Journal: Nucleic Acids Research

Article Title: A transcriptomic and proteomic map of primary human cell types

doi: 10.1093/nar/gkaf1498

Figure Lengend Snippet: Cell type specific genes demonstrated at mRNA level. (A) Heat map of genes with cell type specific expression. (B) Heat map of selected genes with endothelial or epithelial cell type specific expression. (C) Immunohistochemistry staining of protein C1orf116 across human tissues showing its epithelial cell type specific expression. The panel shows a magnified view (20×) from tissue microarrays.

Article Snippet: The Human Protein Atlas (HPA) project provided a tissue-based map of the human proteome through transcriptomics and tissue microarray-based immunohistochemistry analysis [ , ].

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Profiling of Atherosclerotic Lesions by Gene and Tissue Microarrays Reveals PCSK6 as a Novel Protease in Unstable Carotid Atherosclerosis

doi: 10.1161/atvbaha.113.301743

Figure Lengend Snippet: Figure 1. Study design. Carotid plaques (n=34) from asymptomatic and symptomatic patients were sectioned at 2 levels and 3 cores drilled from each level (A) to construct 2 tissue microarray (TMA) blocks with 204 cores in total (B). Based on symptomatic vs asymptom- atic microarray comparisons generated for n=127 plaques (C), highly upregulated genes were chosen for the study (D; in red). Several genes already known to be associated with atherosclerosis were also included (D; in black). TMAs were stained by immunohistochemistry (IHC) with antibodies toward human proteins of interest, and tissue sections were scored (0, 1, 2, or 3) for semiquantitative grading of staining intensity (E). Significantly differentially expressed proteins were validated further by IHC on individual plaques (F).

Article Snippet: From the list of highly upregulated genes obtained by microarray comparisons between symptomatic and asymptomatic carotid lesions, candidates with relatively restricted tissue distribution in humans were chosen for further analysis (based on the Human Protein Atlas).

Techniques: Construct, Microarray, Generated, Staining, Immunohistochemistry

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Profiling of Atherosclerotic Lesions by Gene and Tissue Microarrays Reveals PCSK6 as a Novel Protease in Unstable Carotid Atherosclerosis

doi: 10.1161/atvbaha.113.301743

Figure Lengend Snippet: Figure 4. PCSK6 is the most significantly upregulated proprotein convertase (PC) in symptomatic carotid plaques. Analy- ses of n=127 microarray profiles between carotid plaques (CP) and normal controls (iliac arteries [IA]) show significant down- regulation of Furin and PCSK5 in plaque tissue, upregulation of PCSK6 and PCSK7, whereas PCSK9 and PCSK2 did not exhibit significant variation in expression (A). Posi- tive correlation was found between expres- sion of PCSK6 and PCSK9, PCSK2 and PCSK7, whereas negative was observed with PCSK5 (B). Several PCSK6 isoforms recognized by different Affymetrix probes were significantly upregulated in plaques compared with controls, but most highly the isoform coding for secreted protein (C). This isoform was the only one with signifi- cantly higher expression in comparison between symptomatic (s) and asymptom- atic (as) plaques (D). Results were con- firmed by quantitative real-time polymerase chain reaction (qRT-PCR) analyses for expression of secreted PCSK6 isoform in a separate cohort of n=233 carotid plaques (E and F). Micorarray data expressed as log2 value and qRT-PCR data as fold change compared with control. Data in A, C, and D show mean with SD, whereas median with interquartile range is depicted in E and F. ER indicates endoplasmatic reticulum.

Article Snippet: From the list of highly upregulated genes obtained by microarray comparisons between symptomatic and asymptomatic carotid lesions, candidates with relatively restricted tissue distribution in humans were chosen for further analysis (based on the Human Protein Atlas).

Techniques: Microarray, Expressing, Comparison, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control

Journal: Oncotarget

Article Title: Up-regulation of SERPINA3 correlates with high mortality of melanoma patients and increased migration and invasion of cancer cells

doi: 10.18632/oncotarget.9409

Figure Lengend Snippet: Patient demographics

Article Snippet: Tissue microarray-based immunohistochemistry analysis showed a significant increase in SERPINA3 expression in invasive and metastatic melanomas compared to normal nevi and melanoma-in-situ ( P < 0.001, Chi-square test).

Techniques:

Journal: Oncotarget

Article Title: Up-regulation of SERPINA3 correlates with high mortality of melanoma patients and increased migration and invasion of cancer cells

doi: 10.18632/oncotarget.9409

Figure Lengend Snippet: A tissue microarray was stained using immunohistochemistry and scanned. All samples were scored using a previously described 12 point scale involving intensity and percentage of melanoma cells showing staining for SERPINA3 (see materials and methods). Panel A . Representative images of tissues with high and low scores. Panel B . Increased SERPINA3 expression correlates with progression of melanoma. Sample sizes of each group are shown in bracket. A marked increase in percentage of samples with high SERPINA3 expression was shown between MIS and PM ( P < 0.0001) and further between PM and MM (p<0.0001). NN, Normal nevi; MIS, Melanoma in situ; PM, Primary melanoma; MM, metastatic melanoma.

Article Snippet: Tissue microarray-based immunohistochemistry analysis showed a significant increase in SERPINA3 expression in invasive and metastatic melanomas compared to normal nevi and melanoma-in-situ ( P < 0.001, Chi-square test).

Techniques: Microarray, Staining, Immunohistochemistry, Expressing, In Situ

Journal: Oncotarget

Article Title: Up-regulation of SERPINA3 correlates with high mortality of melanoma patients and increased migration and invasion of cancer cells

doi: 10.18632/oncotarget.9409

Figure Lengend Snippet: Scoring scale: Low=0-4; High=5-12. Panel A . SERPINA3 expression grouped by AJCC stages. Panel B . SERPINA3 expression grouped by gender. Panel C . SERPINA3 expression grouped by melanoma thickness. Panel D . SERPINA3 expression grouped by ulceration status.

Article Snippet: Tissue microarray-based immunohistochemistry analysis showed a significant increase in SERPINA3 expression in invasive and metastatic melanomas compared to normal nevi and melanoma-in-situ ( P < 0.001, Chi-square test).

Techniques: Expressing

Journal: Oncotarget

Article Title: Up-regulation of SERPINA3 correlates with high mortality of melanoma patients and increased migration and invasion of cancer cells

doi: 10.18632/oncotarget.9409

Figure Lengend Snippet: Kaplan-Meier survival analyses on melanoma patients reveals that patients with high SERPINA3 (green line) expression have significantly worse A . overall and B . disease-specific 5-year survival than those with low SERPINA3 expression. ( P < 0.0001 for both).

Article Snippet: Tissue microarray-based immunohistochemistry analysis showed a significant increase in SERPINA3 expression in invasive and metastatic melanomas compared to normal nevi and melanoma-in-situ ( P < 0.001, Chi-square test).

Techniques: Expressing

Journal: Oncotarget

Article Title: Up-regulation of SERPINA3 correlates with high mortality of melanoma patients and increased migration and invasion of cancer cells

doi: 10.18632/oncotarget.9409

Figure Lengend Snippet: SERPINA3 as an independent prognostic marker for melanoma

Article Snippet: Tissue microarray-based immunohistochemistry analysis showed a significant increase in SERPINA3 expression in invasive and metastatic melanomas compared to normal nevi and melanoma-in-situ ( P < 0.001, Chi-square test).

Techniques: Marker

Journal: Oncotarget

Article Title: Up-regulation of SERPINA3 correlates with high mortality of melanoma patients and increased migration and invasion of cancer cells

doi: 10.18632/oncotarget.9409

Figure Lengend Snippet: Panel A . Knockdown of SERPINA3 expression in MMRU cells using synthetic small inhibitor RNA (SiRNA). Realtime PCR verified that SERPINA3 expression was significantly decreased in cultures cells transfected with two individually targeted SiRNA (SiRNA1 and SiRNA2) compared to random scrambled negative control SiRNA. Data represented as mean expression as compared to control +/− standard deviation. A Western blot is also shown from 72hrs after siRNA knockdown. Actin is used as the internal control, and SERPINA3 band seen at ~75kDa is shown. C: control SiRNA, 1: SiRNA1, 2: SiRNA2. Panel B . A Western blot is also shown from 72hrs after siRNA knockdown. Actin is used as the internal control, and SERPINA3 band seen at ~75kDa is shown. C: control SiRNA, 1: SiRNA1, 2: SiRNA2. Panel C . Effect of SERPINA3 silencing on melanoma cell proliferation, measured by MTS assay, represented as mean absorbance +/− standard deviation.

Article Snippet: Tissue microarray-based immunohistochemistry analysis showed a significant increase in SERPINA3 expression in invasive and metastatic melanomas compared to normal nevi and melanoma-in-situ ( P < 0.001, Chi-square test).

Techniques: Knockdown, Expressing, Transfection, Negative Control, Control, Standard Deviation, Western Blot, MTS Assay

Journal: Oncotarget

Article Title: Up-regulation of SERPINA3 correlates with high mortality of melanoma patients and increased migration and invasion of cancer cells

doi: 10.18632/oncotarget.9409

Figure Lengend Snippet: Panel A . Photomicrographs of representative invasion cells. MMRU melanoma cells were transfected with SiRNA for 24 hours then seeded in metrigel coated Boyden chamber (8 μm pore size) in 0.1%FBS media; lower chambers contained 20%FBS media. Membranes were harvested at 24 and 48 hours by swabbing the gel off with cotton swabs and fixing cells that had invaded to the bottom of the membrane using formaldehyde and staining with Toluylene red. Panel B . Effect of SERPINA3 knockdown on cell invasion. Cells were counted after fixing and staining. Represented as mean cell count +/− standard deviation.

Article Snippet: Tissue microarray-based immunohistochemistry analysis showed a significant increase in SERPINA3 expression in invasive and metastatic melanomas compared to normal nevi and melanoma-in-situ ( P < 0.001, Chi-square test).

Techniques: Transfection, Pore Size, Membrane, Staining, Knockdown, Cell Counting, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA inhibits progression of endometriosis by cellular senescence enhancing

doi: 10.1186/s12967-023-04209-0

Figure Lengend Snippet: METTL3 shows decreased expression in endometriosis specimens and primary ESCs. A The mRNA m6A levels in human ectopic with strict match eutopic endometium, and normal control endometrium of 30 paired specimens were detected by ELISA via an m6A RNA methylation colorimetric quantification kit. B The global m6A level in mRNA in human ectopic, eutopic, and normal control endometrium measured by m6A dot blot assay with anti-m6A antibody. C The mRNA level adjusted to GAPDH of methyltransferases, demethylases and N6-methyladenosine readers in EMs and paired tissues by RT-qPCR. D Protein level of METTL3 in EMs and paired specimens by western blotting, using GAPDH as an internal control. E Representative images illuminate METTL3 expression in EMs and paired tissues by immunohistochemistry (IHC) (scale bars = 100 μm and 50 μm). Relative expression of METTL3 in primary Ectopic(ESCs), Eutopic(EuSCs), and Normal control(NESCs) endometrium cells in mRNA level by RT-qPCR (F) and protein level by western blotting (G) , using GAPDH as an internal control. All above result were shown in means ± SD, ns, p ≥ 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Wild-type ESCs and ESCs transfected with the oe-METTL3 plasmid were analysed by Aksomics Company (Shanghai, China) using a human m6A epitranscriptomic microarray and mRNA microarray at Arraystar m6A single-base resolution [ ].

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Methylation, Dot Blot, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA inhibits progression of endometriosis by cellular senescence enhancing

doi: 10.1186/s12967-023-04209-0

Figure Lengend Snippet: METTL3 mediate m6A methylation inhibits the progression of EMs by promoting cellular senescence in vitro and in vivo. A The measurement of the knockdown efficiency of si-METTL3 transfected ESCs in mRNA level by RT-qPCR. B The global m6A levels in si-METTL3 transfected ESCs detected by m6A dot blot assay with anti-m6A antibody. The increasing ability of viability, proliferation, invasion, and migration of ESCs with si-METTL3 transfection was detected by CCK8 C , EdU staining D ), wound healing E and Transwell F assay. G The mRNA level of METTL3 in oe-METTL3 transfected ESCs by RT-qPCR. H The mRNA N6-methyladenosine level in oe-METTL3 transfected ESCs by m6A dot blot. I–L CCK8, EdU, wound healing, and Transwell assay showed the decreasing ability of viability, proliferation, invasion, and migration of ESCs with oe-METTL3 transfection in reverse. M Level of cellular senescence in ESCs transfect with si-NC, si-METTL3, oe-vector and oe-METTL3 by SA-β -Gal staining. N The protein level of METTL3 and senescence biomarkers in ESCs transfect with si-NC, si-METTL3, oe-vector, and oe-METTL3 by western blotting, using GAPDH as an internal control. O Mass morphology of AAV-shNC and AAV-shMETTL3 mice. P The xenografts were isolated and measured by caliper in AAV-shNC and AAV-shMETTL3 mice. Xenografts’ weight Q and volume R were measured and analyzed. S Representative staining images for cellular senescence biomarkers (Lamin b1, p53, and p21) in AAV-shNC(up) and AAV-shMETTL3 group(down) by IHC. (Scale bars = 100 μm and 50 μm). All above result were shown in means ± SD, ns, p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Wild-type ESCs and ESCs transfected with the oe-METTL3 plasmid were analysed by Aksomics Company (Shanghai, China) using a human m6A epitranscriptomic microarray and mRNA microarray at Arraystar m6A single-base resolution [ ].

Techniques: Methylation, In Vitro, In Vivo, Knockdown, Transfection, Quantitative RT-PCR, Dot Blot, Migration, Staining, Transwell Assay, Plasmid Preparation, Western Blot, Control, Isolation

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA inhibits progression of endometriosis by cellular senescence enhancing

doi: 10.1186/s12967-023-04209-0

Figure Lengend Snippet: SIRT1 as a downstream target and negatively regulated by METTL3-mediated m6A modification in vitro and in vivo. A Volcano plots for differentially expressed mRNAs between matched oe-vector and oe-METTL3 transfected ESCs by RNA-seq. (|Fold Change|≥ 1, p < 0.001, Red point: up-regulated mRNAs, blue point: down-regulated mRNAs, the grey point indicated not differential expressed). B Volcano plots for differential methylated m6A modification transcripts between matched oe-vector and oe-METTL3 transfected ESCs by m6A epitranscriptomic microarray.(|Fold Change|≥ 1). Hyper-Up, up-regulated m6A modified mRNAs of |Fold Change|≥ 2; Hyper-Down, down-regulated m6A modified mRNAs of |Fold Change|≥ 2; Hypo-Up, up-regulated m6A modified mRNAs of |Fold Change|< 2; Hypo-Down, down-regulated m6A modified mRNAs of |Fold Change|< 2. C Venn showed SIRT1 at the intersection of RNA-seq and m6A epitranscriptomic microarray. D Top results of KEGG enrichment analysis. KEGG, Kyoto Encyclopedia of Genes and Genomes. E The m6A motif of ESCs identifified by m6A epitranscriptomic microarray. F m6A peak distribution of mRNA in m6A epitranscriptomic microarray. G The decreasing SIRT1 expression level of oe-METTL3 transfected ESCs in RNA-seq. H The increasing m6A mythylation level of oe-METTL3 transfected ESCs in m6A epitranscriptomic microarray. I The mRNA level of SIRT1 in EMs and paired tissues by RT-qPCR. J Representative IF staining of METTL3 and SIRT1 expression in Ectopic, Eutopic, and normal endometrium tissues. (Red fluorescence: METTL3; Green fluorescence: SIRT1; Blue fluorescence: DAPI; scale bar: 100 μm). K mRNA level of SIRT1 in METTL3 knockdown(up) and overexpression(down) by RT-qPCR. L Reduction enrichment level of SIRT1 in ESCs after METTL3 silencing by RIP-qPCR. M Decreasing m6A modification level of SIRT1 transcripts during METTL3 knockdown by Merip-qPCR assay. N Workflow of Endometriosis CKO mice model. O Progression of ectopic lesions (Green rectangular range) on Days 3, 5, and 7 in control and CKO-METTL3 mice. The mRNA expression of METTL3 P and volume on Days 3, 5, and 7 Q of isolated ectopic lesions were measured and analyzed. R Representative IHC images for SIRT1 in Control(up) and CKO-METTL3 group(down). (scale bars = 100 μm and 50 μm). All above result were shown in means ± SD, ns, p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Wild-type ESCs and ESCs transfected with the oe-METTL3 plasmid were analysed by Aksomics Company (Shanghai, China) using a human m6A epitranscriptomic microarray and mRNA microarray at Arraystar m6A single-base resolution [ ].

Techniques: Modification, In Vitro, In Vivo, Plasmid Preparation, Transfection, RNA Sequencing, Methylation, Microarray, Expressing, Quantitative RT-PCR, Staining, Fluorescence, Knockdown, Over Expression, Control, Isolation

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA inhibits progression of endometriosis by cellular senescence enhancing

doi: 10.1186/s12967-023-04209-0

Figure Lengend Snippet: METTL3 reduced the mRNA stability of SIRT1 via YTHDF2 depending on the m6A manner. A SIRT1 mRNA level in ESCs detected undergo si-YTHDF1, si-YTHDF2, si-YTHDF3 transfection by RT-qPCR. B Reduction enrichment level of SIRT1 in ESCs after YTHDF2 depleting by RIP-qPCR. C mRNA level of SIRT1 in YTHDF2 knockdown and overexpression by RT-qPCR. SIRT1 mRNA decay analysis at the indicated times after actinomycin D (Act D, 5 μg/ml) treatment in ESCs under YTHDF2 overexpressed D , METTL3 overexpressed E , and both overexpressed F by RT-qPCR. G The protein level of YTHDF2 and SIRT1 in ESCs transfected with si-NC, si-YTHDF2, oe-METTL3, and si-YTHDF2 + oe-METTL3 H and si-NC, oe-YTHDF2, si-METTL3, and oe-YTHDF2 + si-METTL3 by western blotting, using GAPDH as an internal control. I The protein level of METTL3, SIRT1, and FOXo3a in increasing and exhausting METTL3 treated ESCs. J The protein level of SIRT1 and FOXo3a in si-SIRT1 transfected ESCs, using GAPDH as an internal control. All above result were shown in means ± SD, ns, p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Wild-type ESCs and ESCs transfected with the oe-METTL3 plasmid were analysed by Aksomics Company (Shanghai, China) using a human m6A epitranscriptomic microarray and mRNA microarray at Arraystar m6A single-base resolution [ ].

Techniques: Transfection, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Control

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated m6A modification of SIRT1 mRNA inhibits progression of endometriosis by cellular senescence enhancing

doi: 10.1186/s12967-023-04209-0

Figure Lengend Snippet: Schematic representation mechanisms of METTL3 inhibit the progression of Endometriosis by regulating SIRT1 through m6A-dependent manner

Article Snippet: Wild-type ESCs and ESCs transfected with the oe-METTL3 plasmid were analysed by Aksomics Company (Shanghai, China) using a human m6A epitranscriptomic microarray and mRNA microarray at Arraystar m6A single-base resolution [ ].

Techniques: