Journal: Materials

Article Title: Biological Response Induced in Primary Human Gingival Fibroblasts upon Exposure to Various Types of Injectable Astringent Retraction Agents

doi: 10.3390/ma14082081

Figure Lengend Snippet: The RNA expression levels of: ( A ) SOD1, SOD2, and SOD3; and ( B ) HMOX1 and GPX1 detected by real-time RT-PCR in human gingival fibroblasts after 24 h of incubation with the gingival retraction agents diluted in the cell culture medium to 1 mg/mL concentration; * p < 0.05, ** p < 0.005.

Article Snippet: AceQ qPCR Probe Master Mix (Vazyme Biotech, Nanjing, Jiangsu, China) and specific TaqMan Assays: Hs00533490_m1 (for superoxide dismutase 1; SOD1), Hs00167309_m1 (for superoxide dismutase 2; SOD2), Hs00162090_m1 (for superoxide dismutase 3; SOD3), Hs01110250_m1 (for heme oxygenase 1; HMOX1), Hs00829989_gH (for glutathione peroxidase 1; GPX1) and Hs99999905_m1 (for glyceraldehyde-3-phosphate dehydrogenase; GAPDH) (Thermo Fisher Scientific Waltham, MA, USA) were used to assess RNA expression according to the manufacturers’ instructions.

Techniques: RNA Expression, Quantitative RT-PCR, Incubation, Cell Culture, Concentration Assay

Journal: Genes

Article Title: Microfluidic Quantitative PCR Detection of 12 Transgenes from Horse Plasma for Gene Doping Control

doi: 10.3390/genes11040457

Figure Lengend Snippet: Probe and primer design for microfluidic quantitative PCR (MFQPCR) detection. Two assays (SET1 and SET2) were designed for each transgene. Forward and reverse primers for MFQPCR were designed to target different exons. TaqMan probe for MFQPCR was designed to target exon/exon junctions. Forward and reverse primers for pre-amplification were designed to include the forward and reverse primers for MFQPCR. While forward and reverse primers for pre-amplification and MFQPCR may amplify PCR products, including genomic DNA regions, TaqMan probes do not anneal to the PCR product, including genomic DNA regions ( A ). TaqMan probes specifically anneal to PCR products having exon/exon junction sequences ( B ).

Article Snippet: Genomic DNA of human, monkey, dog, cat, bovine, goat, sheep, porcine, mini-pig, camel, llama, mouse, rabbit, chicken and donkey were purchased from Zyagen (San Diego, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Amplification

Journal: Genes

Article Title: Microfluidic Quantitative PCR Detection of 12 Transgenes from Horse Plasma for Gene Doping Control

doi: 10.3390/genes11040457

Figure Lengend Snippet: Image of microfluidic quantitative PCR (MFQPCR) detection. Positive control samples (PCS_10000, PCS_1000, PCS_100, PCS_10, PCS_5.0 and PCS_2.5); negative control samples (horse genomic DNA and Milli-Q) and plasma at: before administration, 15 min, 3 h, 6 h, 12 h, 24 h, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 14 d, 21 d and 28 d after EPO transgene administering high, middle and low concentration of PCSs for each transgene, and spiked and recovered samples for each transgene were MFQPCR-amplified using SET1 ( A ) and SET2 ( B ) detections. Vertical axis is assays; CKM, EPO, FGF2, FST, GH1, IGF1, MSTN, PCK1, PDK4, PPARD, VEGF and ZFAT for SET1 ( A ) and SET2 ( B ) detections. Light yellow and orange indicate lower cycle thresholds (Cts) (high copy concentrations). Dark purple depicts high Cts (low copy concentrations). Black indicates nonamplification. NCS: negative control sample.

Article Snippet: Genomic DNA of human, monkey, dog, cat, bovine, goat, sheep, porcine, mini-pig, camel, llama, mouse, rabbit, chicken and donkey were purchased from Zyagen (San Diego, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Concentration Assay, Amplification

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Upregulation of ERK-EGR1-heparanase axis by HDAC inhibitors provides targets for rational therapeutic intervention in synovial sarcoma

doi: 10.1186/s13046-021-02150-y

Figure Lengend Snippet: HDACi upregulate heparanase mRNA and protein. a Analysis of HPSE expression by qRT-PCR in SS cells treated with SAHA or FK228 for the indicated times. Data are reported as relative quantification with respect to untreated cells as calibration sample. Relative mRNA values are the mean ± SE from three independent experiments. b Western blot detection of heparanase polypeptides in CME-1 cells exposed to SAHA or FK228 for the indicated times. Image on the right is from a cropped blot (dashed line) from which lanes not of interest have been removed. c N-glycosylation inhibition does not modify electrophoretic mobility of heparanase polypeptides. CME-1 cells were treated with tunicamycin (2 μg/ml for 24 h) and processed for Western blotting. As controls, the shift of PDGFRα bands shows the glycosylation inhibition and BIP upregulation indicates endoplasmic reticulum stress. In ( b ) and ( c ) anti-actin, −vinculin and -GAPDH blots are shown as loading control

Article Snippet: For treatment with human active recombinant heparanase (R&D systems, Minneapolis, MN) (Supplementary Table ), cells plated in complete medium for 24 h were incubated in serum-free medium with or without 5 μg/ml recombinant enzyme for 24 h and 48 h.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Western Blot, Glycoproteomics, Inhibition, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Upregulation of ERK-EGR1-heparanase axis by HDAC inhibitors provides targets for rational therapeutic intervention in synovial sarcoma

doi: 10.1186/s13046-021-02150-y

Figure Lengend Snippet: EGR1 and p53 cooperate to regulate HPSE expression in SS cells in response to HDAC inhibition. a HPSE is enhanced by FK228 treatment in wt p53 expressing EGR1 silenced cells. After 48 h of transfection with an aspecific RNA oligonucleotide (Asp) or EGR1 siRNA (siEGR1), MoJo (wt p53) and Yamato-SS (mut p53) cells were exposed to 10 nM FK228 for 6 h and then processed for Western blot analysis of EGR1 and p53 (upper panels) or qRT-PCR analysis of HPSE mRNA (lower panels). Mean values from three (MoJo) or two (Yamato-SS) independent experiments are reported. b SS18-SSX2 silencing upregulates EGR1, p53 and heparanase. SYO-1 cells were treated with transfection reagent (V), two specific siRNAs targeting the oncogene (siSSX2A and siSSX2B) or an aspecific RNA oligonucleotide (Asp) at 30 nM final concentration. Seventy two hours after transfection, cells were processed for protein and mRNA analysis. On the left, immuno blotting performed on whole cell lysates with the indicated antibodies. Arrows indicate the fusion oncoproteins. On the right, HPSE expression assessed by qRT-PCR. Mean values from three independent experiments are reported. In ( a ) and ( b ) actin and vinculin are shown as protein loading controls. The mean relative HPSE quantification values ± SE are referred to aspecific control

Article Snippet: For treatment with human active recombinant heparanase (R&D systems, Minneapolis, MN) (Supplementary Table ), cells plated in complete medium for 24 h were incubated in serum-free medium with or without 5 μg/ml recombinant enzyme for 24 h and 48 h.

Techniques: Expressing, Inhibition, Transfection, Western Blot, Quantitative RT-PCR, Concentration Assay, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Upregulation of ERK-EGR1-heparanase axis by HDAC inhibitors provides targets for rational therapeutic intervention in synovial sarcoma

doi: 10.1186/s13046-021-02150-y

Figure Lengend Snippet: Heparanase promotes histone acetylation and its inhibition reduces nuclear localization. a Serum starved CME-1 cells were incubated with human active recombinant heparanase (5 μg/ml) for the indicated times. Then, cells were lysed and processed for immunoblotting with the specified antibodies to detect heparanase (r, recombinant 50 kDa heparanase; e, endogenous 65 kDa heparanase) and acetylation of H3 (K27) and H4 (K12). Histone acetylation was also analyzed in cell lysates after transfection with aspecific RNA oligonucleotide (NegCTR) or HPSE siRNA for 72 h (b) and in cells treated with OGT2115 (0.5 μM) for 48 h or SST0001 (0.5 mg/ml) for 24 h (c) . Vinculin, GAPDH and tubulin are shown as controls for protein loading. d Indirect immunofluorescence showing localization of heparanase in control and SST0001-treated (1 mg/ml for 18 h) cells. Nuclei are evidenced with Hoechst 3341 counterstaining (blue). Original magnification, 1000X. e Cytoplasmic and nuclear fractions from cells exposed to 0.5 mg/ml SST0001 or 1.6 μM SAHA for 18 h, were analyzed by Western blotting to examine intracellular distribution of heparanase polypeptides. Lamin B and GAPDH are shown as controls for nuclear-cytoplasmic fractioning and loading. f Biotin-conjugated SST0001 analogue SST0762NA1 enters the nuclei. Serum starved cells treated with SST0762NA1 (1 mg/ml) for 24 h were fixed, permeabilized, and incubated with streptavidin Alexa Fluor 488 conjugate to detect the drug. Cells were stained with Hoechst 33341 to evidence nuclei. Inset, enlarged detail evidencing SST0762NA1 localization in the nucleus and in perinuclear vesicles

Article Snippet: For treatment with human active recombinant heparanase (R&D systems, Minneapolis, MN) (Supplementary Table ), cells plated in complete medium for 24 h were incubated in serum-free medium with or without 5 μg/ml recombinant enzyme for 24 h and 48 h.

Techniques: Inhibition, Incubation, Recombinant, Western Blot, Transfection, Immunofluorescence, Control, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Upregulation of ERK-EGR1-heparanase axis by HDAC inhibitors provides targets for rational therapeutic intervention in synovial sarcoma

doi: 10.1186/s13046-021-02150-y

Figure Lengend Snippet: SS tumor growth inhibition and apoptotic cell death are enhanced by co-treatment with SAHA and SST0001. a Growth curves of CME-1 xenografts grown in the leg muscle of mice. Animals were treated with vehicle (controls), or SST0001 s.c. at 60 mg/kg, 2qdx5/w for 4 weeks, or SAHA by oral gavage at 100 mg/kg, qdx5/w for 4 weeks, or with the two drugs in combination, starting 1 day after tumor cell injection. Each point is the mean tumor volume in 6/8 mice ± SD. * P < 0.05 referred to the entire curves and the last time point. b, d CME-1 cells were treated with SAHA (0.8 and 1.6 μM) and SST0001 (0.5 mg/ml) alone or in combination at the indicated times. The effect of treatments on ERK and AKT activation, EGR1 expression, caspase 3 and PARP cleavage ( b ) and on heparanase expression ( d ) was analyzed by Western blotting. Vinculin and actin are shown as loading controls in immunoblots. Numbers represent the intensity of relevant bands normalized with respect to the respective loading controls. c Cells were exposed to SAHA (1.6 μM) and SST0001 (0.5 mg/ml), alone or in combination for 72 h, to detect caspase 3 cleavage by Western blotting and apoptosis by cytoplasmic histone-associated DNA fragmentation assay. Bars represent mean values referred to control cells ± SE obtained in four independent biological replicates. * P < 0.05 vs single agents and controls

Article Snippet: For treatment with human active recombinant heparanase (R&D systems, Minneapolis, MN) (Supplementary Table ), cells plated in complete medium for 24 h were incubated in serum-free medium with or without 5 μg/ml recombinant enzyme for 24 h and 48 h.

Techniques: Inhibition, Injection, Activation Assay, Expressing, Western Blot, DNA Fragmentation Assay, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Upregulation of ERK-EGR1-heparanase axis by HDAC inhibitors provides targets for rational therapeutic intervention in synovial sarcoma

doi: 10.1186/s13046-021-02150-y

Figure Lengend Snippet: Schematic representation of the proposed HDACi activated auto-sustaining pro-survival loop and its blockade by co-treatment with ERK pathway and heparanase inhibitors in SS cells. This figure was prepared using tools from Servier Medical Art ( http://www.servier.fr/servier-medical-art )

Article Snippet: For treatment with human active recombinant heparanase (R&D systems, Minneapolis, MN) (Supplementary Table ), cells plated in complete medium for 24 h were incubated in serum-free medium with or without 5 μg/ml recombinant enzyme for 24 h and 48 h.

Techniques:

Journal: Aging Cell

Article Title: Galacto‐conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity

doi: 10.1111/acel.13142

Figure Lengend Snippet: Galacto‐conjugation of the senolytic Navitoclax into a new generation senolytic prodrug, namely Nav‐Gal, as an efficient strategy for selective senolysis. (a) Schematic representation of the mechanism of action of Nav‐Gal prodrug. Nav‐Gal is passively taken up by both nonsenescent and senescent cells. In nonsenescent cells, its conjugation with a cleavable galactose renders it inactive and unable to inhibit anti‐apoptotic proteins, such as BCL‐2, preventing the induction of apoptosis. In senescent cells, the increased lysosomal and galactosidase activity, a hallmark of cellular senescence, allows the hydrolysis of the cleavable galactose, resulting in the release of active Navitoclax into the cytoplasm of senescent cells. Free Navitoclax will inhibit anti‐apoptotic BCL‐2 proteins, which are overexpressed in senescent cells, driving specific apoptosis of these cells. (b) Chemical structures of Nav‐Gal prodrug and Navitoclax. The presence of galactopyranoside, covalently linked to the N of bis(sulfonyl)aniline as synthesized in this prodrug, hinders two key interactions: (i) π‐π interaction between the phenylthioether moiety and the bis(sulfonyl)aniline ring; and (ii) the hydrogen bond between morpholine with Tyr‐199 (Liu, Zhang, Huang, Tan, & Zhang, ), thereby preventing the inhibitory effect of the molecule. This moiety, the galactopyranoside, can be hydrolyzed in the presence of β‐galactosidase (cleavable galactose). (c) Chromatograms depicting hydrolysis reaction of Nav‐Gal aqueous solutions in the presence of human β‐galactosidase followed by HPLC‐UV as described in the text

Article Snippet: Human β‐galactosidase (Biotechne, R&D Systems) was then added to Nav‐Gal solutions to a final concentration of 8 ng/µl, and chromatograms were acquired after complete reaction ( λ exc = 365 nm) with a Waters 1525 binary HPLC pump equipped with a Waters 2990 diode array detector.

Techniques: Conjugation Assay, Activity Assay, Synthesized

Journal: Aging Cell

Article Title: Galacto‐conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity

doi: 10.1111/acel.13142

Figure Lengend Snippet: GLB1 transient downregulation prevents the senolytic activity of Nav‐Gal. (a) Representative images of SA‐β‐gal staining of control and cisplatin‐induced senescent A549 cells 48 hr after transfection with scrambled siRNA, siRNA 1 and siRNA 2. Scale bar = 200 μm. (b) Percentage of SA‐β‐gal‐positive cells in conditions presented in (a). Bars represent mean ± SD ( n = 3). (c) GLB1 fold change gene expression in control and senescent A549 cells 48 hr post‐transfection with different siRNAs. (d) Representative images of SA‐β‐gal staining of control and palbociclib‐induced senescent SK‐Mel‐103 cells 48 hr after transfection with scrambled siRNA, siRNA 1 and siRNA 2. Scale bar = 200 μm. (e) Percentage of SA‐β‐gal‐positive cells in conditions presented in (d). Bars represent mean ± SD ( n = 3). (f) GLB1 fold change gene expression in control and senescent SK‐Mel‐103 cells 48 hr post‐transfection with different siRNAs. (g) Quantification of cell viability upon 48 hr Navitoclax treatment of control and cisplatin‐induced senescent A549 cells (10 μM Navitoclax) (left) and control and palbociclib‐induced senescent SK‐Mel103 cells (7.5 μM Navitoclax) (right) previously transfected with different experimental siRNAs against GLB1 transcript. (h) Quantification of cell viability upon 48 hr Nav‐Gal treatment of control and cisplatin‐induced senescent A549 cells (10 μM Nav‐Gal) (left) and control and palbociclib‐induced senescent SK‐Mel103 cells (7.5 μM Nav‐Gal) (right) previously transfected with different experimental siRNAs against GLB1 transcript. Note that siRNA 1 was not functional in all the experiments and hence used as an internal negative control. All bars represent mean ± SEM ( n = 3). One‐way ANOVA followed by Tukey's post‐tests were performed to calculate the significance of the results; * p < .05, ** p < .01, *** p < .001

Article Snippet: Human β‐galactosidase (Biotechne, R&D Systems) was then added to Nav‐Gal solutions to a final concentration of 8 ng/µl, and chromatograms were acquired after complete reaction ( λ exc = 365 nm) with a Waters 1525 binary HPLC pump equipped with a Waters 2990 diode array detector.

Techniques: Activity Assay, Staining, Transfection, Expressing, Functional Assay, Negative Control

Journal: Aging Cell

Article Title: Galacto‐conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity

doi: 10.1111/acel.13142

Figure Lengend Snippet: Concomitant treatment with the prodrug Nav‐Gal and cisplatin significantly inhibits tumour growth in a human lung cancer xenograft mouse model. (a) Representative images of A549 xenografts stained for SA‐β‐Gal activity (in blue) after treatment with cisplatin or vehicle. (b) Schematic representation of concomitant treatment on A549 xenograft‐bearing mice. (c) Tumour volume of A549 xenografts in mice concomitantly treated with cisplatin and Navitoclax or Nav‐Gal (as described in (b); n ≥ 10 tumours per group. Data represent mean ± SEM . (d) Representative histological images of tumours at the end of concomitant treatment, stained for Ki67 and p21 expression, and labelled using TUNEL staining. Scale bar = 100 μm. (e) Percentage of Ki67‐ (top), p21‐ (middle) and TUNEL‐positive (bottom) cells in tumours from animals treated with vehicle, cisplatin, or cisplatin and Navitoclax or Nav‐Gal concomitantly ( n ≥ 5 tumours per group). For quantification, a total of 4 fields per tumour was analyzed, covering most of the total tumour area. Two‐way ANOVA followed by Bonferroni post‐tests was performed to calculate the significance of the results; * p < .05; ** p < .01; *** p < .001

Article Snippet: Human β‐galactosidase (Biotechne, R&D Systems) was then added to Nav‐Gal solutions to a final concentration of 8 ng/µl, and chromatograms were acquired after complete reaction ( λ exc = 365 nm) with a Waters 1525 binary HPLC pump equipped with a Waters 2990 diode array detector.

Techniques: Staining, Activity Assay, Expressing, TUNEL Assay

Journal: Nature Immunology

Article Title: Conserved stromal–immune cell circuits secure B cell homeostasis and function

doi: 10.1038/s41590-023-01503-3

Figure Lengend Snippet: a , B220 and ACTA2 immunostaining of ILNs and mesenteric LNs (MLNs) processed for spatial transcriptomics. b , Spatial expression of Cxcl13 . c , Normalized weights from cell type decomposition projected onto Cxcl13 + spots. d , Dot plot visualizing the fraction of shared spots for each BRC subset with different immune cells averaged across four samples. e , f , Representative histograms ( e ) and quantification ( f ) of TdTomato expression in Lin − Eyfp + cells from Cxcl13-Cre/TdTomato EYFP LN fibroblast cultures 48 h after stimulation with the indicated proteins (Lin − refers to CD45 − CD31 − ). g , IL-6 concentration in supernatants from LN fibroblast cultures 48 h after stimulation with the indicated factors. h – l , In vivo stimulation with predicted maturation cues. h , i , Flow cytometry quantification of FDC frequencies in Lin − PDPN + cells ( h ) and ICAM1 expression in FDCs ( i ). j , k , Flow cytometry quantification of the frequency ( j ) and mean fluorescence intensity (MFI) ( k ) of PE–ICs on CD21/35 + cells after in vivo stimulation. l , Representative confocal microscopy images of PE–IC deposition in LNs from PBS or IL-4-treated mice. m , Schematic representation of BRC-provided niche factors and immune cell-derived BRC activation and differentiation cues. In a – d spatial transcriptome analysis was performed on n = 4 LNs, with a technical replicate for each sample. In a – c one representative sample is shown. In e , f data from n = 8 replicates from two independent experiments are shown. The boxplot midline demarcates the median; the box limits demarcate the upper and lower quartiles; and the whiskers depict the minimum and maximum. In g data from n = 6 replicates from two independent experiments with mean and s.d. are shown. In h , i data from n = 6 PBS-treated mice, n = 7 recombinant IL-4-treated mice, n = 6 recombinant IL-1β-treated mice and n = 4 VEGF-B-treated mice are shown (two independent experiments). In j , k data from n = 7 PBS-treated mice, n = 8 recombinant IL-4-treated mice, n = 6 recombinant IL-1β-treated mice and n = 4 VEGF-B-treated mice are shown (two independent experiments). In h – k the midline of the boxplot demarcates the median; the box limits demarcate the upper and lower quartiles; the whiskers depict the minimum and maximum. In l the representatives of three mice per treatment are shown. In f – k adjusted P values were derived from a Dunnett’s multiple comparison test with a 95% confidence interval using a one-way analysis of variance. m , Created with BioRender .

Article Snippet: To evaluate the differentiation and activation potential, cells were plated at 15,000 cells per cm 2 and stimulated for 48 h with recombinant human TGFβ1 (1 ng ml −1 , catalog no. 7754-BH/CF, R&D Systems), recombinant human PRGN (10 ng ml −1 , catalog no. 2420-PG, R&D Systems) or recombinant human GH (10 ng ml −1 , catalog no. 1067-GH/CF, R&D Systems).

Techniques: Immunostaining, Expressing, Concentration Assay, In Vivo, Flow Cytometry, Fluorescence, Confocal Microscopy, Derivative Assay, Activation Assay, Recombinant, Comparison

Journal: Nature Immunology

Article Title: Conserved stromal–immune cell circuits secure B cell homeostasis and function

doi: 10.1038/s41590-023-01503-3

Figure Lengend Snippet: a , Heatmap visualizing the average expression of conserved receptor-ligand pairs identified as significant interactions in human SLOs by CellPhoneDB analysis. b , c , Fold changes of SOX9 ( b ) and PI16 ( c ) mRNA levels in bulk cultured primary tonsillar fibroblast of OSA patients stimulated with the indicated recombinant proteins for 48 h and measured by qRT-PCR. d , Fold changes of PI16 mRNA levels after in vitro expansion of sorted PI16 + RCs from human tonsils and following stimulation with TGF-β1 for 48 h. b – d , Box plots with whiskers showing the minimum and maximum values. Horizontal lines indicate the median and boxes represent 0.25−0.75 percentiles ( b , c ) q values are derived from Tukey’s test following Kruskal-Wallis test and using Benjamini, Krieger and Yekutieli correction to control the false discovery rate. ( d ) Two-sided Mann Whitney test was used to test for significant differences. In a , data represent 3,450 CXCL13 + cells and 56,887 immune cells sampled from n = 4 patients for human lymph nodes and n = 4 patients for human palatine tonsils. In b , c , cells from n = 8 patients were cultivated and processed in 2 independent experiments. In d , cells from n = 5 patients were cultivated and processed in 2 independent experiments.

Article Snippet: To evaluate the differentiation and activation potential, cells were plated at 15,000 cells per cm 2 and stimulated for 48 h with recombinant human TGFβ1 (1 ng ml −1 , catalog no. 7754-BH/CF, R&D Systems), recombinant human PRGN (10 ng ml −1 , catalog no. 2420-PG, R&D Systems) or recombinant human GH (10 ng ml −1 , catalog no. 1067-GH/CF, R&D Systems).

Techniques: Expressing, Cell Culture, Recombinant, Quantitative RT-PCR, In Vitro, Derivative Assay, Control, MANN-WHITNEY