressions of GYS1, NDUFS1, OXSM, LRPPRC, NDUFA11, NUBPL, NCKAP1, and SLC3A2 were not significantly changed in PTZ group, while the expression of SLC7A11 was significantly increased in PTZ group (n = 6 in each group; Independent sample t-test; #, P < 0.01). Abbreviations: DE-DRMs, differentially expressed disulfidptosis-related molecules; APs, action potentials; GYS1, glycogen synthase 1; SLC3A2, solute carrier family 3 member 2; SLC7A11, solute carrier family 7 member 11; NDUFS1, NADH:ubiquinone oxidoreductase core subunit S1; OXSM, 3-oxoacyl-ACP synthase, mitochondrial; LRPPRC, leucine rich pentatricopeptide repeat containing; NDUFA11, NADH:ubiquinone oxidoreductase subunit A11; NUBPL, NUBP iron‑sulfur cluster assembly factor, mitochondrial; NCKAP1, NCK associated protein 1; PTZ, pentylenetetrazol.

Article Snippet: The paraffin sections were incubated with the primary antibodies (overnight, 4 ◦C): anti-GYS1 antibody (Sangon Biotech, D122431, dilution: 1:100), anti-NDUFS1 antibody (Abcam, ab185733, dilution: 1:100), anti-OXSM antibody (Boster, A12866–1, dilution: 1:100), anti-LRPPRC antibody (Abcam, ab259927, dilution: 1:200), anti-NDUFA11 antibody (Abclonal, A16239, dilution: 1:100) (Yang et al., 2022), anti-NUBPL antibody (Boster, A10634–1, dilution: 1:100), anti-NCKAP1 antibody (Bioworld Technology, BS71703, dilution: 1:50), anti-SLC3A2 antibody (Santa, sc-390,154, dilution: 1:50), anti-SLC7A11 antibody (Abcam, ab307601. dilution: 1:500).

Techniques: Expressing, In Vitro, In Vivo

Journal: Neurobiology of disease

Article Title: Identifying disulfidptosis-related biomarkers in epilepsy based on integrated bioinformatics and experimental analyses.

doi: 10.1016/j.nbd.2025.106789

Figure Lengend Snippet: Fig. 9. Verifying the PPI in seizures models (in vivo and in vitro). (A-B) colocation analysis indicated that SLC7A11 colocalized with SLC3A2, NDUFS1 colocalized with LRPPRC, NDUFS1 colocalized with NUBPL, and NDUFS1 colocalized with NDUFA11 in both primary neurons and hippocampal tissue. (C–D) Pooled quan tification of protein immunoprecipitation showed a significant increment in the pull-down of SLC7A11 and a significant reduction in the pull-down of NDUFA11 in both the Mg2+-free group and PTZ group (n = 6 in each group; Independent sample t-test; #, P < 0.01). Abbreviations: PPI, protein-protein interaction; SLC3A2,solute carrier family 3 member 2; SLC7A11, solute carrier family 7 member 11; NDUFS1, NADH:ubiquinone oxidoreductase core subunit S1; LRPPRC, leucine rich pentatricopeptide repeat containing; NUBPL, NUBP iron‑sulfur cluster assembly factor, mitochondrial; NDUFA11, NADH:ubiquinone oxidoreductase subunit A11; PTZ, pentylenetetrazol.

Article Snippet: The paraffin sections were incubated with the primary antibodies (overnight, 4 ◦C): anti-GYS1 antibody (Sangon Biotech, D122431, dilution: 1:100), anti-NDUFS1 antibody (Abcam, ab185733, dilution: 1:100), anti-OXSM antibody (Boster, A12866–1, dilution: 1:100), anti-LRPPRC antibody (Abcam, ab259927, dilution: 1:200), anti-NDUFA11 antibody (Abclonal, A16239, dilution: 1:100) (Yang et al., 2022), anti-NUBPL antibody (Boster, A10634–1, dilution: 1:100), anti-NCKAP1 antibody (Bioworld Technology, BS71703, dilution: 1:50), anti-SLC3A2 antibody (Santa, sc-390,154, dilution: 1:50), anti-SLC7A11 antibody (Abcam, ab307601. dilution: 1:500).

Techniques: In Vivo, In Vitro, Immunoprecipitation