Journal: PLOS One

Article Title: Differential expression and regulation of FASLG by miR-5195/miR-3941 in age-related hearing loss

doi: 10.1371/journal.pone.0331661

Figure Lengend Snippet: (A). Heat map illustrating differential expression data from RNA-Seq analysis. Heatmap of FASLG gene expression, represented as fold changes in mRNA levels, was generated for the ARHL (n = 12) and NH (n = 12); (B). Expression of FASLG was analyzed using RT-qPCR and confirmed through WB (NH: n = 12, ARHL: n = 14). (C). Expression of miR-5195 was detected using RT-qPCR in WB (NH: n = 6, ARHL: n = 5). (D). Expression of miR-3941 was detected using RT-qPCR in WB (NH: n = 14, ARHL: n = 12). U6 snRNA was used as an internal control (** p < 0.01). Abbreviations used are as follows: Normal hearing group (NH), Age-related hearing loss (ARHL), Whole blood (WB), Fas ligand ( FASLG ).

Article Snippet: For the transfection experiments, the FASLG Human 3’ UTR Clone (#SC210991, OriGene, Rockville, Maryland, USA), miR-5195 (HmiR1468 – MR04, Genecopoeia, Rockville, Maryland, USA), and miR-3941 (HmiR1052 – MR04, Genecopoeia, Rockville, Maryland, USA) were used.

Techniques: Quantitative Proteomics, RNA Sequencing, Gene Expression, Generated, Expressing, Quantitative RT-PCR, Control

Journal: PLOS One

Article Title: Differential expression and regulation of FASLG by miR-5195/miR-3941 in age-related hearing loss

doi: 10.1371/journal.pone.0331661

Figure Lengend Snippet: (A). Identification of 3′-UTR sequences in the FASLG gene predicted to include binding sites for miR-3941; (B). Identification of 3′-UTR sequences in the FASLG gene predicted to harbor binding sites for miR-5195-5p; (C). Dual luciferase reporter assay results for miR-3941, miR-5195, and their corresponding target 3’-UTR of FASLG in HEI-OC-1 cells; (D). Dual luciferase assay results of miR-3941, miR-5195 and their corresponding target 3’-UTR of FASLG in HeLa cell; The significant differences between FASLG and the miRNA target group are represented as ** p < 0.01. Abbreviations used are as follows: Fas ligand ( FASLG ), House Ear Institute-Organ of Corti 1 (HEI-OC-1).

Article Snippet: For the transfection experiments, the FASLG Human 3’ UTR Clone (#SC210991, OriGene, Rockville, Maryland, USA), miR-5195 (HmiR1468 – MR04, Genecopoeia, Rockville, Maryland, USA), and miR-3941 (HmiR1052 – MR04, Genecopoeia, Rockville, Maryland, USA) were used.

Techniques: Binding Assay, Luciferase, Reporter Assay

Journal: PLOS One

Article Title: Differential expression and regulation of FASLG by miR-5195/miR-3941 in age-related hearing loss

doi: 10.1371/journal.pone.0331661

Figure Lengend Snippet: (A). The light blue rectangular node represents the FASLG gene, while the light blue circular nodes represent the 35 selected miRNAs. The nodes with red text indicate the two miRNAs of interest, miR-3941 and miR-5195-5p. The arrows connecting FASLG to the miRNAs indicate that the thickness of the arrow corresponds to the context++ score of the miRNA, with thicker arrows representing lower scores. (B). We utilized blood samples from individuals with ARHL and found that A notable rise in FASLG expression was observed. Additionally, we identified miR-3941 and miR-5195 as potential regulators of FASLG expression. These miRNAs may bind to the 3’ UTR of the elevated FASLG , leading to down-regulation of gene expression. Abbreviations used are as follows: Fas ligand ( FASLG ), Age-related hearing loss (ARHL), Fas receptor (FAS-R), 3’untranslated region (3’UTR).

Article Snippet: For the transfection experiments, the FASLG Human 3’ UTR Clone (#SC210991, OriGene, Rockville, Maryland, USA), miR-5195 (HmiR1468 – MR04, Genecopoeia, Rockville, Maryland, USA), and miR-3941 (HmiR1052 – MR04, Genecopoeia, Rockville, Maryland, USA) were used.

Techniques: Expressing, Gene Expression

Journal: Transplantation

Article Title: Decline in Club Cell Secretory Proteins, Exosomes Induction and Immune Responses to Lung Self-antigens, Kα1 Tubulin and Collagen V, Leading to Chronic Rejection After Human Lung Transplantation

doi: 10.1097/tp.0000000000003428

Figure Lengend Snippet: FIGURE 6. A, Exosome size, verified by nanosight, was <200 nm. B, Exosomes from bronchoalveolar lavage fluid samples from lung transplant recipients (LTxRs) with bronchiolitis obliterans syndrome (BOS) contained increased levels of self-antigens (CIITA, 20S proteasome, NFκB, CD56, NKG2D, FasL and perforin). C, Densitometry analysis of self-antigens (CIITA, 20S proteasome, NFκB). D, Representative densitometry analysis of natural killer (NK) cells related and cytotoxic molecules in LTxRs with BOS (n = 2) vs stable LTxRs (n = 2), demonstrating increased levels of CD56, NKG2D, FasL, and perforin.

Article Snippet: Exosomes isolated from stable LTxRs and from LTxRs with BOS were analyzed with Abs specific to target proteins, including Col-V (ab7046), CIITA (ab49132; Abcam), NF-κB (C22B4), cell-signaling technology, Kα1T (sc-12462-R), 20S proteasome (sc-166205), CD56 (sc-7326), NKG2D (sc-23869), and perforin (sc-373943), FasL (sc-19681; Santa Cruz Biotechnology), per manufacturers’ protocol.

Techniques:

Journal: Transplantation

Article Title: Decline in Club Cell Secretory Proteins, Exosomes Induction and Immune Responses to Lung Self-antigens, Kα1 Tubulin and Collagen V, Leading to Chronic Rejection After Human Lung Transplantation

doi: 10.1097/tp.0000000000003428

Figure Lengend Snippet: FIGURE 7. A, Exosomes isolated from plasma samples of healthy volunteers (HV), stable lung transplant recipients (LTxRs), and LTxRs with bronchiolitis obliterans syndrome (BOS) demonstrate increased levels of CD56, NKG2D, FasL, and perforin in LTxRs with BOS. B, Densitometry analysis of natural killer (NK) cells related and cytotoxic molecules optical density in LTxRs with BOS (n = 14) compared with stable LTxRs (n = 14).

Article Snippet: Exosomes isolated from stable LTxRs and from LTxRs with BOS were analyzed with Abs specific to target proteins, including Col-V (ab7046), CIITA (ab49132; Abcam), NF-κB (C22B4), cell-signaling technology, Kα1T (sc-12462-R), 20S proteasome (sc-166205), CD56 (sc-7326), NKG2D (sc-23869), and perforin (sc-373943), FasL (sc-19681; Santa Cruz Biotechnology), per manufacturers’ protocol.

Techniques: Isolation, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Human dental pulp stem cells modulate pro-inflammatory macrophages both through cell-to-cell contact and paracrine signaling

doi: 10.3389/fimmu.2024.1440974

Figure Lengend Snippet: Specific primers for RT-PCR.

Article Snippet: Primary antibodies were the following: CD80 (dilution 1:2000, TA501575, Thermofisher, USA), CD206 (dilution 1:2000, MA532498, Thermofisher, USA), HLA-DRB1 (dilution 1:400, NBP2-45316, Bio-Techne, USA), FAS-L (dilution 1:1000, sc-6237, Santa Cruz Biotechnology, USA), PD-L1 (dilution 1:1000, NBP2-15791, Bio-Techne, USA), NF-kBp65 (dilution 1:1000, 510500, Thermo Fisher Scientific Inc., USA), IDO (dilution 1:250, 711778, Thermo Fisher Scientific Inc., USA), ICAM-1 (dilution 1:250, MA5407, Thermo Fisher Scientific Inc., USA), IL-6 (dilution 1:1000, 12153, Cell Signaling Technology, USA), COX-2 (dilution 1:200, AF4198, Bio-Techne, USA).

Techniques: Sequencing

Journal: International journal of cancer

Article Title: Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer.

doi: 10.1002/ijc.28076

Figure Lengend Snippet: Figure 2. Signaling through the EP1 receptor upregulates FasL expression in vivo. (a) CT26 cells were treated with increasing concentrations of ONO-8713 for 24 hr, or FasL and EP1 receptor expression was suppressed in CT26FasL shRNA and CT26EP1 shRNA cells, respectively. FasL expression was determined by Western blotting. Expression of the housekeeping gene b-actin was used as an internal control. Results shown are represen- tative of three independent experiments. (b) FasL expression was suppressed in CT26FasL shRNA cells. The efficiency of suppression of FasL was assessed by RT-PCR and Western blotting. RT-PCR data were normalized to b-actin and analyzed using the 22DDCt method. (c) Suppression of FasL expression reduces tumor development in vivo. Cells were subcutaneously inoculated into BALB/c mice. Mice were palpated thrice weekly to detect tumors. Data shown are the percentage of mice remaining tumor free on the indicated day. (d) Suppression of FasL expression reduces tumor growth in vivo. CT26scr shRNA or CT26FasL shRNA cells (2 3 105) were subcutaneously inoculated into BALB/c mice. ONO-8713 was adminis- tered in the basal diet with the day of initiation of treatment indicated. Tumor growth was monitored as described in the Material and Methods section. Data points represent the mean value 6 SEM and show the findings of one tumor challenge experiment. (e) Blocking EP1 signaling suppresses FasL expression in vivo. Tumors were excised after 48 days. FasL expression in the excised tumors was analyzed by RT-PCR and Western blotting. For Western blotting, 30 mg of protein from all tumors per treatment group was pooled. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Generation of FasLlow/negative and EP1low/negative colon cancer cells Cells were transfected with lentiviral particles containing three target-specific shRNAs against FasL (sc-35358-V), EP1 (sc40170-V) or control lentiviral particles containing scrambled shRNA (sc-108080; Santa Cruz Biotechnology, Santa Cruz, CA), according to the manufacturers’ instructions.

Techniques: Expressing, In Vivo, shRNA, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Blocking Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Betulinic Acid Suppresses UBE2T Expression via MAPK / ERK Inhibition to Block FANCI and FANCD2 Monoubiquitination in Glioblastoma

doi: 10.1111/jcmm.71000

Figure Lengend Snippet: Effects of BA on YY1/FAS, ERβ, insulin/IGF‐1, MAPK/ERK, and Notch signalling pathways regulating UBE2T expression in glioma cells. (A) WB assay show that BA suppresses the YY1/FAS axis, decreasing cleaved caspase‐8 and YY1 nuclear accumulation (red arrows); FASL co‐treatment failed to restore UBE2T (black arrows). (B) WB assay show that BA inhibits ERβ nuclear localization and ERK1/2 phosphorylation (red arrows); ERβ activation by DPN re‐stimulated p‐ERK1/2 and elevated UBE2T (red arrows). (C) WB assay show that the insulin/IGF‐1 pathway is unaffected by BA (black arrows), but IGF‐1 treatment enhances IGF‐1Rβ phosphorylation, increases p‐ERK1/2, and up‐regulates UBE2T (red arrows). (D) WB assay show that direct ERK activation (ERK agonist) increases UBE2T expression (red arrows), confirming ERK as a convergent effector. (E) BA does not alter Notch signalling, and DLL4‐Fc–induced Notch activation shows no impact on UBE2T. β‐Actin served as loading controls of total protein, and Lamin B1 for nuclear extracts. (F–H) The relative mRNA levels of UBE2T are detected by qPCR in U87MG (F), LN229 (G) and U251 glioma cell lines (H) additionally treated with agonists of the pathway mentioned above. “*” indicates significant difference compared with BA+CDDP group.

Article Snippet: Recombinant human FasL (MedChemExpress, Cat. No. HY‐P700520) was used at 100 ng/mL for 6 h [ ].

Techniques: Expressing, Phospho-proteomics, Activation Assay

Journal: World Journal of Gastroenterology

Article Title: Fas counterattack in cholangiocarcinoma: A mechanism for immune evasion in human hilar cholangiocarcinomas

doi: 10.3748/wjg.v7.i6.860

Figure Lengend Snippet: FasL positive in human hilar cholangiocarcinom as (brown). SABC × 200

Article Snippet: Detection of FasL expression and CD45 positive cells was performed using a rabbit polyclonal anti-human FasL specific IgG and a mouse anti-human monoclonal antibody on paraffin sections of human cholangiocarcinoma respectively (Boster Biological Technology Company, Wuhan, China).

Techniques:

Journal: World Journal of Gastroenterology

Article Title: Fas counterattack in cholangiocarcinoma: A mechanism for immune evasion in human hilar cholangiocarcinomas

doi: 10.3748/wjg.v7.i6.860

Figure Lengend Snippet: Expression of FasL in cholangiocarcinoma cell line. QBC939 × 200

Article Snippet: Detection of FasL expression and CD45 positive cells was performed using a rabbit polyclonal anti-human FasL specific IgG and a mouse anti-human monoclonal antibody on paraffin sections of human cholangiocarcinoma respectively (Boster Biological Technology Company, Wuhan, China).

Techniques: Expressing

Journal: World Journal of Gastroenterology

Article Title: Fas counterattack in cholangiocarcinoma: A mechanism for immune evasion in human hilar cholangiocarcinomas

doi: 10.3748/wjg.v7.i6.860

Figure Lengend Snippet: Expression of FasL mRNA in human cholangiocarcinoma cells QBC939. M: DL 2000 Marker; 1: FasL; 2: FasL+β-actin

Article Snippet: Detection of FasL expression and CD45 positive cells was performed using a rabbit polyclonal anti-human FasL specific IgG and a mouse anti-human monoclonal antibody on paraffin sections of human cholangiocarcinoma respectively (Boster Biological Technology Company, Wuhan, China).

Techniques: Expressing, Marker

Journal: World Journal of Gastroenterology

Article Title: Fas counterattack in cholangiocarcinoma: A mechanism for immune evasion in human hilar cholangiocarcinomas

doi: 10.3748/wjg.v7.i6.860

Figure Lengend Snippet: Western blotting of FasL protein with mAb from QBC939 cell cultures clone 33 from QBC939 cell cultures

Article Snippet: Detection of FasL expression and CD45 positive cells was performed using a rabbit polyclonal anti-human FasL specific IgG and a mouse anti-human monoclonal antibody on paraffin sections of human cholangiocarcinoma respectively (Boster Biological Technology Company, Wuhan, China).

Techniques: Western Blot

Journal: Mediators of Inflammation

Article Title: Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and Matrix Metalloproteinases

doi: 10.1155/2018/4310419

Figure Lengend Snippet: Neonatal monocytes display elevated pro-AREG surface expression and increased release of AREG in response to E. coli infection. PBMO and CBMO were incubated with E. coli for 1 h, extracellular bacteria were removed, and cells were cultivated for 24 h in total. (a) Immunocytological staining shows AREG protein expression (green) in monocytes. Texas Red-X phalloidin was applied to visualize F-actin in the cytoskeleton (red). (b) Pro-AREG surface expression in uninfected and E. coli -infected monocytes was quantified by using flow cytometry ( n = 5). Representative dot plots show gating strategy and cutoff value for AREG expression. (c) sAREG levels in the supernatant of monocytes were quantified by using ELISA ( n = 5). Data are shown as means + SD. Statistical analysis was performed using one-way ANOVA with Bonferroni's multiple comparisons test (ns: not significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.001).

Article Snippet: For detection of FasL, Human FasL PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, USA) was used according to the manufacturer's instructions.

Techniques: Expressing, Infection, Incubation, Bacteria, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and Matrix Metalloproteinases

doi: 10.1155/2018/4310419

Figure Lengend Snippet: AREG mediates increased shedding of memFasL through engagement with EGFR. PBMO and CBMO were infected as described in . AREG stimulation and EGFR inhibitor treatment was started 1 h prior to infection and was maintained during cultivation. (a) memFasL surface expression in uninfected and E. coli -infected monocytes was analyzed in response to AREG stimulation and neutralization of EGFR by using flow cytometry ( n = 5). AREG stimulation significantly decreased FasL levels in infected PBMO but not CBMO. Neutralization of EGFR resulted in a complete rescue of infection-induced memFasL presentation. Representative dot plots show gating strategy and cutoff value. (b) sFasL levels in the supernatant of uninfected and E. coli -infected monocytes were quantified in response to AREG stimulation and neutralization of EGFR by using ELISA ( n = 3). AREG significantly increased sFasL levels in infected PBMO but not CBMO, while neutralization of EGFR abolished the effect. Data are shown as means + SD. Statistical significance was analyzed using two-way ANOVA with Bonferroni's multiple comparisons test ( ∗∗ p < 0.01, ∗∗∗ p < 0.005, and ∗∗∗∗ p < 0.001).

Article Snippet: For detection of FasL, Human FasL PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, USA) was used according to the manufacturer's instructions.

Techniques: Infection, Expressing, Neutralization, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and Matrix Metalloproteinases

doi: 10.1155/2018/4310419

Figure Lengend Snippet: EGFR-dependent gelatinase activation mediates increased shedding of memFasL. PBMO and CBMO were infected as described in . Stimulation with AREG and gelatinase inhibition by CHX treatment was started 1 h prior to infection and was maintained during cultivation. (a) memFasL surface expression in uninfected and E. coli -infected monocytes was analyzed in response to AREG stimulation and gelatinase inhibition by using flow cytometry ( n = 4). Gelatinase inhibition prevented the decrease in memFasL presentation on monocytes triggered by AREG. (b) sFasL levels in the supernatant of uninfected and E. coli -infected monocytes were quantified in response to AREG stimulation and CHX treatment by using ELISA ( n = 3). Gelatinase inhibition was found to prevent the increase in sFasL levels triggered by AREG. Data are shown as means + SD. Statistical significance was analyzed using two-way ANOVA with Bonferroni's multiple comparisons test ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.001).

Article Snippet: For detection of FasL, Human FasL PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, USA) was used according to the manufacturer's instructions.

Techniques: Activation Assay, Infection, Inhibition, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay