Journal: International journal of molecular medicine

Article Title: Identification of hypo- and hypermethylated genes related to atherosclerosis by a genome-wide analysis of DNA methylation.

doi: 10.3892/ijmm.2014.1692

Figure Lengend Snippet: Figure 1. Overexpression of Drosophila headcase (HECA), early B-cell factor 1 (EBF1), or nucleotide-binding oligomerization domain containing 2 (NOD2) in HEK293 cells. HEK293 cells were transfected with expression vectors for DDK epitope-tagged human (A) HECA, (B) EBF1, or (C) NOD2 or with the empty vector alone (control) for 48 h, after which cell lysates were subjected to immunoblot analysis with antibodies to the corresponding proteins, to DDK, and to β-actin (loading control). Data are representative of eight (HECA, EBF1 and NOD2) or 12 (control) experiments.

Article Snippet: The transfected cells were solubilized with 2X sodium dodecyl sulfate (SDS) sample buffer and subjected to immunoblot analysis with antibodies to human HECA (ab98993), EBF1 (ab126135), or NOD2 (ab31488) (all from Abcam, Cambridge, UK) at a dilution of 1:1,000, 1:1,000 or 1:500, respectively, or with mouse monoclonal antibodies to the DDK epitope (OriGene) at a dilution of 1:6,000.

Techniques: Over Expression, Binding Assay, Transfection, Expressing, Plasmid Preparation, Control, Western Blot

Journal: Journal of Functional Foods

Article Title: Methynissolin confers protection against gastric carcinoma via targeting RIPK2

doi: 10.1016/j.jff.2024.106327

Figure Lengend Snippet: Fig. 1. Increased expression of RIPK2 promotes cachexia in the TgRC mice. (A) The method of generation of RIPK2-tgflox/+ (TgR) mice based on Rosa26 locus (CAG-STOP-RIPK2-EGFP-Rosa26) using the CRISPR/Cas9 system. (B) The generation of TgR/Rosa26-CreERT2 (TgRC) mice using tamoxifen (TAM) injection. (C) The genotype analysis of RIPK2 protein expression in tail tissue of the TgR and TgRC mice. (D) Mouse body weight post tamoxifen (TAM) administration in TgR and TgRC mice (n = 10). (E) Fat mass, lean mass, and weight loss post tamoxifen (TAM) administration in TgR and TgRC mice (n = 10). Mice body composition was analyzed by EchoMRI. (F) Representative images of H&E staining showing the histopathological changes of muscle and WAT tissue in TgR and TgRC mice (n = 5). (G-M) Indicated mice were maintained in metabolic cages. Experimental mice administrated with TAM at the first light cycle. Values are hourly means. The energy balance (G), food intake (H), total energy expenditure (TEE) (I), oxygen consumption (VO2) (J), carbon dioxide production (VCO2) (K), respiratory exchange ratio (RER) (L) and locomotor activity (M) of TgR and TgRC mice post TAM injection were determined (n = 10). (N) The serum albumin, BUN, ALP and GGT contents in the TgR and TgRC mice (n = 10). (O) Kaplan-meier (KM) survival curves of mice. Data were shown as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns., no significance.

Article Snippet: The Ripk2 mouse gene knockout kit (CRISPR) (Cat: KN514841), the Mouse Ripk2 activation kit by CRISPRa (Cat: GA212726), and the primer sequences (Cat: MP212518) for PCR genotyping of TgR mice are sourced from OriGene Technologies in Wuxi, China.

Techniques: Expressing, CRISPR, Injection, Staining, Activity Assay

Journal: Journal of Functional Foods

Article Title: Methynissolin confers protection against gastric carcinoma via targeting RIPK2

doi: 10.1016/j.jff.2024.106327

Figure Lengend Snippet: Fig. 2. Methylnissolin protects against overexpressed RIPK2-associated cachexia in the TgRC mice. (A) A protocol of methylnissolin treatment on TgRC mice with TAM injection. (B) Mouse body weight analysis in TgRC and TgRC mice with increasing dosage of methylnissolin (10 mg/kg, 20 mg/kg and 40 mg/kg) in response to TAM injection (n = 10). (C, D) Fat mass (C) and lean mass (D) in TgRC and TgRC mice (n = 10). (E) Representative images of H&E staining showing the histopathological changes of muscle and WAT tissue in the indicated mice group (n = 5). (F-L) Indicated mice were maintained in metabolic cages. Values are hourly means. The energy balance (F), food intake (G), total energy expenditure (TEE) (H), oxygen consumption (VO2) (I), carbon dioxide production (VCO2) (J), respiratory exchange ratio (RER) (K) and locomotor activity (L) of TgRC + DMSO and TgRC + methylnissolin (40 mg/kg) mice were determined (n = 10). (M) The serum albumin, BUN, ALP and GGT contents in the indicated mice (n = 10). (N) Kaplan-meier (KM) survival curves of mice. Data were shown as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns., no significance.

Article Snippet: The Ripk2 mouse gene knockout kit (CRISPR) (Cat: KN514841), the Mouse Ripk2 activation kit by CRISPRa (Cat: GA212726), and the primer sequences (Cat: MP212518) for PCR genotyping of TgR mice are sourced from OriGene Technologies in Wuxi, China.

Techniques: Injection, Staining, Activity Assay

Journal: Journal of Functional Foods

Article Title: Methynissolin confers protection against gastric carcinoma via targeting RIPK2

doi: 10.1016/j.jff.2024.106327

Figure Lengend Snippet: Fig. 3. Overexpression of RIPK2 impairs lipid homeostasis and blocks lipid biosynthesis. (A) Heatmap showing the serum TG contents alterations in the TgR and TgRC mice in response to fed or fasting conditions (n = 5). (B) Analysis of small (LC-TGs) and large (LC-TGs) levels in fed or fasted TgR and TgRC mice (n = 5). (C) Analysis of small (LC-TGs) and large (LC-TGs) levels in fed or fasted TgR and TgRC mice (n = 5). (D) Heatmap showing the serum TG contents alterations in the fed or fasted TgR and TgRC mice (n = 5). (E) Heatmap showing the serum TG contents in the stomach tissue of Balb/c mice with/without MKN-45-RIPK2 WT or MKN-45-RIPK2 KO tumors (n = 10). (F, G) Lipid biosynthesis analysis in TgR and TgRC mice with TAM administration (F) and in Balb/c mice with/without MKN-45- RIPK2 WT or MKN-45-RIPK2 KO tumors (G) (n = 10). Data were shown as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns., no significance.

Article Snippet: The Ripk2 mouse gene knockout kit (CRISPR) (Cat: KN514841), the Mouse Ripk2 activation kit by CRISPRa (Cat: GA212726), and the primer sequences (Cat: MP212518) for PCR genotyping of TgR mice are sourced from OriGene Technologies in Wuxi, China.

Techniques: Over Expression

Journal: Journal of Functional Foods

Article Title: Methynissolin confers protection against gastric carcinoma via targeting RIPK2

doi: 10.1016/j.jff.2024.106327

Figure Lengend Snippet: Fig. 5. Increased expression of RIPK2 inhibits the ASK signaling pathway. (A) The RNA-seq analysis showing the gene expression profiles in the stomach tissue after TAM administration TgR and TgRC mice (n = 5). (B) KEGG analysis of DEGs by the DAVID database. (C) GSEA enrichment bars for lipid metabolism and ASK signaling pathway. (D) RNA-seq analysis indicating the genes expression alterations of ASK, PPARα, PPARβ, PPARδ, PPARγ, PPARGC1a and PPARGC1b in TgR and TgRC mice (n = 5). (E, F) Analysis of mRNA and protein expression of PPARα, ACLY, CD36, FASN and ACSL1 determined by qPCR (E) and western blotting assay (F) in TgR and TgRC mice (n = 5). (G, H) Analysis of mRNA and protein expression of p-ASK1, PPARα, ACLY, CD36, FASN and ACSL1 determined by qPCR (G) and western blotting assay (H) in TgR and TgRC mice (n = 5). Data were shown as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns., no significance.

Article Snippet: The Ripk2 mouse gene knockout kit (CRISPR) (Cat: KN514841), the Mouse Ripk2 activation kit by CRISPRa (Cat: GA212726), and the primer sequences (Cat: MP212518) for PCR genotyping of TgR mice are sourced from OriGene Technologies in Wuxi, China.

Techniques: Expressing, RNA Sequencing, Gene Expression, Western Blot

Journal: Journal of Functional Foods

Article Title: Methynissolin confers protection against gastric carcinoma via targeting RIPK2

doi: 10.1016/j.jff.2024.106327

Figure Lengend Snippet: Fig. 7. The molecular mechanism diagram by which RIPK2 induces cachexia, and protective effect of methylnissolin on gastric cancer and its associated cachexia progression.

Article Snippet: The Ripk2 mouse gene knockout kit (CRISPR) (Cat: KN514841), the Mouse Ripk2 activation kit by CRISPRa (Cat: GA212726), and the primer sequences (Cat: MP212518) for PCR genotyping of TgR mice are sourced from OriGene Technologies in Wuxi, China.

Techniques:

Journal: PLoS ONE

Article Title: Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

doi: 10.1371/journal.pone.0143518

Figure Lengend Snippet: A , B , Quantitative RT-PCR results of Pla2g3 in cerebrum and cerebellum are shown. Fold changes to the aged wild-type mice on normal diet are indicated. n = 4 in each group. C , Western blots for Pla2g3 and alpha-tubulin are shown. Abbreviation used; WT normal; wild type 29 months old mice fed on normal diet, WT deficient; 29 months old wild type mice fed on vitamin E deficient diet, ttpKO deficient; 29 months old Ttpa -/- mice fed on vitamin E deficient diet. *p<0.05.

Article Snippet: HEK293 cells were used for transient transfection of human Pla2g3 expression vector (Origene).

Techniques: Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

doi: 10.1371/journal.pone.0143518

Figure Lengend Snippet: Double immunostaining of Pla2g3 (green) and GFAP (red) in 29 months old wild type mouse (A, B), 29 months old Ttpa -/- mouse (C, D), Ischemic site (E, F) and traumatic injury site (G, H) of cerebral cortex of wild type mice. Arrow heads indicate the strong Pla2g3 expression in astrocytes of Ttpa -/- cortex. Scale bar: 50 μm.

Article Snippet: HEK293 cells were used for transient transfection of human Pla2g3 expression vector (Origene).

Techniques: Double Immunostaining, Expressing

Journal: PLoS ONE

Article Title: Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

doi: 10.1371/journal.pone.0143518

Figure Lengend Snippet: A , B , Quantitative RT-PCR result of IDE in TR-AST cells and rat primary astrocytes. TR-AST cells and primary astrocytes were treated with hydrogen peroxide in indicated conditions. Fold changes to the non-treated cells as controls are indicated. C , Quantitative PCR results of IDE in Pla2g3 transfected HEK293 cells. Human Pla2g3 was transiently expressed and cells were harvested after 48 hours of transfection. Fold changes to the mock control are indicated. *p<0.05, ***p<0.001.

Article Snippet: HEK293 cells were used for transient transfection of human Pla2g3 expression vector (Origene).

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

Journal: PLoS ONE

Article Title: Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

doi: 10.1371/journal.pone.0143518

Figure Lengend Snippet: A , Pla2g3 expression in normal control brain and AD brain. The images are representative of 6 cases in each group. Frontal cortex was stained with anti-Pla2g3 antibody. B , Analysis of Pla2g3-positive cell numbers in frontal cortex. *p<0.05 C-H , Double immunostaining of Pla2g3 and GFAP in normal control brain (C-E) and AD brain (F-H). Arrowheads indicate the intensive staining of Pla2g3 in astrocytes of AD brain. Scale bars: A , 300 μm; C , 50 μm. Abbreviation used; NC; normal control, AD; Alzheimer’s disease patient, I; Molecular layer, II; External granular layer.

Article Snippet: HEK293 cells were used for transient transfection of human Pla2g3 expression vector (Origene).

Techniques: Expressing, Control, Staining, Double Immunostaining

Journal: bioRxiv

Article Title: Blood originates in hypoblasts during embryonic development

doi: 10.1101/2025.11.07.687183

Figure Lengend Snippet: A. Flow cytometry of hXEN expressed hematopoietic markers CD34 and CD43 upon exposure to hematopoietic cytokines and Activin A culture for 7 days. B. Generation of THP1 monocyte expressing dCas9-VP64 indicated by GFP. Fluorescence microscope showed GFP+ THP1 expressing dCas9-VP64. Scale bar = 100 um. C. CD71 erythroblast marker was up in CRISPRa-pooled gRNA-induced THP1 monocytes. Compared with VP64 that downregulated CD71, addition of pooled gRNA (MOI 0.25 and 1.0, respectively) rescued the CD71 level than WT. D. hXEN expressing dCAS9-VP64 and gRNAs. hXEN (human extraembryonic endoderm).

Article Snippet: The gRNA sequences for 30 genes were selected from the CRISPRa Library (Addgene #60956), with 5 gRNAs for each gene.

Techniques: Flow Cytometry, Expressing, Fluorescence, Microscopy, Marker

Journal: Clinical and Translational Medicine

Article Title: PROM2 overexpression induces metastatic potential through epithelial‐to‐mesenchymal transition and ferroptosis resistance in human cancers

doi: 10.1002/ctm2.1632

Figure Lengend Snippet: ASO sequences used in this study.

Article Snippet: The following day, cells were transfected with the human PROM2 activation kit by crispra (#GA115763; Origene, Germany) using Lipofectamine 3000 (#L3000001; Invitrogen, France), according to the manufacturer's instructions.

Techniques: Sequencing

Journal: Clinical and Translational Medicine

Article Title: PROM2 overexpression induces metastatic potential through epithelial‐to‐mesenchymal transition and ferroptosis resistance in human cancers

doi: 10.1002/ctm2.1632

Figure Lengend Snippet: PROM2 expression level has an impact on melanoma invasion and migration. (A) mRNA expression level of PROM2 in A375 KO (Cq > 40), A375 and A375 PROM2 cell lines (*** p < .001). (B) PROM2 immunofluorescence staining (in green) with nuclei stained with DAPI (in blue), and protein normalised expression using Western blot (** p < .01). (C and D) Count of invading (C) or migrating (D) cells at 24, 48 and 72 h in Boyden chamber (*** p < .001). (E) The left panel displays images illustrating the formation and expansion of spheroids derived from A375 KO, A375 and A375 PROM2 cell lines. On the right, the outcomes of the thresholding process are presented, conducted on the images acquired at day 4 after 24 h of invasion in collagen. Additionally, thresholded images for all analysed spheroids are included in Figure . Scale bars = 100 µm (** p < .01). (F) Left panel shows histological sections of lungs from mice injected with 5 × 10 6 A375 KO, A375 or A375 PROM2 cell lines, using standard haematoxylin and eosin coloration (A, pulmonary alveoli; B, bronchus; M, metastasis). Right panel shows the mean surface area in percentage of lung metastases/total lung surface area quantified 8 weeks after injection (*** p < .001). (G) Mean number of metastases in the five mouse models of patient‐derived melanoma xenografts (** p < .01).

Article Snippet: The following day, cells were transfected with the human PROM2 activation kit by crispra (#GA115763; Origene, Germany) using Lipofectamine 3000 (#L3000001; Invitrogen, France), according to the manufacturer's instructions.

Techniques: Expressing, Migration, Immunofluorescence, Staining, Western Blot, Derivative Assay, Injection

Journal: Clinical and Translational Medicine

Article Title: PROM2 overexpression induces metastatic potential through epithelial‐to‐mesenchymal transition and ferroptosis resistance in human cancers

doi: 10.1002/ctm2.1632

Figure Lengend Snippet: PROM2 overexpression in cells is associated with an EMT phenotype. (A) mRNA expression level of epithelial marker ( CDH1 ), EMT markers ( ZEB1 , ZEB2 , SNAI1 , SNAI2 , TWIST1 , TWIST2 ) and mesenchymal marker ( VIM ) of A375 KO, A375 and A375 PROM2 cell lines (** p < .01). (B) Immunoblotting for ZEB1 and SNAI1, with normalised expression in A375 KO, A375 and A375 PROM2 cell lines (** p < .01). (C) Left panel illustrates the laser‐microdissection of a melanoma lung metastasis tagged with PRAME (green fluorescence, red arrow) from a mice injected with A375 cell line. Right panel shows mRNA quantification of ZEB1 (*** p < .0001), SNAI1 (*** p < .001) and TWIST1 (*** p < .001) in laser‐microdissected lung metastases or tumour xenografts using digital‐droplet PCR. (D) ZEB1 and SNAIL/SLUG immunostaining of tumour xenografts expressing low (PROM2‐) or high (PROM2+) PROM2 (*** p < .0001). (E) PROM2, ZEB1 and SNAIL/SLUG immunoblotting of tumour xenografts expressing low (PROM2−) or high (PROM2+) PROM2 (** p < .01).

Article Snippet: The following day, cells were transfected with the human PROM2 activation kit by crispra (#GA115763; Origene, Germany) using Lipofectamine 3000 (#L3000001; Invitrogen, France), according to the manufacturer's instructions.

Techniques: Over Expression, Expressing, Marker, Western Blot, Laser Capture Microdissection, Fluorescence, Injection, Immunostaining

Journal: Clinical and Translational Medicine

Article Title: PROM2 overexpression induces metastatic potential through epithelial‐to‐mesenchymal transition and ferroptosis resistance in human cancers

doi: 10.1002/ctm2.1632

Figure Lengend Snippet: PROM2 overexpression is associated with ferroptosis resistance. (A) Quantification of ferrous iron (Fe 2+ ) in A375 KO, A375 and A375 PROM2 cell lines without treatment, or after 24 h of treatment with deferoxamine (DFO, ferroptosis inhibitor), or with RSL3 or Erastin (ferroptosis activators) (*** p < .0001). (B) Cell viability of A375 KO, A375 and A375 PROM2 cell lines after the addition of DFO, RSL3 or Erastin during 24 h (*** p < .0001). (C) Quantification of Fe 2+ (bar histograms) and cell viability (dash‐dotted line) of A375 PROM2 cell line treated for 24 h with increasing concentrations of RSL3 or Erastin. (D) Left panel shows spheroids obtained from A375 cell lines and cultured with or without Erastin [using white light (left) or FerroOrange fluorescence (right)]. Right panel shows Fe 2+ quantification through FerroOrange fluorescence quantification (** p < .01). (E) Immunoblotting and quantification of TSG101 (multivesicular bodies) in A375 KO (white), A375 (grey) and A375 PROM2 (black) cell lines (** p < .01). (F) Quantification of Fe 2+ in patient‐derived melanoma xenografts, with the correlation with PROM2 mRNA expression in tumours (*** p < .0001). (G) Quantification of 4HNE (lipid peroxidation) in A375 KO, A375 and A375 PROM2 cell lines (top panel, *** p < .0001), and the correlation with PROM2 mRNA expression in patient‐derived melanoma xenografts (bottom panel, *** p < .001).

Article Snippet: The following day, cells were transfected with the human PROM2 activation kit by crispra (#GA115763; Origene, Germany) using Lipofectamine 3000 (#L3000001; Invitrogen, France), according to the manufacturer's instructions.

Techniques: Over Expression, Cell Culture, Fluorescence, Western Blot, Derivative Assay, Expressing

Journal: Clinical and Translational Medicine

Article Title: PROM2 overexpression induces metastatic potential through epithelial‐to‐mesenchymal transition and ferroptosis resistance in human cancers

doi: 10.1002/ctm2.1632

Figure Lengend Snippet: Targeting PROM2 with an antisense oligonucleotide (ASO) prevents metastases in human melanoma xenografts. (A) PROM2 mRNA expression of A375 PROM2 cells treated during 4 days with different concentrations of an anti‐PROM2. (B) Counts of invading A375 PROM2 cells treated for 72 h with two concentrations of the ASO (*** p < .0001). (C) Counts of migrating A375 PROM2 cells treated for 72 h with two concentrations of the ASO (*** p < .0001). (D) Quantification of Fe 2+ of A375 PROM2 cell line treated for 4 days with two concentrations of the ASO (*** p < .0001). (E) mRNA expression level of epithelial marker ( CDH1 ), EMT markers ( ZEB1 , ZEB2 , SNAI1 , SNAI2 , TWIST1 , TWIST2 ) and mesenchymal marker ( VIM ) of A375 PROM2 cell line treated for 4 days with 0 or 50 nM of the ASO (* p = .04). (F) Mean surface area of lung metastases determined 8 weeks after injection of 5 × 10 6 A375 PROM2 cell line, untreated or treated in vitro for 4 days with 50 or 500 nM of the ASO before injection (*** p < .001).

Article Snippet: The following day, cells were transfected with the human PROM2 activation kit by crispra (#GA115763; Origene, Germany) using Lipofectamine 3000 (#L3000001; Invitrogen, France), according to the manufacturer's instructions.

Techniques: Expressing, Marker, Injection, In Vitro

Journal: Clinical and Translational Medicine

Article Title: PROM2 overexpression induces metastatic potential through epithelial‐to‐mesenchymal transition and ferroptosis resistance in human cancers

doi: 10.1002/ctm2.1632

Figure Lengend Snippet: PROM2 is implicated in a pathophysiological loop of increased metastatic potential. (A) PROM2 mRNA expression after detachment of A375 cell line for 0, 30, 60 and 120 min (*** p < .0001). (B) PROM2 mRNA expression in A375 and A375 PROM2 cell lines, and in subcutaneous xenografts and lung metastases derived from A375 PROM2 cell line and obtained 8 weeks after grafting (*** p < .0001). (C) Schematic of experimental enrichment loop (BioRender and SMART Servier Medical Art). (D) Mean number of metastases between round 1 and round 2 (*** p < .001). (E) PROM2 , CAV1 and EMT marker mRNA expression in cultured cells after successive rounds (** p < .01 from round 0 to 3).

Article Snippet: The following day, cells were transfected with the human PROM2 activation kit by crispra (#GA115763; Origene, Germany) using Lipofectamine 3000 (#L3000001; Invitrogen, France), according to the manufacturer's instructions.

Techniques: Expressing, Derivative Assay, Marker, Cell Culture

Journal: Clinical and Translational Medicine

Article Title: PROM2 overexpression induces metastatic potential through epithelial‐to‐mesenchymal transition and ferroptosis resistance in human cancers

doi: 10.1002/ctm2.1632

Figure Lengend Snippet: PROM2 pathophysiological loop is linked to metastatic progression in patients. (A) PROM2 and EMT marker mRNA expression in sequential biopsies of three patients with metastatic melanoma (Patient 1: Mel1, Patient 2: Mel2, Patient 3: Mel3). (B) PROM2 and EMT marker mRNA expression in sequential biopsies of two patients with renal cell carcinoma (Patient 1: RCC1, Patient 2: RCC2). Ipi, ipilimumab; nivo, nivolumab; pembro, pembrolizumab.

Article Snippet: The following day, cells were transfected with the human PROM2 activation kit by crispra (#GA115763; Origene, Germany) using Lipofectamine 3000 (#L3000001; Invitrogen, France), according to the manufacturer's instructions.

Techniques: Marker, Expressing