Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 4: FAIM deficiency suppressed the effects of PACAP on alleviating hepatic lipid accumulation in HFD-fed mice. (A) FAIM protein level of mice liver was subjected to western blot. Statistical analysis of FAIM/b-actin was shown in a bar chart (right) (*P < 0.05, **P < 0.01, ***P < 0.001 vs. CD group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HFD group). (B) Schematic diagram of HFD mouse model with PACAP treatment (0.4 mg/kg) or control saline solution and lentivirus mediated Faim knockdown. (C) Hepatic Faim mRNA level was determined by qRT-PCR, n ¼ 3. (DeH) Modulation of Faim level in the liver influenced HFD-induced hepatic steatosis, (D) bodyweight, liver weight, and WAT weight; (E) liver TG and TC contents; (F) plasma TG, TC, HDL-c, and LDL-c levels; (G) fasting blood glucose level; blood glucose concentrations were measured on day 12 after lentivirus injection; (H) representative photomicrographs of Oil Red O and H&E staining of liver tissues in different groups (scale bars: 50 or 200 mM). (I and J) Ablation of Faim impaired glucose tolerance and insulin sensitivity in mice. IPGTT (I) and IPITT (J), blood glucose was measured at different time points after glucose or insulin injection (left), and quantification of the AUC (right). Data are presented as mean SEM, n ¼ 5 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Western Blot, Control, Saline, Knockdown, Quantitative RT-PCR, Clinical Proteomics, Injection, Staining

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 5: PACAP activates the FAIM-AMPK-IRb axis to govern the SREBP and lipid synthetic gene program. (A and B) PACAP activated AMPK-IRb signaling pathway and down- regulated the expression of lipogenesis genes in HFD-fed mice, such effects were suppressed following knockdown of Faim. Western blot analysis were used to detect the protein level of indicated antibodies. (C and D) The overexpression of FAIM activated AMPK-IRb signaling pathway, the protein levels of FAIM, p-AMPK, AMPK, p-IRb, IRb, and lipid synthesis genes SREBP1, SREBP2, FAS, SCD1, HMGCR were determined by western blot with quantifications. Data are presented as mean SEM, n ¼ 3 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Expressing, Knockdown, Western Blot, Over Expression

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 6: Knockdown of Faim inhibited the effects of PACAP on decreasing lipid accumulation and activating AMPK-IRb signaling pathway. AML12 cells were transiently transfected with Faim siRNA or scramble siRNA in cell culture medium containing 200 mM PA or 10% BSA (as control of PA solution) for 24 h, then the cells were treated with PACAP (1 mM) and Max.D.4 (10 mM) for another 24 h as indicated. (A) Representative for Oil Red O staining of AML12 (Scale bars, 50 mM). (B and C) Impacts of Faim knockdown on cellular TG and TC level (B), glucose uptake and glycogen content (C) of AML12. (D and E) Faim knockdown impaired the activation of AMPK-IRb pathway in AML12 treated with PACAP, the protein levels were analyzed by western blot and quantitative measurements (right). (F and G) AML12 were exposed to PA for 24 h, then treated with PACAP (1 mM), Max.D.4 (10 mM) and Compound C (10 mM) (F) or AICAR (0.5 mM) (G) for another 24 h. The protein level of FAIM was detected by western blot analysis and quantitative measurements (down). Data are presented as mean SEM. n ¼ 3 (&P < 0.05, &&P < 0.01, &&&P < 0.001 vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. PA group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. PA þ PACAP group, OP < 0.05, OOP < 0.01, OOOP < 0.001 vs. PA þ PACAP þ Max.D.4 group).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Knockdown, Transfection, Cell Culture, Control, Staining, Activation Assay, Western Blot

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 7: PACAP activates the PAC1-PKA-CREB signaling pathway to stimulate FAIM expression. (A) mRNA level of Faim in PA-induced AML12 treated with PACAP, determined by qRT-PCR (n ¼ 3). (B) The effect of PACAP on the activation of PKA and CREB in PA induced AML12 cells, the protein levels of PKA, CREB and FAIM, and phosphorylation of PKA and CREB were determined by western blot. (*P < 0.05, **P < 0.01, ***P < 0.001 vs. PA group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. PACAP (1 mM) group). (C) Schematic of the sequence of two CRE binding sites that CREB targets the WT or mutated promoter region of Faim mRNA. (DeF) Luciferase activities in AML12 transiently transfected with either wild type (WT), truncated, or mutant Faim promoter-luciferase reporter constructs. Then the cells were treated with PACAP (1 mM), or co- transfected with CREB cDNA or empty vectors. (G) Luciferase activities of FAIM WT reporter plasmids in AML12 cells with the treatment of KG-501 (a specific CREB inhibitor) and PACAP (1 mM) as indicated. Data are shown as mean SEM, n ¼ 3 or 5 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Expressing, Quantitative RT-PCR, Activation Assay, Phospho-proteomics, Western Blot, Sequencing, Binding Assay, Luciferase, Transfection, Mutagenesis, Construct

Journal: Cell Reports Medicine

Article Title: A PD-1-targeted, receptor-masked IL-2 immunocytokine that engages IL-2Rα strengthens T cell-mediated anti-tumor therapies

doi: 10.1016/j.xcrm.2024.101747

Figure Lengend Snippet:

Article Snippet: CD45 (TIL) MicroBeads, mouse , Miltenyi Biotec , Cat# 130-110-618.

Techniques: Purification, Blocking Assay, Control, Recombinant, Staining, Luciferase, Isolation, Cell Isolation, Biomarker Discovery, Amplification, Knock-In, Plasmid Preparation, Expressing, Software

Journal: Communications Biology

Article Title: Identifying the lungs as a susceptible site for allele-specific regulatory changes associated with type 1 diabetes risk

doi: 10.1038/s42003-021-02594-0

Figure Lengend Snippet: The locus tagged by rs6679677 was cloned within the 3′ UTR of a luciferase gene driven by a minimal promoter and transiently transfected into A549 and HepG2 cells. The relative enhancer activity for the ref and alt versions of the rs6679677 locus was calculated compared to the empty control vector (pMRAdonor2). Relative luminescence units (RLU) for the luciferase assay were normalised using the absorbance values for the beta-galactosidase assay. Results were plotted as the percentage of the ref_alleles enhancer activity (mean ± SD). Transfection experiments were repeated three times across n = 3 or n = 9 technical replicates in HepG2 (liver) and A549 (lung) cells, respectively. Representative results are shown for one transient transfection of A549 and HepG2 cells. Two-sided t -test was used for independent mean evaluation.

Article Snippet: On the day of transfection (24 following cell plating), ~75% confluent wells were co-transfected with luciferase plasmid DNA (i.e. 800 ng of pMPRA1:luc_rs6679677_ref, pMPRA1:luc_rs6679677_alt, or pMPRAdonor2 luciferase control) and a beta-galactosidase control plasmid (200 ng) using lipofectamine 3000 (ThermoFisher; according to the manufacturer’s instructions).

Techniques: Clone Assay, Luciferase, Transfection, Activity Assay, Control, Plasmid Preparation, β-Gal Assay

Journal: Nature Materials

Article Title: Parenchymal and stromal tissue regeneration of tooth organ by pivotal signals reinstated in decellularized matrix

doi: 10.1038/s41563-019-0368-6

Figure Lengend Snippet: Fig. 4 | Alx3-restored adult MSC-induced angiogenesis and VEGF signalling. a,d, Angiogenesis in regenerated tissues with magnifications. d, native dentin. b,e, Human von Willebrand factor immunohistochemistry with magnifications. Green arrowheads, endothelial cells of human origin (e); black arrowheads, endothelial cells of host (mouse) origin (e); red arrowheads chimeric blood vessels (e). c,f, Human mitochondria staining; red arrowheads, human cells; black arrowheads, host (mouse) cells. a–f, n = 5 independent biological samples. g, Blood vessel (bv) quantification. n = 5 independent biological samples, presented as the mean ± s.d. P values calculated by one-way ANOVA with Bonferroni: **P < 0.01, ***P < 0.001. h, CCK8 of HUVECs treated with conditioned medium (CM) by vector control or Alx3-restored MSCs. n = 4 independent biological samples, presented as the mean ± s.d. P values calculated by one-way ANOVA with Bonferroni: *P < 0.05). i–o, Transwell migration assay by Alx3 conditioned medium or with neutralizing antibody or VEGFR2 inhibitor (i–n) and quantification (o). n = 4 independent biological samples, presented as mean ± s.d. P values calculated by one-way ANOVA with Bonferroni: *P < 0.05, **P < 0.01, ***P < 0.001). p, Mouse VEGF A promoter luciferase reporter with two Alx3-binding elements in the dashed red boxes (left) and VEGF luciferase assay on Alx3 transfection (right). n = 3 independent biological samples, presented as median with range. P values calculated by Kruskal–Wallis tests: *P < 0.05). BE, binding element. q,r, VEGF production and p-VEGFR2 activation by western blot (q) and quantification (r). n = 4 independent biological samples, presented as the mean ± s.d. P values calculated by one-way ANOVA with Bonferroni: *P < 0.05, **P < 0.01, ***P < 0.001. s–x: The tube formation of HUVECs treated by Alx3 conditioned medium with or without NAb or VEGFR2 inhibitor. n = 3 independent biological samples. y, Tube length quantification. n = 3 independent biological samples, presented as the median with range. P values calculated by Kruskal–Wallis tests: *P < 0.05, **P < 0.01. z, rtPCR of VE-cadherin, PECAM1, VEGF and Flk1. n = 4 independent biological samples, presented as the mean ± s.d. P values calculated by one-way ANOVA with Bonferroni: *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The 1.6 kB VEGF luc plasmid was a gift from P. D’Amore (Addgene plasmid no. 29667).

Techniques: Immunohistochemistry, Staining, Plasmid Preparation, Control, Transwell Migration Assay, Luciferase, Binding Assay, Transfection, Activation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer, pSi-shUSP22-1 or pSi-shUSP22-2 plasmid. ( A ) Western blot analysis of USP22 expression; β-actin was used as endogenous control. ( B ) Cell viability was measured using MTT assay at 1, 2, 3, and 4 d after transfection. ( C ) EdU staining used to examine the proliferation. Scale bar: 5 μm. ( D ) Percentage of EdU-positive staining in (C). ( E ) Effect of USP22 knockdown on cell proliferation evaluated by colony formation assay. After 21 d of transfection, cells were stained with Giemsa solution. ( F ) Histograms representing the colony formation number in (E). ( G ) Western blot conducted to analyze the expression of USP22 and proliferation-related proteins including Rb, p-Rb, Akt, p-Akt, and cyclin D2. β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pSilencer group (B, D, and F).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Control, MTT Assay, Staining, Knockdown, Colony Assay

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer or pSi-shUSP22-1 plasmid. ( A and B ) After 48 h of transfection, the scratch wound-healing assay was performed to evaluate the effect of USP22 on cell migration. The representative images of cell migration were shown in (A), and the width of wounds at the indicated times was shown in (B). ( C ) Invasion of CAL-62 and 8505C cells analyzed by transwell assay. The invaded cells were stained with DAPI. Scale bar: 5 μm. ( D ) Number of DAPI-positive cells per field counted under a fluorescent microscopy. ( E ) Immunofluorescence staining was performed to analyze the effect of USP22 on the expression of E-cadherin and vimentin in 8505C cells. Scale bar: 5 μm. ( F and G ) Expression of invasion-related proteins containing BMI-1, E-cadherin, vimentin, and snail in ATC cells was measured by qPCR (F) and western blot (G). GAPDH and β-actin were used as endogenous controls, respectively. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with the group at 0 h after transfection (B). ## P < 0.01, as compared with mock or pSilencer group at 24 h after transfection (B). * P < 0.05, as compared with mock or pSilencer group (D and F).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Transfection, Plasmid Preparation, Wound Healing Assay, Migration, Transwell Assay, Staining, Microscopy, Immunofluorescence, Expressing, Western Blot

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer or pSi-shUSP22-1 plasmid. ( A ) Apoptosis of 8505C cells analyzed by flow cytometry. ( B ) Apoptosis rate in (A) was calculated. ( C ) Fluorescent TUNEL assay conducted to determine the apoptosis of 8505C cells. Scale: 10 μm. ( D ) Percentage of TUNEL-positive cells in (C). ( E ) CAL-62 and 8505C cell apoptosis evaluated by nucleosomal fragmentation assay. ( F ) Quantification of caspase-3 activity in CAL-62 and 8505C cells. ( G ) Western blot analyses of apoptosis-related protein (Bid, Bax, cl-caspase-3, caspase-3, and Bcl-2) expressions in CAL-62 and 8505C cells. β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pSilencer group (B and D–F).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, TUNEL Assay, Activity Assay, Western Blot, Control

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: Female six-week-old SCID mice were inoculated subcutaneously into right hind flanks or injected via tail vein with stably expressed pLKO.1 or pLKO.1-shUSP22 8505C-luc cells. Mock-treatment was used as control. ( A ) Tumor growth progression was measured by in vivo luciferase imaging of the xenografts at days 1, 5, 9, and 12 d after inoculation. ( B ) Representative gross photos of tumors 22 d after subcutaneous xenografting ( n = 6). ( C ) Tumor volumes of subcutaneous implantation models of ATC were monitored and calculated after 0, 7, 11, 13, 17, 20, and 22 d of inoculation. D–F. After 28 d of injection through tail vein, the lungs were removed and photographed ( D ), the number of metastatic nodules in lungs was counted ( E ), and the weight of lungs was measured ( F ). ( G ) Fluorescence TUNEL assay was carried out to determine cell apoptosis in the same tumor tissues as indicated above. The rate of TUNEL-positive cells was calculated. Scale bar: 10 μm. ( H ) qPCR assay was performed to detect the mRNA expression of USP22 in tumor tissues from (B). ( I ) Protein expression of USP22 in tumor tissues was analyzed by western blot. ( J ) Representative results of western blot analyses of cyclin D2, Akt, p-Akt, E-cadherin, vimentin, Bax, Bcl-2, cl-caspase-3, and caspase-3 in tumor tissues. (I and J) β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pLKO.1 group (C and E–H).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Injection, Stable Transfection, Control, In Vivo, Luciferase, Imaging, Fluorescence, TUNEL Assay, Expressing, Western Blot

Journal: Stem Cells and Development

Article Title: MicroRNAs Involved in Asthma After Mesenchymal Stem Cells Treatment

doi: 10.1089/scd.2015.0339

Figure Lengend Snippet: miRNAs differently expressed during asthma and after BM-MSCs treatment. (A) Hierarchical clustering analysis of all the aberrant miRNAs that were differently expressed either between the model ( n = 3) and control groups ( n = 3) or between the model+BM-MSCs ( n = 3) and model groups (total n = 9, greater than twofold; P < 0.05). (B) The miRNA development patterns after asthma induction and then with the BM-MSCs. (a) The first lines in the boxes represent increased or decreased miRNA expression after asthma induction compared with the control groups. The second lines in the boxes represent increased or decreased miRNA expression after the BM-MSCs treatment compared with the model group. The numbers in the boxes represent the number of miRNAs that obey the development pattern of that box. Two patterns with a total 14 aberrant miRNAs were selected and marked with orange for next experiments. (b) A detailed pattern of 10 miRNAs in the upper orange box and 4 miRNAs in the lower orange box in (B, a ) for the three groups. (C) Hierarchical clustering analysis of the said 14 miRNAs marked with orange in (B, a ) . Red , expression above the median; green , expression below the median. miRNAs, microRNAs.

Article Snippet: For the miRNA array, the Exiqon miRCURY LNA TM Universal RT miRNA PCR profiling kit with two 384-well plates (Ready-to-Use Mouse & Rat Panel I+II V3, Cat. No. 20379; Exiqon; www.exiqon.com ), which covered 752 murine miRNAs, was used with quantitative real-time PCR (qRT-PCR) technology.

Techniques: Control, Expressing

Journal: Stem Cells and Development

Article Title: MicroRNAs Involved in Asthma After Mesenchymal Stem Cells Treatment

doi: 10.1089/scd.2015.0339

Figure Lengend Snippet: Mmu-miR-21a-3p , mmu-miR-449c-5p , and mmu-miR-496a-3p were significantly differentially expressed in asthma and with BM-MSCs treatment. (A) The fold changes of the 14 selected miRNAs with the log ratio of the mean value are shown. The arrows indicate the miRNAs that were selected for the next experiments. The mmu-miR-21a-3p (B) , mmu-miR-449c-5p (C), and m mu-miR-496a-3p (D) expression levels were confirmed with real time PCR in subsequent lung tissue samples from the control ( n = 14), model ( n = 12), and model+BM-MSC ( n = 14) groups. ** P < 0.01; *** P < 0.001. PCR, polymerase chain reaction.

Article Snippet: For the miRNA array, the Exiqon miRCURY LNA TM Universal RT miRNA PCR profiling kit with two 384-well plates (Ready-to-Use Mouse & Rat Panel I+II V3, Cat. No. 20379; Exiqon; www.exiqon.com ), which covered 752 murine miRNAs, was used with quantitative real-time PCR (qRT-PCR) technology.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Polymerase Chain Reaction

Journal: Stem Cells and Development

Article Title: MicroRNAs Involved in Asthma After Mesenchymal Stem Cells Treatment

doi: 10.1089/scd.2015.0339

Figure Lengend Snippet: The mRNA profile in asthma with the treatment of BM-MSCs and the relationship between mmu-miR-21a-3p and Acvr2a . (A) A hierarchical clustering analysis of all the aberrant protein-coding genes in the PCR array that were differentially expressed either between the model and control groups or between the model+BM-MSCs and model groups (≥1.5-fold; P < 0.05). (B) A hierarchical clustering analysis of 19 aberrant protein-coding genes in the PCR array. Red , expression above the median; green , expression below the median in (A) . (C) Schematic diagrams showing the interaction sites between the mmu-miR-21a-3p , mmu-miR-449c-5p , and mmu-miR-496a-3p and the 3′ UTR of their corresponding mRNAs. (D) Mmu-miR-21a-3p was negatively correlated with the Acvr2a gene in miRNAs array and mRNA PCR array. y , the mean Acvr2a Ct value in the three groups. x , the mean mmu-miR-21a-3p Ct value in the three groups. (E) qRT-PCR analysis of Acvr2a in lung tissue samples from the control, model, and model+BM-MSC groups. n = 6 in each group. (F) Dual luciferase assay of 293T cells cotransfected with firefly luciferase vector containing the Acvr2a 3′ UTRs and the mmu-miR-21a-3p mimics or scrambled oligonucleotides as the miRNA negative control. * P < 0.05; ** P < 0.01. Acvr2a, activin A receptor, Type IIA; mRNA, messenger RNA; UTR, untranslated region; qRT-PCR, quantitative real-time PCR.

Article Snippet: For the miRNA array, the Exiqon miRCURY LNA TM Universal RT miRNA PCR profiling kit with two 384-well plates (Ready-to-Use Mouse & Rat Panel I+II V3, Cat. No. 20379; Exiqon; www.exiqon.com ), which covered 752 murine miRNAs, was used with quantitative real-time PCR (qRT-PCR) technology.

Techniques: Control, Expressing, Quantitative RT-PCR, Luciferase, Plasmid Preparation, Negative Control, Real-time Polymerase Chain Reaction

Journal: Genome Biology

Article Title: RNA methylomes reveal the m 6 A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening

doi: 10.1186/s13059-019-1771-7

Figure Lengend Snippet: Dynamics of DNA methylation (5mC) and mRNA m 6 A methylation in tomato fruit ripening. a Images of wild-type (WT) fruit at different ripening stages and Cnr fruit at 42 DPA. DPA, days post-anthesis; scale bar = 1 cm. b Relative 5mC levels of WT and Cnr fruit shown in a . For 5mC assay, 100 ng of genomic DNA was detected in each sample by MethylFlash™ methylated DNA quantification kit. 5mC level in each sample was normalized to that of the positive control according to the manufacturer’s instructions. The plus sign represents the average in each box. c LC-MS/MS assay showing the amount of mRNA m 6 A in WT and Cnr fruit shown in a . Data are presented as mean ± standard deviation ( n = 3). Asterisks indicate significant differences (* P < 0.05, ** P < 0.01; Student’s t test). NS, no significance

Article Snippet: In brief, genomic DNA was extracted from the agroinfiltrated N. benthamiana leaves, and 500 ng of purified DNA was treated with bisulfite to produce mutations from cytosine (C) to thymine (T) using EZ DNA methylation-gold kit (ZYMO Research, D5005).

Techniques: DNA Methylation Assay, Methylation, Positive Control, Liquid Chromatography with Mass Spectroscopy, Standard Deviation

Journal: Genome Biology

Article Title: RNA methylomes reveal the m 6 A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening

doi: 10.1186/s13059-019-1771-7

Figure Lengend Snippet: SlALKBH2 is transcriptionally regulated by DNA methylation. a The 5mC levels in the differentially methylated region (DMR) of SlALKBH2 promoter in wild-type (WT) and Cnr mutant fruit based on the Tomato Epigenome Database ( http://ted.bti.cornell.edu/epigenome/ ). b The 5mC levels in the DMR of SlALKBH2 promoter in WT and sldml2 mutant fruit based on the DNA methylome database . a , b The numbers indicate the cytosine positions relative to the start codon. Black represents the methylation frequency of cytosines at the indicated positions. DPA, days post-anthesis. c Schematic of the dual-luciferase system used for promoter activity assay. The SlALKBH2 promoter was cloned into the dual-luciferase reporter vector to activate the expression of firefly luciferase (Fluc). The renilla luciferase (Rluc) driven by the CaMV 35S promoter served as an internal control. LB, left border; RB, right border; Ter, terminator. d – f Co-expression of SlDML2 (SlDML2-HA) with the dual-luciferase reporter vector in the Nicotiana benthamiana leaves increased the relative Fluc activity ( d ), facilitated the Fluc gene expression ( e ), and reduced the 5mC level in SlALKBH2 promoter ( f ) compared with the empty plasmid control (HA). d The representative image from a total of six images (left panel). The Fluc activity was normalized against the Rluc activity, followed by normalization against the control (right panel). Data are presented as means ± standard deviation ( n = 6). Asterisks indicate significant differences (*** P < 0.0001; Student’s t test). e Gene expression was determined by quantitative RT-PCR analysis. Error bars represent the standard deviation of three independent experiments. Asterisks indicate significant differences (* P < 0.05; Student’s t test). f The box plot showing 5mC levels of all cytosines ( n = 41) in the DMR analyzed by Sanger bisulfite sequencing. The plus sign represents the average level in each box

Article Snippet: In brief, genomic DNA was extracted from the agroinfiltrated N. benthamiana leaves, and 500 ng of purified DNA was treated with bisulfite to produce mutations from cytosine (C) to thymine (T) using EZ DNA methylation-gold kit (ZYMO Research, D5005).

Techniques: DNA Methylation Assay, Methylation, Mutagenesis, Luciferase, Activity Assay, Clone Assay, Plasmid Preparation, Expressing, Standard Deviation, Quantitative RT-PCR, Methylation Sequencing

Journal: Genome Biology

Article Title: RNA methylomes reveal the m 6 A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening

doi: 10.1186/s13059-019-1771-7

Figure Lengend Snippet: SlALKBH2 is necessary for normal tomato fruit ripening. a Genotyping of mutations mediated by CRISPR/Cas9 gene-editing system in slalkbh2-23 , slalkbh2-25 , and slalkbh2-28 mutants. Diagram showing the single guide RNAs (sgRNAs) containing different target sequences (T1, T2, and T3), which were designed to specifically target the exons of SlALKBH2 . The red letters indicate the protospacer adjacent motif (PAM). The transgenic plants in the second generation were genotyped by sequencing genomic regions flanking the target sites. Red arrows indicate the editing sites. Two mutants ( slalkbh2-23 and slalkbh2-28 ) have a homozygous 1-bp insertion, and one ( slalkbh2-25 ) has a homozygous 5-bp deletion caused by target T2 in the fourth exon of SlALKBH2 . b Ripening phenotype of slalkbh2 mutants. Fruit from wild-type (WT) and slalkbh2 mutants ( slalkbh2-23 , slalkbh2-25 , and slalkbh2-28 ) at 39, 42, 47, and 52 days post-anthesis (DPA) are shown. c LC-MS/MS assay showing the amount of mRNA m 6 A in WT and slalkbh2 mutant fruit at 39 DPA. Data are presented as mean ± standard deviation ( n = 3). d m 6 A-IP-PCR assay showing the relative m 6 A enrichment in SlDML2 mRNA in WT and slalkbh2 mutant fruit at 39 DPA. e SlDML2 gene expression in WT and slalkbh2 mutant fruit at 39 and 42 DPA. The ACTIN gene was used as an internal control. d , e Error bars represent the standard deviation of three independent experiments. Asterisks indicate significant differences (* P < 0.05, ** P < 0.01; Student’s t test). f Model for the relationship between DNA methylation and m 6 A mRNA methylation in fruit ripening. DNA methylation negatively regulates SlALKBH2 to mediate overall m 6 A mRNA methylation. The m 6 A modification promotes SlDML2 mRNA decay, thereby affecting DNA methylation and fruit ripening

Article Snippet: In brief, genomic DNA was extracted from the agroinfiltrated N. benthamiana leaves, and 500 ng of purified DNA was treated with bisulfite to produce mutations from cytosine (C) to thymine (T) using EZ DNA methylation-gold kit (ZYMO Research, D5005).

Techniques: CRISPR, Transgenic Assay, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Standard Deviation, Expressing, DNA Methylation Assay, Methylation, Modification

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 1 S100A9 promotes prostate cancer cell invasion and β1 integrin expression through interaction with TLR4. PC-3 and DU-145 cells were treated with S100A9 (20 µg/ml) for 48 h. (A) PC-3 and DU-145 cells invasion was measured by transwell invasion assay. (B) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. PC-3 and DU-145 cells were treated with S100A9 (20 µg/ml) for 48 h. (C, D) Cell extracts were immunoprecipitated (IP) with control mouse IgG, mouse anti-S100A9 antibody. Immunoblot (IB) was used to detect S100A9, TLR4 and RAGE. PC-3 and DU-145 cells were transfected with TLR4 or control siRNA. (E) TLR4 expression was examined by Western blot after 48 h siRNA transfection. PC-3 and DU-145 cells were transfected with TLR4 or control siRNA followed by stimulation with S100A9. (F) Tumor cell invasion was measured by transwell invasion assay. (G) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. Scale bar 50 μ. Magnifcation×200. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Expressing, Transwell Invasion Assay, Western Blot, Immunoprecipitation, Control, Transfection

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 2 NF-κB mediates S100A9-induced prostate cancer cell β1 integrin up-regulation. PC-3 and DU-145 cells transfected with or without TLR4 siRNA or control siRNA, were transfected with NF-κB-luciferase reporter plasmid, and treated with S100A9 (20 µg/ml) for 48 h. (A, B) Activity of NF-κB was detected by measuring the relative activity of luciferase. PC-3 and DU-145 cells were treated with or without BAY11-7082 (5µM) for 30 min. Then cells were treated with or without S100A9 (20 µg/ ml) for 48 h. (C) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Transfection, Control, Luciferase, Plasmid Preparation, Activity Assay, Western Blot

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 3 S100A9 promotes prostate cancer cell invasion via integrin β1/FAK signaling. PC-3 and DU-145 cells were treated with or without BAY11-7082 (5µM) for 30 min. Then cells were treated with or without S100A9 (20 µg/ml) for 48 h. (A) Fibronectin expression was determined by Western blot. PC-3 and DU-145 cells were treated with or without S100A9 (20 µg/ml) for 48 h. (B) Supernatant fibronectin (FN) concentration was determined by ELISA. PC-3 and DU-145 cells were treated with or without S100A9 (20 µg/ml) for 30 min. (C) The phosphorylation of FAK was measured by Western blot. PC-3 and DU-145 cells were transfected with control siRNA or integrin β1-specific siRNA for 48 h. Then cells were treated with or without S100A9 (20 µg/ml) for 30 min, the expression of integrin β1 (D) or phosphorylation of FAK (E) was measured by Western blot. PC-3 and DU-145 cells were transfected with control siRNA or integrin β1-specific siRNA for 48 h. Then cells were treated with or without S100A9 (20 µg/ml) for 48 h. (F) The invasion activity were measured by transwell invasion assay. (G) PC-3 and DU- 145 cells were treated with β1 integrin functional blocking antibody MAB13 (50 μg/mL) or control IgG (50 μg/mL) for 30 min and then treated with or without S100A9 (20 µg/ml) for 30 min and the phosphorylation of FAK was measured by Western blot. (H) PC-3 and DU-145 cells were treated with MAB13 (50 μg/mL) or control IgG (50 μg/mL) for 30 min and then treated with or without S100A9 (20 µg/ml) for 48 h for invasion. (I) PC-3 and DU-145 cells were pretreated for 30 min with FAK inhibitor, PF562271 (100 nM) followed by stimulation with S100A9 (20 µg/ml) for 48 h for invasion. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Transfection, Control, Activity Assay, Transwell Invasion Assay, Functional Assay, Blocking Assay

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 4 S100A9 induces prostate cancer cell metastasis in vivo. DU-145 cells were transfected with pcDNA3.1 or pc DNA-S100A9 plasmid. (A, B) The expression or secretion of S100A9 was determined by Western blot and ELISA. The cells were injected to nude mice via tail vein. 30 days after inoculation, nude mice were sacrificed. (C) Expressions of S100A9 and integrin β1 in xenograft tumors were detected by immunohistochemistry. (D) Expressions of FAK, p-FAK, NF-kB p65 and p-NF-kB p65 in xenograft tumors were determined by Western blot. (E) Micrometastatic tumors in the lungs of mouse xenografts were counted and subjected to H and E staining. Scale bar 50 μ. Magnifcation×200. Data are represented as the mean ± S.E.M.*p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Immunohistochemistry, Staining