Journal: Frontiers in Immunology

Article Title: GNA15 predicts poor outcomes as a novel biomarker related to M2 macrophage infiltration in ovarian cancer

doi: 10.3389/fimmu.2025.1512086

Figure Lengend Snippet: GNA15 is a differentially expressed gene for drug resistance. (A) Differential gene expression analysis on the GSE33482 database, red points represent upregulated genes, and green points represent downregulated genes. (B) The intersection of 1598 drug resistance related DEGs and 18 prognostic M2-like TAM-related genes. (C, D) GNA15 expression between cisplatin-resistant cells of ovarian cancer cell and corresponding OC cells by qRT-PCR analysis. *p < 0.05; **: p < 0.01.

Article Snippet: Antibodies against GNA15 (1:350 dilution; NBP2-16557, Novus), CD163 (1:100 dilution, Gene, China) were used in this study.

Techniques: Gene Expression, Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: GNA15 predicts poor outcomes as a novel biomarker related to M2 macrophage infiltration in ovarian cancer

doi: 10.3389/fimmu.2025.1512086

Figure Lengend Snippet: GNA15 expression in normal and cancer tissues. (A) RNA-Seq analysis using TCGA-OV samples and matched normal control samples from the GTEx project. (B) ROC analysis of GNA15 expression for the discrimination between OC and normal controls using TCGA-OC samples and matched normal control samples from the GTEx project. (C) qRT-PCR analysis using OC cells and normal ovarian cells. (D) qRT-PCR analysis using clinical OC and normal control samples. (E) IHC was performed to detect the expression of GNA15 in OC and normal tissues. (F–I) Survival analysis comparing the high and low expression of GNA15 in OC patients based on TCGA-OC database, GSE3149, GSE63885 and our clinical samples. *p < 0.05; **: p < 0.01; *** p < 0.001.

Article Snippet: Antibodies against GNA15 (1:350 dilution; NBP2-16557, Novus), CD163 (1:100 dilution, Gene, China) were used in this study.

Techniques: Expressing, RNA Sequencing, Control, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: GNA15 predicts poor outcomes as a novel biomarker related to M2 macrophage infiltration in ovarian cancer

doi: 10.3389/fimmu.2025.1512086

Figure Lengend Snippet: Down-regulating GNA15 in vitro decreased ovarian cancer cell proliferation. (A) knock down of GNA15 in OVCAR5 and OVCAR8 using ShRNA and CRISRP-Cas9. (B) Down-regulating GNA15 significantly decreased ovarian cancer cell proliferation. The protein data were normalized to β-actin. *** p < 0.001.

Article Snippet: Antibodies against GNA15 (1:350 dilution; NBP2-16557, Novus), CD163 (1:100 dilution, Gene, China) were used in this study.

Techniques: In Vitro, Knockdown, shRNA

Journal: Nature Communications

Article Title: Histone H3K36me2 and H3K36me3 form a chromatin platform essential for DNMT3A-dependent DNA methylation in mouse oocytes

doi: 10.1038/s41467-022-32141-2

Figure Lengend Snippet: a Representative images of germinal vesicles of FGOs immunostained for H3K36me2 ( n = 14, from two independent experiments). Other representative images are also shown in Supplementary Fig. . An arrowhead indicates relatively strong H3K36me2 signals near the nuclear envelope. The maximum intensity projection images are shown. DAPI 4’,6-diamidino-2-phenylindol. Scale bar, 10 μm. b Genome browser snapshots showing H3K36me2 and H3K36me3 enrichment and CG methylation in FGOs. The upper and lower panels show the representative regions of chromosomes 15 and X, respectively. H3K36me2 and H3K36me3 enrichment are indicated by ChIP/input. c Violin plots showing H3K36me2 enrichment in 5 Mb bins in autosomes ( n = 502) and X chromosome ( n = 35). Boxplots show median value and 25–75th percentiles, and whiskers show 1.5× interquartile range from the box. Source data are provided as a Source Data file. d Violin plots showing CG methylation levels of 10 kb bins in autosomes ( n = 238,084) and the X chromosome ( n = 15,494). The methylation level was determined in 10 kb bins. Horizontal bars indicate mean values. Pie charts show percentages of 10 kb bins categorized as HMRs, MMRs, and LMRs. e Violin plots showing CG methylation levels of 10 kb bins across the genome ( n = 253,578), H3K36me2-enriched regions (ChIP/input ≥ 1.4, n = 30,234), and H3K36me3-enriched regions (ChIP/input ≥ 1.5, n = 28,655). Horizontal bars indicate mean values. Pie charts show percentages of 10 kb bins categorized as HMRs, MMRs, and LMRs.

Article Snippet: The total number of reads mapped to the spike-in chromatin (SNAP-ChIP-Kmet Stat Panel, EpiCypher) was used to normalize the read counts in samples of different genotypes (Supplementary Table ). (The number of reads from the spike-in was not sufficient for H3K36me2 ChIP-seq replicate 2; therefore, normalization was performed for replicate 1 only).

Techniques: Methylation

Journal: Nature Communications

Article Title: Histone H3K36me2 and H3K36me3 form a chromatin platform essential for DNMT3A-dependent DNA methylation in mouse oocytes

doi: 10.1038/s41467-022-32141-2

Figure Lengend Snippet: a Representative images of control and K36M FGOs immunostained for H3K36me2 (left) and plots showing their signal intensities (right). Expression of HA-tagged H3.3K36M (H3.3K36M-HA) was confirmed by HA immunostaining. Signal intensity was measured in the control ( n = 10) and K36M FGOs ( n = 10) from two independent experiments. Scale bar, 10 μm. P -values (two-tailed Mann–Whitney U tests) are indicated. Error bars indicate the mean ± SD. Source data are provided as a Source Data file. b Heatmaps showing genome-wide H3K36me2 (left) and H3K36me3 enrichment (right) in 10 kb bins in control and K36M FGOs. Enrichment values were normalized using the spike-in control. c Genome browser snapshots showing H3K36me2 and H3K36me3 enrichment and CG methylation in control and K36M FGOs. A representative region on the X chromosome. CG methylation is lost upon H3K36me2 depletion in the regions highlighted in violet. The enrichment values were ChIP/input. d Scatter plots showing CG methylation levels in control and K36M FGOs. Fifty thousand randomly selected 10 kb bins were plotted with a color gradient for H3K36me2 enrichment in control FGOs. e Violin plots showing CG methylation levels in control and K36M FGOs across the genome (left), autosomes (middle), and X chromosome (right). Horizontal bars indicate mean values. Pie charts show percentages of 10 kb bins categorized as HMRs, MMRs, and LMRs. f Boxplots showing CG methylation differences between K36M and control FGOs in individual chromosomes. CG methylation differences were determined in 10 kb bins ( n = 19,191, 17,638, 15,612, 15,010, 14,673, 14,592, 13,734, 12,541, 12,115, 12,685, 11,859, 11,646, 11,673, 11,848, 10,067, 9495, 9155, 8737, and 5813 bins for chromosome 1, 2, …, and 19, respectively, and n = 15,494 bins for chromosome X). The box shows the median value and 25–75th percentiles, and whiskers show 1.5× interquartile range from the box. g Genome browser snapshots showing H3K36me2 enrichment in control FGOs and changes in CG methylation in K36M FGOs. CG methylation differences between K36M and control FGOs (∆CGme) are indicated. The upper and lower panels indicate the representative regions of chromosomes 15 and X, respectively. H3K36me2 enrichment is indicated by ChIP/input.

Article Snippet: The total number of reads mapped to the spike-in chromatin (SNAP-ChIP-Kmet Stat Panel, EpiCypher) was used to normalize the read counts in samples of different genotypes (Supplementary Table ). (The number of reads from the spike-in was not sufficient for H3K36me2 ChIP-seq replicate 2; therefore, normalization was performed for replicate 1 only).

Techniques: Control, Expressing, Immunostaining, Two Tailed Test, MANN-WHITNEY, Genome Wide, Methylation

Journal: Nature Communications

Article Title: Histone H3K36me2 and H3K36me3 form a chromatin platform essential for DNMT3A-dependent DNA methylation in mouse oocytes

doi: 10.1038/s41467-022-32141-2

Figure Lengend Snippet: a Heatmaps showing H3K36me3, H3K36me2, and H3K27me3 enrichment and CG methylation levels in control and K36M FGOs. CG methylation differences between K36M and control FGOs (ΔCGme). 10 kb bins from the whole genome were grouped into five clusters based on H3K36me2 and H3K36me3 enrichment statuses in control FGOs. b Plots showing H3K36me2 enrichment around genic regions in control and K36M FGOs. The analysis was performed for all genes (left, n = 23,081) and those with H3K36me2 loss (right, n = 2407). c Plots showing H3K27me3 enrichment around genic regions in control and K36M FGOs. The analysis was performed for all genes (left, n = 23,081) and those with H3K36me2 loss (right, n = 2407). d MA plots showing changes in gene expression between K36M and control FGOs (left, n = 3) and between K36M oocyte-derived and control late two-cell embryos (right, n = 8). Differentially expressed genes with false discovery rate (FDR) < 0.05, are colored in red. CPM, counts per million. e Boxplots showing expression levels of all genes ( n = 23,081) and genes with H3K36me2 loss ( n = 2407) in control FGOs (left) and two-cell embryos (right). Genes with H3K36me2 loss included weakly expressed genes, such as olfactory receptor ( Olfr ) and vomeronasal receptor ( Vmnr ) genes (Supplementary Data ). The box shows the median value and 25–75th percentiles, and whiskers show 1.5× interquartile range from the box.

Article Snippet: The total number of reads mapped to the spike-in chromatin (SNAP-ChIP-Kmet Stat Panel, EpiCypher) was used to normalize the read counts in samples of different genotypes (Supplementary Table ). (The number of reads from the spike-in was not sufficient for H3K36me2 ChIP-seq replicate 2; therefore, normalization was performed for replicate 1 only).

Techniques: Methylation, Control, Gene Expression, Derivative Assay, Expressing

Journal: Nature Communications

Article Title: Histone H3K36me2 and H3K36me3 form a chromatin platform essential for DNMT3A-dependent DNA methylation in mouse oocytes

doi: 10.1038/s41467-022-32141-2

Figure Lengend Snippet: a Scatter plots showing CG methylation levels in control and Setd2 KO FGOs. Fifty thousand randomly selected 10 kb bins were plotted. b Heatmaps showing H3K36me3, H3K36me2, H3K27me3, and H3K4me3 enrichment and CG methylation levels in 10 kb bins in control and Setd2 KO FGOs , . CG methylation differences between Setd2 KO and control FGOs (ΔCGme). The data were sorted in the same order as in Fig. . c Heatmaps showing H3K4me3 enrichment in 10 kb bins in control and Mll2 KO FGOs . d Violin plots showing CG methylation differences in 10 kb bins of cluster 4 ( n = 45,161) between Setd2 KO and control FGOs (left) or between Mll2 KO and control FGOs (right) . Boxplots show median value and 25–75th percentiles, and whiskers show 1.5× interquartile range from the box. e Scatter plots showing CG methylation levels in control and Setd2 KO FGOs across the genome (left), autosomes (middle), and X chromosome (right). Fifty thousand (left) or ten thousand randomly selected 10 kb bins (middle and right) were plotted with a color gradient for H3K36me2 enrichment in Setd2 KO FGOs .

Article Snippet: The total number of reads mapped to the spike-in chromatin (SNAP-ChIP-Kmet Stat Panel, EpiCypher) was used to normalize the read counts in samples of different genotypes (Supplementary Table ). (The number of reads from the spike-in was not sufficient for H3K36me2 ChIP-seq replicate 2; therefore, normalization was performed for replicate 1 only).

Techniques: Methylation, Control

Journal: Nature Communications

Article Title: Histone H3K36me2 and H3K36me3 form a chromatin platform essential for DNMT3A-dependent DNA methylation in mouse oocytes

doi: 10.1038/s41467-022-32141-2

Figure Lengend Snippet: a Scatter plots showing CG methylation levels in the control and DM FGOs. Fifty thousand randomly selected 10 kb bins were plotted. b Violin plots showing CG methylation levels in 10 kb bins across the genome in control, Setd2 KO, DM , and Dnmt3a KO FGOs. Horizontal bars indicate mean values. c Scatter plot showing CG methylation levels in Dnmt3a KO and DM FGOs. Fifty thousand randomly selected 10 kb bins were plotted. d Genome browser snapshots showing CG methylation levels in control, Setd2 KO, DM , and Dnmt3a KO FGOs. The upper and lower panels show representative regions from chromosomes 13 and X, respectively. e Western blotting detecting DNMT3A and DNMT3L proteins in DM FGOs. Representative images from three biological replicates. Pan-H3 was used as the loading control. Source data are provided as a Source Data file. f Violin plots showing CG methylation differences between Dnmt3a KO and control FGOs in 10 kb bins of DM affected ( n = 59,487) and DM unaffected regions ( n = 450). Boxplots show median value and 25–75th percentiles, and whiskers show 1.5× interquartile range from the box. g Boxplots showing CG methylation levels in nongrowing oocytes in 10 kb bins of DM affected ( n = 59,487) and DM unaffected regions ( n = 450). The box shows the median value and 25–75th percentiles, and whiskers show 1.5× interquartile range from the box. h A model summarizing H3K36me2/3-dependent CG methylation in mouse oocytes. H3K36me2 and H3K36me3 facilitate CG methylation in MMRs and HMRs, respectively (top). Upon H3K36me2 depletion, CG methylation is lost in MMRs (second), but upon H3K36me3 depletion, CG methylation is lost in H3K36me3-enriched regions and gained in H3K36me2-enriched regions (third). When both H3K36me2 and H3K36me3 are lost, CG methylation is severely lost (bottom). Open and filled circles indicate unmethylated and methylated CG sites, respectively.

Article Snippet: The total number of reads mapped to the spike-in chromatin (SNAP-ChIP-Kmet Stat Panel, EpiCypher) was used to normalize the read counts in samples of different genotypes (Supplementary Table ). (The number of reads from the spike-in was not sufficient for H3K36me2 ChIP-seq replicate 2; therefore, normalization was performed for replicate 1 only).

Techniques: Methylation, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

doi: 10.3390/ijms21124383

Figure Lengend Snippet: Identification of the KLK14 hydrolysis sites within the CleavEx proMMP protein. CleavEx proMMP fusion proteins were separated using SDS-PAGE and electrotransferred for N-terminal sequencing. Identified sequences are represented in the bold font and the underscore denotes where the location of the expected activation cleavage P1-P1′ in the proMMP-derived sequence.

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 0.5 μg proMMP14 (catalog no. 918-MP-010, R&D Systems), 1 μg proMMP15 (catalog no. 916-MP-010, R&D Systems), 0.5 μg proMMP16 (catalog no. 1785-MP-010, R&D Systems), and 1 μg proMMP17 (catalog no. 7796-MP-010, R&D Systems) were separately incubated in 10 μL with a range of KLK14 concentrations (25–250 nM, with molar ratios from around 1:65 to 1:10 KLK14:MMP) in the presence of 5 μM batimastat (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS.

Techniques: Sequencing, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

doi: 10.3390/ijms21124383

Figure Lengend Snippet: N-terminal identification of KLK14-mediated processing of recombinant proMMPs. The KLK14 hydrolysis product sequences were analyzed by N-terminal sequencing using Edman degradation. The bold font denotes the amino acid sequences identified. The underscored residues represent changes to the native protein sequence, as reported by the manufacturer (R&D Systems, Abingdon, United Kingdom). KLK14 recognized the sequence 3-aa upstream of the native MMP17 activation site, likely because the native site was modified by the manufacturer. All residues are numbered according to the Uniprot reported sequence of the full-length proteins. Bands are labeled according to the notation explained at Figure 2 .

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 0.5 μg proMMP14 (catalog no. 918-MP-010, R&D Systems), 1 μg proMMP15 (catalog no. 916-MP-010, R&D Systems), 0.5 μg proMMP16 (catalog no. 1785-MP-010, R&D Systems), and 1 μg proMMP17 (catalog no. 7796-MP-010, R&D Systems) were separately incubated in 10 μL with a range of KLK14 concentrations (25–250 nM, with molar ratios from around 1:65 to 1:10 KLK14:MMP) in the presence of 5 μM batimastat (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS.

Techniques: Recombinant, Sequencing, Activation Assay, Modification, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

doi: 10.3390/ijms21124383

Figure Lengend Snippet: Gelatin zymography of proMMPs by KLK14-mediated processing. Activation of proMMPs by KLK14 results in a fully functional mature enzyme. Each proMMP was incubated with the indicated concentrations of KLK14 for 1 h at 37 °C. The reaction was stopped by the addition of KLK14-specific inhibitors, and the reaction mixture was analyzed by SDS-PAGE, followed by a zymogram with gelatin as a substrate. The proMMP2 ( A ) negative control was not activated. ProMMP14 ( B ), proMMP15 ( C ), and proMMP16 ( D ) were activated, whereas proMMP17 ( E ) did not show hydrolysis of gelatin; yet a shift corresponding to the loss of the profragment was observed (note that an amino acid substitution was introduced in proMMP17 by the manufacturer (R&D Systems, Abingdon, United Kingdom)). KLK = kallikrein-related peptidase; MMP = matrix metalloproteinase.

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 0.5 μg proMMP14 (catalog no. 918-MP-010, R&D Systems), 1 μg proMMP15 (catalog no. 916-MP-010, R&D Systems), 0.5 μg proMMP16 (catalog no. 1785-MP-010, R&D Systems), and 1 μg proMMP17 (catalog no. 7796-MP-010, R&D Systems) were separately incubated in 10 μL with a range of KLK14 concentrations (25–250 nM, with molar ratios from around 1:65 to 1:10 KLK14:MMP) in the presence of 5 μM batimastat (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS.

Techniques: Zymography, Activation Assay, Functional Assay, Incubation, SDS Page, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

doi: 10.3390/ijms21124383

Figure Lengend Snippet: Primers used for generating the proMMP CleavEx fusion proteins using mutagenesis.

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 0.5 μg proMMP14 (catalog no. 918-MP-010, R&D Systems), 1 μg proMMP15 (catalog no. 916-MP-010, R&D Systems), 0.5 μg proMMP16 (catalog no. 1785-MP-010, R&D Systems), and 1 μg proMMP17 (catalog no. 7796-MP-010, R&D Systems) were separately incubated in 10 μL with a range of KLK14 concentrations (25–250 nM, with molar ratios from around 1:65 to 1:10 KLK14:MMP) in the presence of 5 μM batimastat (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS.

Techniques: Mutagenesis, Sequencing