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: 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

Journal: Journal of Biological Chemistry

Article Title: A Single-nucleotide Deletion Leads to Rapid Degradation ofTAP-1 mRNA in a Melanoma Cell Line

doi: 10.1074/jbc.m300954200

Figure Lengend Snippet: FIG. 1. Deficiency of surface HLA class I expression in mela- noma cell line SK-MEL-19 was due to the TAP-1 down-regula- tion. a, HLA class I expression in three melanoma cell lines, SK-MEL- 19, 1102, and 1195. Bold black lines depict the staining by PE- conjugated anti-human HLA-A, -B, and -C antibody in untreated cells; dotted lines represent the staining by PE-conjugated mouse IgG1 as isotype control; and red lines represent anti-HLA-A, -B, and -C antibody staining after stimulation with 1000 units/ml IFN- for 72 h. b, expres- sion of HLA class I heavy chain (MHC I), 2M, TAP-1, TAP-2, LMP-2, and LMP-7 in each cell line with or without IFN- induction (1000 units/ml for 72 h). Total RNA loading to each well was shown as 28 S rRNA (28S) and 18 S rRNA (18S). c, transfection with wild type TAP-1, but not vector alone, restored HLA class I expression in the SK-MEL-19 cells. SK-MEL-19 cells were transfected with either vector alone (top panels) or vector with TAP-1 cDNA insert (bottom panels). These stable clones from each group were stimulated with or without IFN- and analyzed for cell surface HLA-A, -B, and -C, as detailed in a.

Article Snippet: For induction of HLA class I expression, cells were cultured in medium supplemented with recombinant human IFN- (1000 units/ml; R&D Systems, Minneapolis, MN).

Techniques: Expressing, Staining, Control, Transfection, Plasmid Preparation, Clone Assay

Journal: Journal of Biological Chemistry

Article Title: A Single-nucleotide Deletion Leads to Rapid Degradation ofTAP-1 mRNA in a Melanoma Cell Line

doi: 10.1074/jbc.m300954200

Figure Lengend Snippet: FIG. 2. Posttranscriptional mechanisms are responsible for poor accumulation of TAP-1 mRNA. a, a single-nucleotide polymorphism, adjacent to the first transcription start site (427), was identified at 446 in the bidirectional promoter shared by the TAP-1 and LMP-2 genes. The G 3 T change results in the loss of the AflIII restriction site. Southern blot hybridization was performed using AflIII and detected by a DNA probe that encompasses the downstream region of the polymorphism site. SK-MEL-19 cells showed one 5.6-kb band that represents homozygous T allele, as did the breast cancer cell line SK-BR-3, which has significant cell surface HLA class I surface expression (data not shown). HeLa cells, in contrast, are homozygous for the G allele. b, activities of T and G alleles of TAP-1 promoter (pTAP1/T and pTAP1/G, respectively) in SK-MEL-19 cells. The two allelic forms of TAP-1 promoter were cloned into pGL2-basic vector (basic) that did not contain any promoter or enhancer but encoded firefly luciferase. The pGL2-SV40 construct (SV40) that had both SV40 promoter and SV40 enhancer as well as the firefly luciferase reporter gene was used as positive control. After transfection, IFN- was added to the cell culture at 1000 units/ml. Cells were lysed 48 h after transfection, and luciferase expression was tested using a luminometer. Data shown are representative of at least five independent experiments. c, the TAP-1 gene was actively transcribed in SK-MEL-19 cells in the presence and absence of IFN- in nuclear run-on assay. Endogenous GAPDH expression was used as a positive control, and the pcDNA3.1/Hyg() vector was used as a negative control. The run-on experiments were repeated three times with similar results.

Article Snippet: For induction of HLA class I expression, cells were cultured in medium supplemented with recombinant human IFN- (1000 units/ml; R&D Systems, Minneapolis, MN).

Techniques: Southern Blot, Hybridization, Expressing, Clone Assay, Plasmid Preparation, Luciferase, Construct, Positive Control, Transfection, Cell Culture, Nuclear Run-on Assay, Negative Control

Journal: PLoS ONE

Article Title: High EMT Signature Score of Invasive Non-Small Cell Lung Cancer (NSCLC) Cells Correlates with NFκB Driven Colony-Stimulating Factor 2 (CSF2/GM-CSF) Secretion by Neighboring Stromal Fibroblasts

doi: 10.1371/journal.pone.0124283

Figure Lengend Snippet: Calu-1 and NCI-H1437 tumor cells were co-cultured with HDFs and two patient-derived NF/CAF pairs (NF1/CAF1, NF2/CAF2) for 24 h. Supernatants from mono- (green) and co-cultures (purple: Calu-1 and orange: NCI-H1437) were collected and tested for the indicated cytokines with at least three biological replicates, each representing two technical replicates (Cytokine Array), or three biological replicates, each representing three technical replicates (ELISA). (A) Cytokine Array: Relative pixel counts on the Y-axis. (B) ELISA: The cytokine level is depicted on the Y-axis [pg/ml]. Statistical analysis was performed by using Student’s t-test (for p-values see ).

Article Snippet: The following ELISA kits were purchased from R&D Systems for cross-validation of identified hits: IL-6 (D6050), IL-8 (D8000C), GCP-2 (DGC00), CXCL1 (DGR00), GM-CSF (DGM00) and VEGF (DVE00), respectively.

Techniques: Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: High EMT Signature Score of Invasive Non-Small Cell Lung Cancer (NSCLC) Cells Correlates with NFκB Driven Colony-Stimulating Factor 2 (CSF2/GM-CSF) Secretion by Neighboring Stromal Fibroblasts

doi: 10.1371/journal.pone.0124283

Figure Lengend Snippet: (A) Luciferase-based NFκB reporter activity in co-cultures of Calu-1 or NCI-H1437 with various FBs. 24 h after transfection of the respective cell type (depicted in the title of each graph) the NFκB-reporter plasmid-containing cell line was incubated with a non-transfected cell line as indicated on the X-axis for additional 24 h. The upper three graphs display studies with transfected FBs co-cultured with non-transfected tumor cell lines or with the non-transfected parental FB line, respectively. The lower two graphs exhibit the experiment with transfected tumor cell lines. Bars indicate fold changes of the Luciferase reporter signal mean values. For FBs lipofectamine transfection, for tumor cell lines electroporation transfection was applied. (B) NCI-H157 co-cultures were treated with trametinib and BI5700 for 24 h (4 μM each). The different FBs had been transfected prior with an NFκB-reporter plasmid. Luciferase units are depicted on the Y-axis. (C) BI5700 dose-dependent reduction of CSF2 transcription in HDFs. Relative CSF2 transcripts are depicted (RT-qPCR). Supernatant from a 24 h HDF mono- or Calu-1/HDF co-culture was transferred to an HDF mono-culture for another hour with or without BI5700 treatment. Incubation with RPMI 10% FCS medium served as a control. The level of CSF2 transcripts in the respective non BI5700-treated HDFs is indicated by [-∞]. Statistical analyses were performed on the mean values by unpaired comparisons using Student’s t-test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; n.s.: not significant). (D) ELISA for CSF2 (Y-axis [pg/ml]) of supernatants derived from mono- and co-cultures of invasive (Calu-1, NCI-H157) and non-invasive tumor cell lines (NCI-H1437) with HDFs. Supernatants were taken and replaced with fresh medium at the indicated time points (X-axis; time [h]). All experiments were performed in triplicates (n = 3).

Article Snippet: The following ELISA kits were purchased from R&D Systems for cross-validation of identified hits: IL-6 (D6050), IL-8 (D8000C), GCP-2 (DGC00), CXCL1 (DGR00), GM-CSF (DGM00) and VEGF (DVE00), respectively.

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Incubation, Cell Culture, Electroporation, Quantitative RT-PCR, Co-Culture Assay, Control, Enzyme-linked Immunosorbent Assay, Derivative Assay

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: Fragments were digested into single-cell suspensions using the Miltenyi tumor isolation kit (130-096-730).

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