Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: CD36 is overexpressed in human CRC. (A,B) Immunoreactivity score of CD36 expression was analyzed in matched normal colon mucosa and tumor tissues from patients diagnosed with Stage I–IV CRC (TMA: n = 56, * p < 0.001 vs. normal tissue). (C) CD36 staining in matched normal colon mucosa, primary CRC, and CRC metastasis to liver and lung [representative images are shown; liver ( n = 12) and lung metastasis ( n = 5)]. (D) Correlations between FASN and CD36 was determined based on RNASeq data of CRC patient samples ( n = 22 of normal tissues and n = 215 of tumors) from The Cancer Genome Atlas. (E) Expression of FASN and CD36 in human normal colon mucosa and tumor tissues. N, normal mucosa; T, primary tumor; L, normal liver tissue; M, liver metastasis.

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Expressing, Staining

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: Expression of CD36 is selectively regulated by the level of de novo fatty acid synthesis in CRC. (A) shRNA-mediated knockdown of FASN leads to upregulation of CD36 mRNA expression in HCT116 cells (* p < 0.05). (B) TVB-3664 treatment of CRC tissue slices (18 h) selectively upregulates CD36 mRNA expression (* p < 0.05). (C) TVB-3664 treatment of Pt 93 and Pt 130 primary CRC cells increases CD36 mRNA and protein expression. (D) shRNA mediated knockdown of FASN increases CD36 protein expression in HCT116 and HT29 cells. (E) Relative mRNA expression of FASN and CD36 in intestinal tumors collected from APC/Cre and FASN +/− /APC/Cre mice. (F) FASN and CD36 protein expression in intestinal mucosa collected from Apc/Cre and Apc/Cre mice with hetero- and homo-zygous deletion of FASN. (G,H) Inhibition of FASN increases membrane-associated expression of CD36. (G) Confocal images of FASN and CD36 in control and 0.2 μM TVB-3664 treated (6 days) Pt 93 primary CRC cells. (H) Flow cytometry analysis of Pt 93 and Pt 130 primary CRC cells treated with 0.2 μM TVB-3664 (6 days) in normal and serum free media conditions. Mean fluorescence for CD36 is shown for representative data from three different experiments (** p < 0.01, * p < 0.05). (I) FA uptake in HCT116, NTC, and FASN shRNA. Cells were pre-treated with anti-CD36 antibody or vehicle for 24 h and then treated with BODIPY FL for 10 min.

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Expressing, shRNA, Knockdown, Inhibition, Membrane, Control, Flow Cytometry, Fluorescence

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: Inhibition of CD36 is associated with decreased cellular proliferation. (A) Primary Pt 93 and Pt 130 CRC cells treated with 100 μM SSO for 6 days. Cellular proliferation assays were performed via cell count. Representative data from three experiments is shown (* p < 0.05). (B) Confocal images of FASN and CD36 in Pt 93 cells in normal and serum free media (6 days). (C) Expression of proteins associated with apoptosis and survival in HCT116 transfected with CD36 shRNAs and analyzed via western blot. (D) Cellular proliferation assay with HCT116, NTC, and CD36 shRNA (* p < 0.05 for normal medium, # p < for SFM). (E) qRT-PCR confirmation of CD36 knockdown using CD36 shRNA #2 (73%) and CD36 shRNA #4 (67%).

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Inhibition, Cell Counting, Expressing, Transfection, Western Blot, Proliferation Assay, shRNA, Quantitative RT-PCR, Knockdown

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: SSO treatment and CD36 knockdown inhibit tumor growth in vivo . (A) Tumor volume, tumor weight, and mouse weight of control and SSO treated (20 mg/kg) mice are shown. SSO was dissolved in 10% PEG and administered in 200 μl dosages via oral gavage daily. 1.0 × 10 6 cells were injected into NU/NU mice. Treatment was initiated when tumors reached ~100 mm 3 (day 0). (B) RT-PCR analysis of HCT116 tumors showing the effect of SSO treatment on CD36, FASN, and survivin mRNA expression. (C) Tumor volume of HCT116 NTC and CD36 shRNA #2 and #4 xenografts is shown. 1.0 × 10 6 cells were injected into NU/NU mice and tumor growth was measured every 3 days. (D) H&E and Ki67 staining of HCT116 NTC and CD36 shRNA tumors. (E) Tumor volume and tumor weight of HT29 LuM3 NTC and CD36 shRNA #4 xenografts are shown. (F) mRNA expression of survivin in HT29 LuM3 xenografts (analysis of tumors from 2 mice per group is show).

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Knockdown, In Vivo, Control, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, shRNA, Staining

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: High expression of CD36 is associated with an increase in pAkt and survivin in CRC. (A) Diagram of Pt 2402 propagation after flow cytometry sorting for CD36 high and CD36 low cells. (B) Numbers of CD36 high and CD36 low Pt 2402 cells for first and second flow cytometry sorts. (C) Protein expression levels of FASN, CD36, and survivin in CD36 high and CD36 low Pt 2402 primary cells from first flow cytometry sort. (D) IHC staining for Ki67 in Pt 2402 CD36 high and CD36 low tumors. (E) Protein expression levels of FASN, CD36, pAkt, cleaved PARP, and survivin in HCT116 CRC cells, control, and CD36 overexpression.

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Expressing, Flow Cytometry, Immunohistochemistry, Control, Over Expression

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: Inhibition of CD36 and FASN have a synergetic effect in reducing cell proliferation. (A) Pt 93, Pt 130, and HCT116 cells were treated with SSO and TVB-3664 alone or in combination for 6 days and cell number was counted. Representative data from three experiments is shown (* p < 0.05). (B) Western blot analysis of cells treated with TVB-3664, SSO, or TVB-3664 and SSO in combination.

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Inhibition, Western Blot

Journal: Cancers

Article Title: Fatty Acid Synthase as a Potential Metabolic Vulnerability in Ocular Adnexal Sebaceous Carcinoma

doi: 10.3390/cancers18020349

Figure Lengend Snippet: FASN-mediated viability, clonogenicity, and proliferation in vitro. ( A ) HMGECs (black) were the most sensitive to C75 treatment across all concentrations evaluated, while SebCA01 (blue) was the most resistant to changes in viability resulting from incubation with C75. * p < 0.05, ** p < 0.01,*** p < 0.001, **** p ≤ 0.0001. ( B ) The number of anchorage-dependent colonies formed was reduced following a 14 d incubation with C75. ( C ) Quantification of stained colonies demonstrated the most significant reduction in clonogenicity in SebCA03 in response to C75 treatment. * p < 0.05, ** p < 0.01, **** p ≤ 0.0001. ( D ) Fewer EdU-positive nuclei (yellow green, merged) in FASN-inhibited cells were observed relative to DMSO control. EdU: Alexa Fluor 488, green. Hoechst: blue. Scale bar: 25 µm for all panels. ( E ) Quantification of EdU-positive nuclei demonstrated a significant reduction in mean proliferation rate in C75-treated cells relative to DMSO only in HMGECs. * p < 0.05.

Article Snippet: Expression of MYC , FASN , and PLN2 was quantified using 2 −ΔΔcT normalized to polR2α using predesigned probes: Hs00153408_m1 (human MYC ), Mm00487804_m1 (murine MYC ), Hs01005622_m1 (human FASN ), Mm00662319_m1 (murine FASN ), Hs00605340_m1 (human PLIN2 ), Mm00475794_m1 (murine PLIN2 ), Hs00172187_m1 (human polR2α ), and Mm01309448_m1 (murine polR2α ) (ThermoFisher).

Techniques: In Vitro, Incubation, Staining, Control

Journal: Cancers

Article Title: Fatty Acid Synthase as a Potential Metabolic Vulnerability in Ocular Adnexal Sebaceous Carcinoma

doi: 10.3390/cancers18020349

Figure Lengend Snippet: Differential protein and transcript expression following FASN inhibition in vitro. ( A ) Cytoplasmic FASN expression (Alexa Fluor 555, red) was reduced in C75-treated cells relative to vehicle control (DMSO) across all cell lines. Phalloidin: Alexa Fluor 488, green. DAPI: blue. Scale bar: 10 µm for all panels. ( B ) Semi-quantitative evaluation of FASN expression by H-score determination revealed significant downregulation in response to C75 treatment in all cell lines. **** p ≤ 0.0001. ( C ) Relative FASN expression was significantly upregulated in all cell lines following C75 treatment. * p < 0.05, ** p < 0.01, *** p < 0.001. ( D ) Assessment of c-MYC protein concentration by ELISA showed significant decreases in MYC concentration in all SebCA lines treated with C75 relative to vehicle control. * p < 0.05, ** p < 0.01, *** p < 0.001. ( E ) Relative expression of MYC was significantly induced in response to C75 treatment in SebCA02 and SebCA03. ** p < 0.01. 2 −ΔΔcT was utilized to normalize target transcript expression to polR2α.

Article Snippet: Expression of MYC , FASN , and PLN2 was quantified using 2 −ΔΔcT normalized to polR2α using predesigned probes: Hs00153408_m1 (human MYC ), Mm00487804_m1 (murine MYC ), Hs01005622_m1 (human FASN ), Mm00662319_m1 (murine FASN ), Hs00605340_m1 (human PLIN2 ), Mm00475794_m1 (murine PLIN2 ), Hs00172187_m1 (human polR2α ), and Mm01309448_m1 (murine polR2α ) (ThermoFisher).

Techniques: Expressing, Inhibition, In Vitro, Control, Protein Concentration, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Cancers

Article Title: Fatty Acid Synthase as a Potential Metabolic Vulnerability in Ocular Adnexal Sebaceous Carcinoma

doi: 10.3390/cancers18020349

Figure Lengend Snippet: Lipidomic alterations secondary to FASN inhibition in vitro. ( A ) The relative abundance of each lipid class across cell lines in response to incubation with an FASN inhibitor. ( B ) The relative abundance of saturated (SFAs) and unsaturated fatty acids (UFAs) from each lipid class across treatments in each cell line. TG: triacylglycerol; PC: phosphatidylcholine; PE: phosphatidylethanolamine; LPC and LPE: lysophosphatidylcholine and lysophosphatidylethanolamine; CE: cholesterol ester; SM: sphingomyelin; Cer: ceramide.

Article Snippet: Expression of MYC , FASN , and PLN2 was quantified using 2 −ΔΔcT normalized to polR2α using predesigned probes: Hs00153408_m1 (human MYC ), Mm00487804_m1 (murine MYC ), Hs01005622_m1 (human FASN ), Mm00662319_m1 (murine FASN ), Hs00605340_m1 (human PLIN2 ), Mm00475794_m1 (murine PLIN2 ), Hs00172187_m1 (human polR2α ), and Mm01309448_m1 (murine polR2α ) (ThermoFisher).

Techniques: Inhibition, In Vitro, Incubation

Journal: Cancers

Article Title: Fatty Acid Synthase as a Potential Metabolic Vulnerability in Ocular Adnexal Sebaceous Carcinoma

doi: 10.3390/cancers18020349

Figure Lengend Snippet: Histopathological and morphometric alterations resulting from MYC and FASN modulation in vivo. ( A ) Representative H&E-stained FFPE sections of murine tissues. Conditionally MYC -overexpressing transgenic (TG) Meibomian glands exhibited basal meibocyte hyperplasia which was reduced following topical FASN inhibition. The cytoplasmic volume was reduced in both TG and wildtype (wt) mice in response to topical administration of C75. Scale bar: 50 µm. ( B ) The mean cross-sectional area C75-treated meibocytes was significantly reduced relative to vehicle control. * p < 0.05, **** p ≤ 0.0001.

Article Snippet: Expression of MYC , FASN , and PLN2 was quantified using 2 −ΔΔcT normalized to polR2α using predesigned probes: Hs00153408_m1 (human MYC ), Mm00487804_m1 (murine MYC ), Hs01005622_m1 (human FASN ), Mm00662319_m1 (murine FASN ), Hs00605340_m1 (human PLIN2 ), Mm00475794_m1 (murine PLIN2 ), Hs00172187_m1 (human polR2α ), and Mm01309448_m1 (murine polR2α ) (ThermoFisher).

Techniques: In Vivo, Staining, Transgenic Assay, Inhibition, Control

Journal: Cancers

Article Title: Fatty Acid Synthase as a Potential Metabolic Vulnerability in Ocular Adnexal Sebaceous Carcinoma

doi: 10.3390/cancers18020349

Figure Lengend Snippet: MYC modulatory, differentiative and apoptotic effects of FASN inhibition in vivo. ( A ) Representative images of chromogenic and fluorescently immunolabeled FFPE sections of murine tissues. Vehicle-treated MYC -overexpressing transgenic (TG) Meibomian glands exhibited pronounced upregulation of MYC (DAB: brown) relative to wildtype (wt) littermates and compared to contralateral C75-treated eyelids. Scale bar: 50 µm. ( B ) Adipophilin (DAB: brown) expression was diminished in TG mice relative to wt littermates and in C75-treated eyelids relative to vehicle controls. Scale bar: 50 µm. ( C ) MYC -overexpressing TG glands exhibited upregulated FASN expression (Alexa Fluor 555, red) relative to wt littermates and when compared to C75-treated eyelids. C75-treated eyelids exhibited increased distribution of apoptotic cells (TUNEL: Alexa Fluor 488, green) when compared to vehicle controls. DAPI: blue. Scale bar: 50 µm. ( D ) FASN immunolabeling was assessed by H scoring, demonstrating significant downregulation in response to C75 treatment in both wt and TG mice and in wt when compared to TG littermates. **** p ≤ 0.0001. ( E ) Quantification of TUNEL-positive meibocytes demonstrated a significant increase in mean apoptotic rates in C75-treated eyelids relative to contralateral controls and in wt mice relative to TG. * p < 0.05; **** p ≤ 0.0001.

Article Snippet: Expression of MYC , FASN , and PLN2 was quantified using 2 −ΔΔcT normalized to polR2α using predesigned probes: Hs00153408_m1 (human MYC ), Mm00487804_m1 (murine MYC ), Hs01005622_m1 (human FASN ), Mm00662319_m1 (murine FASN ), Hs00605340_m1 (human PLIN2 ), Mm00475794_m1 (murine PLIN2 ), Hs00172187_m1 (human polR2α ), and Mm01309448_m1 (murine polR2α ) (ThermoFisher).

Techniques: Inhibition, In Vivo, Immunolabeling, Transgenic Assay, Expressing, TUNEL Assay

Journal: Cancers

Article Title: Fatty Acid Synthase as a Potential Metabolic Vulnerability in Ocular Adnexal Sebaceous Carcinoma

doi: 10.3390/cancers18020349

Figure Lengend Snippet: MYC and FASN-mediated differential transcript expression in vivo. ( A ) Relative MYC and ( B ) FASN expression were significantly upregulated in vehicle-treated TG mice relative to other groups. ( C ) Relative PLIN2 expression was significantly downregulated in TG mice relative to wt controls and in C75-treated eyelids relative to contralateral vehicle treatment. ** p < 0.01, *** p < 0.001, **** p ≤ 0.0001. 2 −ΔΔcT was utilized to normalize target transcript expression to polR2α.

Article Snippet: Expression of MYC , FASN , and PLN2 was quantified using 2 −ΔΔcT normalized to polR2α using predesigned probes: Hs00153408_m1 (human MYC ), Mm00487804_m1 (murine MYC ), Hs01005622_m1 (human FASN ), Mm00662319_m1 (murine FASN ), Hs00605340_m1 (human PLIN2 ), Mm00475794_m1 (murine PLIN2 ), Hs00172187_m1 (human polR2α ), and Mm01309448_m1 (murine polR2α ) (ThermoFisher).

Techniques: Expressing, In Vivo

Journal: Cancers

Article Title: The lncRNA H19-Derived MicroRNA-675 Promotes Liver Necroptosis by Targeting FADD

doi: 10.3390/cancers13030411

Figure Lengend Snippet: Negative miR-675/FADD (Fas-Associated Protein with Death Domain) correlation in liver samples from human and Mdr2-KO mice. ( A ) qRT-PCR analysis of FADD performed on RNA extracted from human non-NAFLD liver (NNL) and human cirrhotic liver (cirrhosis). ( B – F ) miR-675/FADD correlation in ( B ) in human cirrhotic liver samples; ( C ) combined human samples taken from normal and cirrhotic adult liver, fetal liver, and non-tumor and tumor samples of hepatic adenoma and HCC; ( D ) human HCC samples only; ( E ) RNA samples extracted from tumor and non-tumor liver tissues of Mdr2-KO mice (as in E); and ( F ) combined Mdr2-KO mice liver RNA samples (as in F). RNA was isolated and analyzed by qRT-PCR. FADD levels were normalized to HPRT, and miR-675 levels were normalized to RNU48. Expression correlation was calculated using Spearman’s correlation coefficient.

Article Snippet: All experiments were performed in triplicate. qRT-PCR of RNA isolated from human samples was performed using the following specific TaqMan (TAqMan, Invitrogen, CA, USA) predesigned probes: Hs00399294_g1 ( H19 ),Hs04187499_m1 (FADD), and hsa-miR-675-002005 (miR-675).

Techniques: Quantitative RT-PCR, Isolation, Expressing

Journal: Cancers

Article Title: The lncRNA H19-Derived MicroRNA-675 Promotes Liver Necroptosis by Targeting FADD

doi: 10.3390/cancers13030411

Figure Lengend Snippet: Regulation of FADD by miR-675 in vitro and in vivo. ( A ) The effect of miR-675 on target gene expression in Hep3B cells. qRT-PCR on RNA extracted from Hep3B cells 48 h after transfection with miR-675 or controls (mutated miR-675 and siLuc) (50 nM). ( B , C ) Hypoxia upregulates H19 and miR-675 and downregulates FADD expression in vitro. FLC4 cells were incubated under hypoxic conditions (0.1% O 2 ) for the indicated times. At each time point, RNA and proteins were extracted. ( B ) qRT-PCR for H19 , miR-675, and FADD ( n = 3). ( C ) Western blot analysis of FADD protein expression (top, scanned images of blot; bottom, densitometric quantification). ( D ) miR-675 overexpression downregulates FADD in mice. Balb/C mice were tail-vein-injected with miR-675 (5 μg) or control (siCtrl) and liver RNA extracted 24 h later ( n = 3). qRT-PCR was performed for FADD, RUNX1 (positive control), and TLR2 (negative control). mRNA levels were normalized to HPRT, and miR-675 levels were normalized to RNU-6. Error bars = SD. * p < 0.05; ** p < 0.01. RAD1 = a DNA damage repair complex protein; TBK1 = TANK Binding Kinase 1; RUNX1 = Runt-Related Transcription Factor 1; TLR2 = Toll Like Receptor 2.

Article Snippet: All experiments were performed in triplicate. qRT-PCR of RNA isolated from human samples was performed using the following specific TaqMan (TAqMan, Invitrogen, CA, USA) predesigned probes: Hs00399294_g1 ( H19 ),Hs04187499_m1 (FADD), and hsa-miR-675-002005 (miR-675).

Techniques: In Vitro, In Vivo, Targeted Gene Expression, Quantitative RT-PCR, Transfection, Expressing, Incubation, Western Blot, Over Expression, Injection, Control, Positive Control, Negative Control, Binding Assay

Journal: Cancers

Article Title: The lncRNA H19-Derived MicroRNA-675 Promotes Liver Necroptosis by Targeting FADD

doi: 10.3390/cancers13030411

Figure Lengend Snippet: The effect of miR-675 on the inflammatory response in mice. Balb/C mice were tail-vein-injected with miR-675 or control miR (siCtrl) (5 µg), and 24 h later, the mice were injected intraperitoneal (IP) with LPS (25 µg) or saline. Liver RNA and proteins were extracted 24 h after IP injections ( n = 5). ( A ) qRT-PCR performed for FADD RNA. ( B ) Fold induction of FADD RNA calculated by dividing the qRT-PCR result for LPS with the result for saline. ( C ) Western blot analysis of FADD protein expression following saline or LPS treatment (top, scanned images of blot; bottom, densitometric quantification). ( D ) miR-675 promotes macrophage infiltration to the liver. Representative images of f4/80 immunohistochemical macrophage staining of liver sections (magnification ×20). ( E ) qRT-PCR analysis of liver RNA for Tumour Necrosis Factor Alpha (TNFα), Interferon Gamma (IFNγ), and Regulated on Activation, Normal T cell Expressed and Secreted (RANTES). mRNA levels were normalized to HPRT. Error bars = SD. * p < 0.05; ** p < 0.01.

Article Snippet: All experiments were performed in triplicate. qRT-PCR of RNA isolated from human samples was performed using the following specific TaqMan (TAqMan, Invitrogen, CA, USA) predesigned probes: Hs00399294_g1 ( H19 ),Hs04187499_m1 (FADD), and hsa-miR-675-002005 (miR-675).

Techniques: Injection, Control, Saline, Quantitative RT-PCR, Western Blot, Expressing, Immunohistochemical staining, Staining, Activation Assay

Journal: Cancers

Article Title: The lncRNA H19-Derived MicroRNA-675 Promotes Liver Necroptosis by Targeting FADD

doi: 10.3390/cancers13030411

Figure Lengend Snippet: Our schematic model for induction of necroptosis by miR-675. Necroptosis occurs following the rupture of the cell membrane, which involves phosphorylated MLKL molecules. Phosporylation of MLKL requires prior assembly of the necrosome, which contains RIP1 and RIP3 molecules. Caspase-8, which is activated by FADD, induces apoptosis and inhibits necrosome formation due to cleavage of RIP1 and RIP3. Administration of miR-675, which targets the FADD gene, leads to inhibition of caspase-8 activation, thus enabling the assembly of the necrosome, resulting in necroptosis. TNFα = Tumour Necrosis Factor Alpha; RIP = kinase receptor-interacting proteins; MLKL = Mixed Lineage Kinase Domain-Like Pseudokinase.

Article Snippet: All experiments were performed in triplicate. qRT-PCR of RNA isolated from human samples was performed using the following specific TaqMan (TAqMan, Invitrogen, CA, USA) predesigned probes: Hs00399294_g1 ( H19 ),Hs04187499_m1 (FADD), and hsa-miR-675-002005 (miR-675).

Techniques: Membrane, Inhibition, Activation Assay

Journal: Mediators of Inflammation

Article Title: Apoptosis through Death Receptors in Temporal Lobe Epilepsy-Associated Hippocampal Sclerosis

doi: 10.1155/2016/8290562

Figure Lengend Snippet: Array target genes: apoptosis through death receptors in TLE(HS).

Article Snippet: FASLG Fas ligand Hs00181225_m1 , CD178, FasL , Cytokine ligand for FAS , 5.46.

Techniques: Activation Assay, Inhibition, Over Expression, Transduction, Binding Assay

Journal: The American Journal of Pathology

Article Title: Mechanisms of Retinal Damage after Ocular Alkali Burns

doi: 10.1016/j.ajpath.2017.02.005

Figure Lengend Snippet: Ocular protection with anti–TNF-α treatment. A–C: Ocular tissue sections (10 μm thick) of mice burned with 1 mol/L sodium hydroxide followed by copious irrigation with saline solution for 15 minutes. A: TNF-α expression (green) is elevated in all intraocular tissues 24 hours after the burn, with diffuse anterior uveal expression persisting up to 7 days after the burn. B: Conversely, anti–TNF-α–treated mice (infliximab, 6.25 mg/kg), administered immediately after irrigation, exhibit reduced TNF-α (green) expression in all ocular tissues. C: No TNF-α expression is present in the eyes of naive control mice. D and E: Quantification of TNF-α fluorescence shows up-regulation in the cornea and retina 24 hours after the burn, which is sustained for 45 days and then gradually decreases. TNF-α expression is undetectable in the corneal and retina of anti–TNF-α–treated mice 45 days after the burn. Anti–TNF-α treatment inhibits central (F) and peripheral (G) retinal cell apoptosis, as shown using TUNEL assay (red) 24 hours after corneal burn. H: Systemic administration of 6.25 mg/kg IgG isotype control does not inhibit retinal cell apoptosis 24 hours after corneal burn. I: Untreated eyes have elevated TUNEL expression in the retina 24 hours after corneal burn. J: Naive control tissue has no TUNEL signal. K: Ten-minute DNase treatment of naïve tissue causes TUNEL-positive expression in cells. L: Corneal alkali burn causes significant apoptosis in the retinal ganglion cell layer (GCL), inner nuclear layer (INL)/inner plexiform cell layer (IPL), and outer nuclear layer (ONL)/outer plexiform cell layer (OPL) 24 hours after the burn. Prompt TNF-α inhibition with anti–TNF-α antibody significantly reduces the relative TUNEL expression in all retinal layers (P < 0.0001). Anti–TNF-α treatment suppresses TNF-α, TNF-R1, TNF-R2, IL-1β, Fas, and MMP9 expression in the iris and ciliary body (M) and reduces TNF-α and IL-1β expression in the retina (N). Anti–TNF-α therapy causes a moderate (nonsignificant) increase in TNF-R1, TNF-R2, Fas, and FasL expression in the retina but significantly suppresses IL-1β expression in the retina. Data are expressed as means ± SD (D, E, L–N). n = 10 (D and E). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001 compared to untreated eyes. Scale bar = 50 μm (F–K).

Article Snippet: TaqMan primers were used to assess the expression levels of the following genes: TNFA (Mm99999068_m1; Life Technologies, Woburn, MA), TNFR1 (Mm01182929_m1; Life Technologies), TNFR2 (Mm00441889_m1; Life Technologies), FAS (Mm01204974_m1; Life Technologies), FASLG (Mm00438864_m1; Life Technologies), IL1B (MmMm00434228_m; Life Technologies), INFG (Mm01168134_m1; Life Technologies), and MMP9 (Mm00442991_m1; Life Technologies).

Techniques: Saline, Expressing, Control, Fluorescence, TUNEL Assay, Inhibition

Journal: Mediators of Inflammation

Article Title: Apoptosis through Death Receptors in Temporal Lobe Epilepsy-Associated Hippocampal Sclerosis

doi: 10.1155/2016/8290562

Figure Lengend Snippet: Array target genes: apoptosis through death receptors in TLE(HS).

Article Snippet: FAS Fas cell surface death receptor Hs00531110_m1 , APO-1, CD95, and “TNF receptor superfamily member 6” , Receptor with death domain for FASLG , 1.65.

Techniques: Activation Assay, Inhibition, Over Expression, Transduction, Binding Assay