Journal: Frontiers in Immunology

Article Title: Deciphering hub genes and immune landscapes related to neutrophil extracellular traps in rheumatoid arthritis: insights from integrated bioinformatics analyses and experiments

doi: 10.3389/fimmu.2024.1521634

Figure Lengend Snippet: Validation of pivotal gene expression in the mouse model. (A) H&E-stained images of synovial tissues of a mouse model. Expression of CRYBG1 (B) , RRM2 (C) , MMP1 (D) and SLC19A2 (E) proteins in synovial tissues of mouse knee joints was detected by IHC (standard bar = 50 μm). Data are expressed as mean ± standard deviation. *** P < 0.001, **** P < 0.0001.

Article Snippet: Next, primary antibodies CRYBG1 (1:300, bs-9093R, bioss), RRM2 (1:300, bs-7133R, bioss), MMP1 (1:100, ab52631, abcam), and SLC19A2 (1:300, bs-10738R, bioss) were added dropwise to the sections and placed in the incubator at 37°C for 60 min.

Techniques: Expressing, Staining, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Deciphering hub genes and immune landscapes related to neutrophil extracellular traps in rheumatoid arthritis: insights from integrated bioinformatics analyses and experiments

doi: 10.3389/fimmu.2024.1521634

Figure Lengend Snippet: Validation of NETs formation and hub gene expression in a clinical cohort. (A, B) Immunofluorescence microscopy examination revealed the presence of NETs in RA, defined as MPO (green) and NE (red) (standard bar = 50 μm). RT-qPCR was applied to detect the expression levels of hub genes CRYBG1 (C) , RRM2 (D) , MMP1 (E) and SLC19A2 (F) in neutrophils. *** P < 0.001, **** P < 0.0001.

Article Snippet: Next, primary antibodies CRYBG1 (1:300, bs-9093R, bioss), RRM2 (1:300, bs-7133R, bioss), MMP1 (1:100, ab52631, abcam), and SLC19A2 (1:300, bs-10738R, bioss) were added dropwise to the sections and placed in the incubator at 37°C for 60 min.

Techniques: Expressing, Immunofluorescence, Microscopy, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Deciphering hub genes and immune landscapes related to neutrophil extracellular traps in rheumatoid arthritis: insights from integrated bioinformatics analyses and experiments

doi: 10.3389/fimmu.2024.1521634

Figure Lengend Snippet: NETs-related hub genes are clinically relevant. (A) Forest plot of logistic regression analysis of hub genes in RA prediction. (B) The clinical RA prediction nomogram model is based on 4 NETs-related hub genes. (C) ROC curves of the nomogram model and the characteristic variables within the model. Calibration curves (D) , DCA (E) and CIC (F) were predicted by the nomogram model. (G-P) Correlation analysis between CRYBG1, RRM2, MMP1, and SLC19A2 and laboratory markers in RA patients. * P < 0.05, ** P < 0.01.

Article Snippet: Next, primary antibodies CRYBG1 (1:300, bs-9093R, bioss), RRM2 (1:300, bs-7133R, bioss), MMP1 (1:100, ab52631, abcam), and SLC19A2 (1:300, bs-10738R, bioss) were added dropwise to the sections and placed in the incubator at 37°C for 60 min.

Techniques:

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 1. Pivotal role of Glu246 and Asp247 in the conformation of the RRM2 domain of TDP-43. A. a, Scheme showing the alignment of RRM2 subdomains (residues 232–270 in human TDP-43) in multiple species. Glu246 (E246) and Asp247 (D247) are preserved across all species. b, Chemical properties of amino acid substitution mutants of E246 or D247. E246Q and D247N were used to create substitution mutants with minimal alteration of side chains. E246G and D247G were designed so that the effects of the side chains were eliminated, and the flexibility of the amide bonds was increased. B. Western blot analysis of recombinant RRM2 proteins of wild-type (WT), E246Q/D247N (QN), and E246G/D247G (GG), using an anti-TDP-43 rabbit polyclonal antibody that recognizes the RRM2 domain (Proteintech). The E246G/D247G mutant RRM2 showed marked oligomerization even when incubated at 4uC (vertical line). Note that considerable RRM2 dimers or oligomers were dissociated into monomers in the presence of DTT. Arrowhead and double arrowheads indicate RRM2 monomer and dimers, respectively. Asterisk possibly indicates heat-related high molecular complexes comprising RRM2 domain. C. Thioflavin T (ThT) fluorescence assay showing amyloid fibril formation of RRM2 with mutations in E246 and D247. Each value indicates averaged RFU of ThT with standard error of mean from triplicates. *p,0.01 vs. RRM2 WT at RT by one-way ANOVA with Newman-Keuls test. D. Superdex 75 size exclusion chromatography of recombinant RRM2 domain of WT (a), E246Q/D247N (QN, b), and E246G/D247G (GG, c). a, WT RRM2 alone was exclusively monomeric in its native condition (unfilled circle). Heat denaturation at 70uC for 10 min induced higher molecular assembly (filled circle). Arrowhead indicates the RRM2 oligomer. b, E246Q/D247N (QN) mutant RRM2 without stress was predominantly monomeric (unfilled circle). Heat denaturation markedly increased the ratio of oligomers to monomers (filled circle). c, E246G/D247G (GG) mutant existed as a mixture of monomer and oligomers at the baseline condition (unfilled circle). Higher molecular species were prominently

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Western Blot, Recombinant, Mutagenesis, Incubation, Fluorescence, Size-exclusion Chromatography

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 2. D247 predominantly governs the conformation of RRM2. A. Coomassie staining of recombinant RRM2 protein with either single or double mutation(s) at E246 and D247 incubated for 24 hr at 4uC by denaturing SDS-PAGE. The effects of oligomerization were greater with the single mutant at D247 than at E246. Oligomerization is most prominent in the double E246G/D247G mutant. B. Size exclusion chromatography of RRM2 proteins of E246G and D247G incubated at 4uC (a) and 70uC for 10 min with 24 hr post incubation at 4uC. (b). The effect of oligomerization is more pronounced in the D247G mutant RRM2 than in the E246G in both the 4uC and 70uC conditions. Molecular size markers are as follows: bovine serum albumin (66 kDa), ovalbumin (43 kDa), superoxide dismutase 1 (32 kDa), myoglobin (17.6 kDa), and aprotinin (6.5 kDa). doi:10.1371/journal.pone.0052776.g002

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Staining, Recombinant, Mutagenesis, Incubation, SDS Page, Size-exclusion Chromatography

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 3. Substitution mutants of full-length TDP-43 at E246 and D247 are readily misfolded. A. Confocal micrographs of HEK293A cells overexpressing EGFP-fused full-length TDP-43 (a, b, wild-type (WT), c, d, E246G, e, f, D247G, g, h, E246G/D247G). i, j, Percentages of transfected HEK293A cells harboring multiple puncta or inclusions (i, arrowheads) or displaying nucleus-excluded TDP-43 (unfilled arrowhead). (j). Scale bar indicates 30 mm. Data were expressed as the mean 6 SEM (N = 7–10). *p,0.05 vs. WT, #p,0.05 vs. E246G by one-way ANOVA with Newman-Keuls test. NS indicates not significant vs. WT. B. a, Western blotting showing the increased detergent-insolubility of TDP-43 with mutations at E246/D247, with defective nucleus localizing signal (mNLS), or devoid of RRM2 domain (DRRM2). Lysates from HEK293A cells transiently transfected with TDP-43- EGFP were separated into 1% TritonX100-soluble or -insoluble fractions. Top panel, anti-GFP; middle panel, anti-actin; bottom panel, anti-GAPDH. The GAPDH blot validates the successful separation between detergent-soluble and -insoluble components. b, Quantified insolubility of TDP-43-EGFP proteins with or without mutation at E246/D247 to glycine. Relative TDP-43-EGFP in the detergent-soluble or -insoluble fraction was obtained from the ratio of the GFP density to actin density from the densitometric value in each fraction (designated as insoluble TDP or soluble TDP, respectively). Insolubility index was obtained from the ratio of insoluble TDP to soluble TDP, and each value was standardized by the average ratio of WT. Data were expressed as the mean 6 SEM of four experiments.*p,0.05 vs. WT TDP-43-EGFP by one-way ANOVA with Newman-Keuls test. C. Size exclusion chromatography and Western blotting indicating the existence of oligomeric and monomeric states of full-length TDP-43 in cells. WT and E246G/ D247G (GG) TDP-43-FLAG genes were expressed in HEK293A cells. Cells were sonicated in PBS, and the supernatants were fractionated by a Superose 12 column (10/300) at a flow rate of 0.5 mL/min in PBS. Fractionated cell extracts were applied to Western blotting by anti-TDP-43 (Proteintech). Mutant TDP-43 (GG) proteins were collected in a larger fraction than 88–440 kDa. The molecular size markers thyroglobulin (669 kDa), ferritin (440 kDa), Mn-SOD (88 kDa), ovalbumin (43 kDa), and RNase (13.7 kDa) were eluted under the same conditions. Abs280 is presented to show the equal amount of proteins between WT and the GG mutant in each fraction. doi:10.1371/journal.pone.0052776.g003

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Transfection, Western Blot, Mutagenesis, Size-exclusion Chromatography, Sonication

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 4. Oligomerization affects the nucleotide interaction and RNA splicing efficiency of TDP-43. A. Exon 9 skipping assay showing that mutations at E246 and D247 affect the RNA splicing activity of full-length TDP-43. a, Agarose gel electrophoresis of PCR products (top). Western blot analysis of the total cell lysates using anti-EGFP (middle) and -actin (bottom) antibodies was also shown. b, Quantification of spliced and unspliced fragments using densitometry. Each value is the ratio of spliced to unspliced PCR products. Data is mean 6 standard error of mean from triplicates. *p,0.01 vs. Wild-type (WT) TDP-43 by one-way ANOVA with Newman-Keuls test. B. Size exclusion chromatography for recombinant RRM2 proteins of WT (a), or mutants with E246Q/D247N (QN, b) or E246G/D247G (GG, c), and for (TG)12 oligonucleotides. Mixtures of RRM2 mutants and (TG)12 oligonucleotides were centrifuged at 15,0006g for 20 min and subjected to a Superdex75 (10/300) column at a flow rate of 0.5 mL/min in PBS. Only the RRM2 monomer showed a molecular shift with the (TG)12 oligonucleotides to a single peak, indicating their association (arrowheads). Molecular size markers are as follows: bovine serum albumin (66 kDa), ovalbumin (43 kDa), superoxide dismutase 1 (32 kDa), myoglobin (17.6 kDa), and aprotinin (6.5 kDa). *1 indicates free monomeric RRM2, *2 indicates oligomeric RRM2. Note that there is no peak for free (TG)12, indicating all the (TG)12 was bound to RRM2 monomers. doi:10.1371/journal.pone.0052776.g004

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Activity Assay, Agarose Gel Electrophoresis, Western Blot, Size-exclusion Chromatography, Recombinant

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 5. 3B12A recognizes cytosol-redistributed TDP-43. A–D, SHSY-5Y cells were transiently transfected with TDP-43-EGFP of wild type (WT), mutants with defective NLS (mNLS), or deletion mutant of RRM2 deletion (DRRM2) (EGFP shown as green). At 48 h after transfection, cells were fixed and stained with 3B12A (red). DAPI was used for counterstaining (blue). A. a–f, Transfected or endogenous WT TDP-43 was rarely stained by 3B12A (unfilled arrowheads). Occasionally, cells with very high fluorescence were labeled (arrowhead). B. Cytosolic redistributed TDP-43 (mNLS) was preferentially stained by 3B12A (arrowheads). 3B12A recognized the mNLS mutant of TDP-43-EGFP even at moderate expression levels, regardless of aggregate formation. c–e are high power fields of a–b. C. Nuclear-excluded WT TDP-43 is recognized by 3B12A. WT TDP-43-EGFP expressing SHSY-5Y cells exposed to 5 mM lactacystin were fixed and stained with 3B12A (arrowheads). D. No reactivity of 3B12A to TDP-43-EGFP devoid of RRM2 (DRRM2) (unfilled arrowheads). E. Immunoprecipitation experiment showing that 3B12A preferentially recognized NLS-defective TDP-43 in cell lysates. HEK293A cells were transiently transfected with WT, mNLS, or FALS mutant (A315T and Q331K) forms of TDP-43-FLAG. Total lysates were immunoprecipitated with the 3B12A. Western blot analysis using a rabbit polyclonal anti-FLAG antibody showed that 3B12A predominantly recognized the defective NLS, but more weakly recognized the WT and FALS-linked mutant forms of TDP-43. doi:10.1371/journal.pone.0052776.g005

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Transfection, Mutagenesis, Staining, Fluorescence, Labeling, Expressing, Immunoprecipitation, Western Blot

Journal: Diagnostic Pathology

Article Title: RRM2 expression in different molecular subtypes of breast cancer and its prognostic significance

doi: 10.1186/s13000-021-01174-4

Figure Lengend Snippet: Clinical and pathological characteristics of patient cohort

Article Snippet: Rabbit polyclonal primary antibody against RRM2 (NBP1–31661), obtained from NOVUS Biological, Centennial, Co 80,112 USA was used at a dilution rate of 1:50. (DAB) was used as a chromogen, after that, hematoxylin was applied for counterstaining.

Techniques:

Journal: Diagnostic Pathology

Article Title: RRM2 expression in different molecular subtypes of breast cancer and its prognostic significance

doi: 10.1186/s13000-021-01174-4

Figure Lengend Snippet: Intense RRM2 expression in cancer breast ×200

Article Snippet: Rabbit polyclonal primary antibody against RRM2 (NBP1–31661), obtained from NOVUS Biological, Centennial, Co 80,112 USA was used at a dilution rate of 1:50. (DAB) was used as a chromogen, after that, hematoxylin was applied for counterstaining.

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: RRM2 expression in different molecular subtypes of breast cancer and its prognostic significance

doi: 10.1186/s13000-021-01174-4

Figure Lengend Snippet: Correlation between RRM2 protein expression and other clinicopathological variables

Article Snippet: Rabbit polyclonal primary antibody against RRM2 (NBP1–31661), obtained from NOVUS Biological, Centennial, Co 80,112 USA was used at a dilution rate of 1:50. (DAB) was used as a chromogen, after that, hematoxylin was applied for counterstaining.

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: RRM2 expression in different molecular subtypes of breast cancer and its prognostic significance

doi: 10.1186/s13000-021-01174-4

Figure Lengend Snippet: Kaplan-Meier disease free survival for RRM2 score in whole series

Article Snippet: Rabbit polyclonal primary antibody against RRM2 (NBP1–31661), obtained from NOVUS Biological, Centennial, Co 80,112 USA was used at a dilution rate of 1:50. (DAB) was used as a chromogen, after that, hematoxylin was applied for counterstaining.

Techniques:

Journal: Diagnostic Pathology

Article Title: RRM2 expression in different molecular subtypes of breast cancer and its prognostic significance

doi: 10.1186/s13000-021-01174-4

Figure Lengend Snippet: Kaplan-Meier DFS for RRM2 score in ER positive group

Article Snippet: Rabbit polyclonal primary antibody against RRM2 (NBP1–31661), obtained from NOVUS Biological, Centennial, Co 80,112 USA was used at a dilution rate of 1:50. (DAB) was used as a chromogen, after that, hematoxylin was applied for counterstaining.

Techniques:

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Schematic description of the filtering process for identification of therapeutically relevant target candidates. b) Analysis of RRM2 mRNA expression levels in 50 EwS primary tumors compared to 929 normal tissues samples from 71 tissue types. Data are shown as log2 fold increase normalized to expression values of normal tissues. The dotted line indicates the cut-off value of 2 for candidate selection. c) Analysis of overall survival time of 166 EwS patients stratified for candidate gene expression. P -values (–log10) were determined in Kaplan-Meier analyses using a Mantel-Haenszel test (Bonferroni-adjusted for multiple testing). The dotted line indicates a significance value of 1.3. d) Kaplan-Meier survival analysis of 166 EwS patients stratified by the 78th percentile RRM2 expression. P -value determined by log-rank test.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: Expressing, Selection, Gene Expression

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Left: Heat map for gene expression which is positively or negatively correlated with RRM2 expression in 166 EwS. Right: Gene ontology (GO) enrichment analysis of RRM2 and its co-expressed genes derived from gene expression data sets of 166 EwS tumors. Pearson correlation coefficients between RRM2 and other genes were determined, of which those with |r Pearson | > 0.5 were further analyzed by GO enrichment analysis. b) Representative images of immunohistochemical RRM2 staining. Scale bar = 50 µm. c) Kaplan-Meier survival analysis of 122 EwS patients stratified by RRM2 protein expression (low IRS≤2, high IRS >2). P -values were determined by log-rank test. d) WGCNA of downregulated genes upon RRM2 silencing in A-673 and ES-7 cells harboring Dox-inducible shRRM2 constructs. NES, normalized enrichment score. e) Analysis of tumor growth of EwS cell lines A-673 and TC-71 harboring Dox-inducible shRRM2 constructs or non-targeting shRNA (shControl) xenografted in NSG mice. Once tumors were palpable, animals were randomized in Dox (+) or Dox (–) group. Tumor growth on time course and f) Tumor weight at the experimental endpoint. Arrows indicate treatment start. Values are normalized to shControl. Horizontal bars represent means and whiskers SEM. FC, fold change. P -values were calculated at the experimental endpoint with two-sided (tumor growth) or one-sided (tumor weight) Mann-Whitney test. g) Representative micrographs of xenografts immunohistochemically stained for RRM2, cleaved caspase-3 (CC3) or γH2A.X (scale bar=250 µm, 50 µm, 250 µm, respectively). h) quantification of positive cells for cleaved caspase-3 (CC3) (left) and γH2A.X (right). Values were normalized to shControl. Horizontal bars represent means and whiskers SEM. FC, fold change. P -values were calculated at the experimental endpoint using a two-sided Mann-Whitney test.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: Gene Expression, Expressing, Derivative Assay, Immunohistochemical staining, Staining, Construct, shRNA, MANN-WHITNEY

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Analysis of proliferation assays upon shRNA-mediated RRM2 silencing in EwS cell lines. Upper: Cell proliferation over 120h upon RRM2 silencing in A-673. Viable cells upon RRM2 silencing (middle) and dead cells (lower) in EwS cell lines (A-673, ES7, TC-71) harboring Dox-inducible shRRM2 constructs or non-targeting shRNA (shControl). Values are normalized to shControl. Horizontal bars represent means and whiskers SEM. FC, fold change. Two-sided Mann-Whitney test. b) Analysis of clonogenic growth upon shRNA-mediated RRM2 silencing in EwS cell lines (A-673, ES7, TC-71) harboring Dox-inducible shRRM2 constructs or non-targeting shRNA (shControl). Horizontal bars represent means and whiskers SEM. Two-sided Mann-Whitney test at the experimental endpoint.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: shRNA, Construct, MANN-WHITNEY

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Integrative Gene Ontology (GO) enrichment analysis of gene expression microarray data generated in A-673 and ES7 cells after RRM2 silencing or pharmacological RRM2 inhibition by triapine (corresponding IC50 of 0.44 µM or 0.65 µM, respectively). b) Correlation of gene expression between RRM2 and CHEK1 or WEE1 in 166 EwS. Each dot represents an individual expression value. Solid red lines indicate a trend line created by a simple linear regression. P -values were calculated by a two-tailed t-test. c) Drug interaction and combination efficiency analysis between triapine and CHEK1 inhibitor (CCT245737) or WEE1 inhibitor (MK-1775) in 4 EwS cell lines (A-673, ES7, EW-7, TC-71) assessed by combination index. CI value < 1 indicative of synergistic, CI = 1 additive, and CI > 1 antagonistic d) Drug interaction and combination efficiency estimation between triapine and CHEK1 inhibitor (CCT245737) or WEE1 inhibitor (MK-1775) in A-673 EwS cell line assessed by SynergyFinder 2.0. ZIP synergy score > 10, likely to be synergistic; between -10 and 10, likely to be additive; < –10, likely to be antagonistic.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: Gene Expression, Microarray, Generated, Inhibition, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

doi: 10.1101/2021.03.04.433896

Figure Lengend Snippet: a) Distribution analysis of RRM2 mRNA expression in 166 EwS patients. Each dot represents individual RRM2 expression. b) Correlation analysis of CNVs at the RRM2 locus with RRM2 mRNA expression levels in primary EwS tumors (n=32). The solid line indicates a trend line estimated by a simple linear regression model. b) Correlation analysis of promoter methylation on five CpG sites with RRM2 expression levels in primary EwS tumors (n=40). The solid lines indicate trend lines estimated by a simple linear regression model.

Article Snippet: Slides were incubated with a polyclonal anti-RRM2 primary antibody (rabbit, 1:500, atlas antibodies, HPA056994) for 60 min at room temperature followed by incubation with a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody with AEC+ as chromogen, counterstained by hematoxylin Gill’s formula (H-3401, Vector Laboratories, Germany).

Techniques: Expressing, Methylation

Journal: Cancer

Article Title: Molecular classification of nonsmall cell lung cancer using a 4-protein quantitative assay.

doi: 10.1002/cncr.26450

Figure Lengend Snippet: Figure 1. Antibody validation process. The dynamic range of CK13 (A), CK5 (B), epidermal growth factor receptor (EGFR) (C), and transcription factor 1 (TTF1) (D) expression by Western Blot (WB) in cell lines controls was consistent with AQUA analysis. Immunofluorescence for A431 and HCC193 shown; quantitative immunofluorescence (AQUA) scores displayed in insets.

Article Snippet: Cytokeratin 5 NCL-L-CK5/m 5.2 lg/mL 1 hour RT A431 Novocastra, Newcastle, UK Cytokeratin 13 DE-K13/m IgG2a, kappa 57 lg/mL ON 4 C A431 Dako, Carpinteria, CA Cytokeratin 14 LL002/m IgG3 0.2 lg/mL ON 4 C A431 Thermo Fisher, Fremont, CA Cytokeratin 17 2D10/m IgG1 0.1 lg/mL ON 4 C A431 Abnova, Walnut, CA EGFR 31G7/m IgG1 3 lg/mL ON 4 C EGFR transfected CHO, A431 Zymed/Invitrogen, Carlsbad, CA HER2 r polyclonal 25 lg/mL ON 4 C HER2 transfected CHO Dako, Carpinteria, CA HER3 r polyclonal 0.2 lg/mL ON 4 C HER3 transfected BaF3 Santa Cruz, Santa Cruz, CA HER4 SPM338/m IgG2b 0.4 lg/mL ON 4 C HER4 transfected CHO Santa Cruz, Santa Cruz, CA AKT1 2H10/m 1/200a ON 4 C A431 Cell Signaling, Danvers, MA ERK m polyclonal 1/100a ON 4 C A431 Cell Signaling, Danvers, MA DUSP6 3G2/m IgG1, kappa 0.3 lg/mL ON 4 C Pancreatic carcinoma Novus Biologicals, Littleton, CO STAT1 42H3/r IgG 1/750a ON 4 C Colon carcinoma Cell Signaling, Danvers, MA STAT2 Y141/r IgG 1/200a ON 4 C Breast carcinoma Novus Biologicals, Littleton, CO STAT3 124H6/m 1/500a ON 4 C H1650, HCC2279 Cell Signaling, Danvers, MA mTOR 7C10/r 1/1000a ON 4 C Breast carcinoma, A431, H1299 Cell Signaling, Danvers, MA pS6K 1A5/m 1/200a ON 4 C H1299 Cell Signaling, Danvers, MA pS6 91B2/r IgG 1/400a ON 4 C Colon carcinoma, HCC193 Cell Signaling, Danvers, MA TTF1 8G7G3/m IgG1, kappa 20 lg/mL ON 4 C H2126 Dako, Carpinteria, CA E2F4 SPM179/m IgG1, kappa 2 lg/mL ON 4 C Tonsil Novus Biologicals, Littleton, CO BCL2 124/m IgG1, kappa 2.6 lg/mL ON 4 C Lymphocytes Dako, Carpinteria, CA CC3 5A1/r 1/500* ON 4 C Pancreatic carcinoma Cell Signaling, Danvers, MA MENA 21/m IgA 1 lg/mL ON 4 C A431 BD Biosciences RRM2 1E1/m IgG1, kappa 0.05 lg/mL ON 4 C Pancreatic carcinoma Novus Biologicals, Littleton, CO PTEN 6H2.1/m IgG2b, kappa 0.96 lg/mL ON 4 C H1666 Dako, Carpinteria, CA Abbreviations: M, mouse; ON, overnight; r, rabbit; RT, room temperature. a Antibody concentration not provided by the manufacturer.

Techniques: Biomarker Discovery, Expressing, Western Blot, Immunofluorescence

Journal: Heliyon

Article Title: Tanshinone IIA attenuates fluoride-induced spinal cord injury by inhibiting ferroptosis and inflammation

doi: 10.1016/j.heliyon.2024.e40549

Figure Lengend Snippet: Primers used in qRT-PCR study.

Article Snippet: The membranes were then incubated overnight at 4 °C with primary antibodies against CXCL2 (26791-1-AP, Proteintech), PCK2 (67676-1-lg, abcam), RRM2 (DF7248, Affinity), SLC7A11 (BM5318, BOSTER), or β-actin (BA2305, BOSTER).

Techniques:

Journal: Heliyon

Article Title: Tanshinone IIA attenuates fluoride-induced spinal cord injury by inhibiting ferroptosis and inflammation

doi: 10.1016/j.heliyon.2024.e40549

Figure Lengend Snippet: Tanshinone IIA improved spinal cord injury by inhibiting ferroptosis (A) Photomicrographs of spinal cord sections from Tanshinone IIA control, fluoride-exposed rats, and Tanshinone IIA + high fluoride group visualized by transmission electron microscopy (5000X, white box). Fibrous myelin integrity (indicated by red arrows) is maintained in both Tanshinone IIA control and Tanshinone IIA-treated groups, while in the fluorosis group, it appears loosely arranged with an increased gap and disordered structure. (B) Scatter plot exhibited the expression of 15 ferroptosis-related genes in Tan IIA control, high fluoride group, and Tan IIA + high fluoride group. (C–D) Western blots showed that Tan IIA treatment restored the reduced protein expression of SLC7A11 and RRM2 observed in the NaF group. Additionally, Tan IIA treatment decreased the elevated protein levels of CXCL2 and PCK2 in the NaF group. Uncropped and unadjusted original images of the Western blots are provided in . Data are represented as the mean ± SEM (n = 6). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Article Snippet: The membranes were then incubated overnight at 4 °C with primary antibodies against CXCL2 (26791-1-AP, Proteintech), PCK2 (67676-1-lg, abcam), RRM2 (DF7248, Affinity), SLC7A11 (BM5318, BOSTER), or β-actin (BA2305, BOSTER).

Techniques: Control, Transmission Assay, Electron Microscopy, Expressing, Western Blot