Journal: Molecular Cancer Research

Article Title: The Ubiquitin-Specific Peptidase USP18 Promotes Lipolysis, Fatty Acid Oxidation, and Lung Cancer Growth

doi: 10.1158/1541-7786.mcr-20-0579

Figure Lengend Snippet: Figure 2. USP18 upregulated ATGL protein by removing the ISG15 conjugate from this substrate. A, Knockdown of USP18 by indi- vidual siRNAs was independently achieved in ED1 andA549 cancer cell lines and this led to decreased ATGL expression, with expression profiles quantified relative to actin expression and normalized to control (inactive) siRNAs independently trans- fected into these respective lung cancer cell lines. Real-time qPCR assays were done to validate siRNA knockdown of USP18. B, Gain of USP18 expression was achieved independently in murine ED1 lung cancer and KHC2 leiyomyosarcoma cell lines and this led to increased ATGL protein expres- sion. C, Gain of expression of the E1-like ubiquitin activating ligase, UBE1L, con- ferred reduced ATGL protein expression. Quantification was relative to actin expres- sion and normalized to the empty vector transfected into these respective lung can- cer cell lines. D, Immunoprecipitation assays confirmed that ISG15 and ATGL form conjugated protein. IB, immunoblot; IP, immunoprecipitation; NS, nonspecific. The quantification of immunoblots shows the average of three independent experiments. , P < 0.05; , P < 0.01. Representative immunoblots are displayed.

Article Snippet: Primary antibodies usedwere: ATGL (#2138; Cell Signaling Technology), USP18 (#4813; Cell Signaling Technology), b-actin (#3700; Cell Signaling Technology), HA tag (#3724S; Cell Signaling Technology), UBE1L (#61266; Cell Signaling Technology), Tubulin, UCP1 (#PA1-24894; Thermo Fisher Scientific), Myc-tag (#2276S; Cell Signaling Technology), and ISG15 (#2743; Cell Signaling Technology).

Techniques: Knockdown, Expressing, Control, Ubiquitin Proteomics, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot

Journal: Molecular Cancer Research

Article Title: The Ubiquitin-Specific Peptidase USP18 Promotes Lipolysis, Fatty Acid Oxidation, and Lung Cancer Growth

doi: 10.1158/1541-7786.mcr-20-0579

Figure Lengend Snippet: Figure 5. Combined USP18, ATGL, and UCP1 expression profiles in human lung can- cers were interrogated using TCGA database. A, Comparisons of USP18, ATGL, and UCP1 mRNA levels were between normal and malignant (AD, adenocarcinoma; SCC, squamous cell carcinoma) lung tissues using TCGA database. B, Kaplan–Meier analysis of survival in human lung adenocarcino- mas and squamous cell carcinomas is displayed. Cases with higher com- bined USP18, ATGL, and UCP1 expres- sion levels had an unfavorable survival as compared with those cases with lower USP18, ATGL, and UCP1 expres- sion profiles. TPM, transcripts per million.

Article Snippet: Primary antibodies usedwere: ATGL (#2138; Cell Signaling Technology), USP18 (#4813; Cell Signaling Technology), b-actin (#3700; Cell Signaling Technology), HA tag (#3724S; Cell Signaling Technology), UBE1L (#61266; Cell Signaling Technology), Tubulin, UCP1 (#PA1-24894; Thermo Fisher Scientific), Myc-tag (#2276S; Cell Signaling Technology), and ISG15 (#2743; Cell Signaling Technology).

Techniques: Expressing

Journal: Molecular Cancer Research

Article Title: The Ubiquitin-Specific Peptidase USP18 Promotes Lipolysis, Fatty Acid Oxidation, and Lung Cancer Growth

doi: 10.1158/1541-7786.mcr-20-0579

Figure Lengend Snippet: Figure 6. Combined USP18, ATGL, and UCP1 expression profiles independently determined in prostate cancers, kidney chromophobes, and low-grade gliomas. Comparisons of USP18, ATGL, and UCP1 mRNA levels were assessed between normal prostate and malignant prostate (A), normal kidney versus kidney chromophobe (B), and low- grade gliomas (C). Kaplan–Meier analysis of survival was determined in prostate cancer (D), kidney chromophobe (E), and low-grade glioma (F) cases comparing high USP18, ATGL, and UCP1 expression profiles versus all other cases. TPM, transcripts per million.

Article Snippet: Primary antibodies usedwere: ATGL (#2138; Cell Signaling Technology), USP18 (#4813; Cell Signaling Technology), b-actin (#3700; Cell Signaling Technology), HA tag (#3724S; Cell Signaling Technology), UBE1L (#61266; Cell Signaling Technology), Tubulin, UCP1 (#PA1-24894; Thermo Fisher Scientific), Myc-tag (#2276S; Cell Signaling Technology), and ISG15 (#2743; Cell Signaling Technology).

Techniques: Expressing

Journal: Arthritis Research & Therapy

Article Title: α2AP regulates vascular alteration by inhibiting VEGF signaling in systemic sclerosis: the roles of α2AP in vascular dysfunction in systemic sclerosis

doi: 10.1186/s13075-017-1227-y

Figure Lengend Snippet: Effect of α2AP on the VEGF signaling through ATGL/SHP2 axis in endothelial cells (ECs). a ECs were pretreated with 4 nM α2AP for 30 minutes and then stimulated with 100 pg/mL VEGF for the indicated periods. Phosphorylation of each protein was examined by western blot analysis. b ECs were stimulated with 4 nM α2AP for the indicated periods. Phosphorylation of SHP2 was examined by western blot analysis. c ECs were cultured for 30 minutes in the absence or presence of 4 nM α2AP or 100 μM NSC87877, and then stimulated with 100 pg/mL VEGF for 15 minutes. Phosphorylation of each protein was examined by western blot analysis. d ECs were transfected with control or ATGL siRNA. At 24 hours after transfection, the cells were stimulated with 4 nM α2AP for 5 minutes. The expression of each protein was examined by western blot analysis. e ECs were pretreated with 10 μM BEL for 30 minutes and then stimulated with 4 nM α2AP for 5 minutes. Phosphorylation of SHP2 was examined by western blot analysis. f ECs were pretreated with 4 nM α2AP or 10 μM BEL for 30 minutes and then stimulated with 100 pg/mL VEGF for 15 minutes. Phosphorylation of each protein was examined by western blot analysis. g The proposed mechanism of α2AP-attenuated vascular endothelial functions. VEGF induced Akt, ERK1/2, and p38 activation and led to pro-angiogenic effects, such as tube formation, cell proliferation, and endothelial junction-associated protein production in ECs. On the other hand, α2AP activated SHP2 through ATGL, and then α2AP-induced SHP2 activation inhibited VEGF signaling in ECs. α2AP inhibited VEGF signaling through the ATGL/SHP2 axis, and may cause impairment of vascular functions

Article Snippet: SSc dermal fibroblasts were transfected with ATGL siRNA (Santa Cruz Biotechnology, CA, USA) using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Western Blot, Cell Culture, Transfection, Control, Expressing, Activation Assay

Journal: Foods

Article Title: Sea Buckthorn Pericarp Flavonoids Improve Diet-Induced Hyperlipidemia via Coordinated Modulation of Hepatic Lipid Metabolism and Gut Microbiota

doi: 10.3390/foods15061049

Figure Lengend Snippet: TFSP modulates the expression of ACC, FAS, CPT-1α, PPARα and ATGL proteins in the livers of HFD mice. n = 6 per group. Data are presented as mean ± SD. Different letters above bars indicate statistically significant differences ( p < 0.05) by one-way ANOVA followed by LSD post hoc test.

Article Snippet: Peroxisome proliferator-activated receptor alpha (PPARα) primary antibody (Cat. No. A00600-2), carnitine palmitoyltransferase-1 alpha (CPT-1α) primary antibody (Cat. No. A00917-3), acetyl-CoA carboxylase (ACC) primary antibody (Cat. No. M01802-2), fatty acid synthase (FAS) primary antibody (Cat. No. BA0484), adipose triglyceride lipase (ATGL) primary antibody (Cat. No. A01800-1), and GAPDH primary antibody (Cat. No. BM1623) were obtained from BOSTER Biological Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: Primers Used for qRT-PCR

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: Generation of RPE-specific PNPLA 2-cKO mice. ( A ) Scheme of Pnpla 2 floxed and Cre-mediated recombined allele. The loxP sites flank exon 1. P1 and P2 are the primers homologous to sequences outside the floxed region (flanked by the loxP sites) used to detect Cre-mediated recombination (generating recombined alleles) on genomic DNA. The sizes of the amplicons obtained by PCR using P1 and P2 are indicated. ( B ) Gel electrophoresis of PCR reaction products obtained using primers P1 and P2 and genomic DNA isolated from mouse eyecups from either cKO or control (Ctr) mice ( Pnpla 2 f/+ ); lane 1 (MW) corresponds to molecular weight markers (GeneRuler DNA Ladder Mix). One eyecup per lane from a 4-month-old mouse ( n = 2 cKO, n = 2 Ctr). ( C ) Pnpla 2 expression (vs. HPRT ) in RPE from 1-month-old cKO mice ( Pnpla 2 f/f/Cre ) relative to control littermates ( Pnpla 2 f/f ). Each data point corresponds to the average of six PCR reactions per eyecup, six eyes from three cKO mice and six eyes from three control mice at 5 to 7 months old. ( D ) Cre ( red ) and phalloidin ( yellow ) labeling of RPE/choroid flatmounts from control ( Pnpla2 f/f ) ( left ) and littermate cKO (Pnpla2 f/f/Cre ) ( right ) mice ( n = 2 images from individual mouse eyecup at 11–14 months old). Scale bar : 20 µm. (E) Plot of percentage of Cre-positive RPE cells in cKO animals ( Pnpla2 f/f/Cre ; n = 10; age, 10.5–18.5 months old) as indicated in the x -axis. Each data point corresponds to percentage of Cre-positive RPE cells from an ROI, each bar corresponds to a flatmount of an individual cKO mouse, and the bar for control ( Pnpla2 f/f ) has data from 10 mice.

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Nucleic Acid Electrophoresis, Isolation, Control, Molecular Weight, Expressing, Labeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: Lipid accumulation in the RPE of Pnpla2 -cKO mice. Electron microscopy micrographs showing the RPE structure of 3-month-old ( A ) and 13-month-old ( B ) cKO mice and control animals. Scale bar : 2 µm. The representative images were selected among examinations of micrographs from eight eyes of cKO mice ( PNPLA2 f/f/Cre+ ), from seven eyes of PNPLA2 f/f control mice at 1.75 to 3.75 months old, and from three eyes of cKO mice and three eyes of control mice at 12.5 to 13 months old. LD, lipid droplets; BI, basal infoldings.

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Electron Microscopy, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: Phagocytosis and β-hydroxybutyrate production in the RPE of Pnpla2 -cKO mice. ( A ) Representative ROIs of the eyecup from one control and one cKO animal isolated at 2 hours (8 AM) and 5 hours (11 AM) after light onset (6 AM) after immunolabeling for rhodopsin ( green ), phalloidin ( yellow ), and Cre ( red ). The column to the right shows magnification of an area. The mean of rhodopsin immunolabel intensity in micrographs ( n ≥ 6 ROIs) from flatmounts (as indicated in the x -axis) relative to control at 2 hours was determined among three mice per condition and is shown in the plot. Age of mice was 10.5 to 18.5 months. ( B ) Ex vivo β-HB release by the RPE of Pnpla 2-cKO eyecups upon ingestion of OSs in comparison to that of controls. Eyecups were isolated at 5 hours (11 AM) and 8 hours (2 PM) after light onset (6 AM). Statistical significance was calculated using two-way ANOVA for the two groups (controls and cKO mice) with and without treatment (second variance) for each time after light onset. * P = 0.02, ** P = 0.006, *** P = 0.0001; ns, not significant; n = 6 eyecups from three control (f/+) mice at 3.5 months; n = 4 eyecups from two control (f/f/Cre–) mice at 3.5 months; n = 10 eyecups from five mice (f/f/Cre+) at 2.75 to 3.5 months. ( C ) The OS-mediated increase in β-HB release above base levels of the cKO RPE/choroid explants was calculated from the data in panel C and plotted.

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Control, Isolation, Immunolabeling, Ex Vivo, Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: RPE and retinal functionality in RPE- Pnpla2- cKO mice. ( A ) ERG amplitude graphs of scotopic a- and b-waves and photopic b-waves, as a function of light intensity ( x -axis) of 3- and 12-month-old cKO mice ( open circles ) and littermate controls ( Pnpla2 f/f , closed circles ) ( n = 3 per genotype). ( B ) Bar graph showing the amplitude (mean, SD) of the c-wave, fast oscillation (FO), light peak (LP), and off-response (OFF) measured by DC-ERG of 11-week-old cKO mice ( n = 4, open bars ) and Pnpla2 f/f and Pnpla2 f/+ control mice ( n = 5, closed bars ).

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: Knockdown of PNPLA2 in ARPE-19 cells. ARPE-19 cells were transfected with Scr or siRNAs targeting PNPLA2 , and mRNA levels and protein were tested. ( A ) RT-qPCR to measure PNPLA2 mRNA levels in ARPE-19 cells 72 hours after transfection with Scr and six different siRNAs (as indicated on the x -axis) was performed, and a plot is shown. PNPLA2 mRNA levels were normalized to 18S. All siRNA are represented as the percentage of the scrambled siRNA control ( n = 3). ( B ) A plot is shown for a time course of PNPLA2 mRNA levels following transfection with Scr and siPNPLA2-C ( n = 3 ( C ) RT-qPCR of mock-transfected cells, cells transfected with Scr, and siPNPLA2-C ( x -axis) at 72 hours after transfection. mRNA levels were normalized to the 18S RNA ( y -axis) ( n = 3). ( D ) Total protein was obtained from cells harvested 72 hours after transfection and resolved by SDS-PAGE followed by western blotting with anti-PNPLA2 and anti-GAPDH (loading control). The siRNAs used in the transfections are indicated at the top, and migration positions for PEDF-R and GAPDH are to the right of the blot. Data are presented as mean ± SD. ** P < 0.01, *** P < 0.001, *** P < 0.001.

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Control, SDS Page, Western Blot, Migration

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-Derived Factor Receptor (PEDF-R)

doi: 10.1167/iovs.62.2.30

Figure Lengend Snippet: Phagocytosis and fatty acid metabolism in si PNPLA2 cells. ARPE-19 cells were transfected with Scr or siRNAs targeting PNPLA2. At 72 hours after transfection, ARPE-19 cells were incubated with POSs (1 × 10 7 units/mL) in 24-well tissue culture plates for pulse–chase experiments. ( A ) Representative immunoblot of total lysates of ARPE-19 cells at 0.5 hour, 1 hour, and 2.5 hours of POS pulse and at 16-hour and 24-hour chase periods, as indicated at the top of the blot. Proteins in cell lysates were subjected to immunoblotting with anti-rhodopsin followed by reprobing with anti-GAPDH as the loading control. ( B ) Quantification of rhodopsin from duplicate samples and three blots of cell lysates from pulse–chase experiments and time periods (indicated in the x -axis) as from panel. Data are presented as mean ± S.D; ** P < 0.01; ns, not significant. Intensities of the immunoreactive bands were determined, and the percentages of the remaining rhodopsin after 16-hour and 24-hour chase periods relative to rhodopsin at 2.5-hour pulse are plotted ( y -axis). ( C , D ) Levels of secreted free fatty acids ( C ) and β-HB ( D ) were measured in culture media of cells transfected with Scr or siPNPLA2 following incubation with POS for the indicated periods of times ( x -axis). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01 ( n = 3). Duplex si PNPLA2 C was used to generate the data (see Table 3 for sequences of duplexes).

Article Snippet: Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Transfection, Incubation, Pulse Chase, Western Blot, Control

Journal: Nature Communications

Article Title: Lipid droplet availability affects neural stem/progenitor cell metabolism and proliferation

doi: 10.1038/s41467-021-27365-7

Figure Lengend Snippet: a Scheme illustrating the different manipulations. Carnitine palmitoyl transferase 1 (CPT1A), adipose triglyceride lipase (ATGL), fatty acid synthase (FASN). b Representative images of proliferating (pH3, red) NSPCs treated with Etomoxir. PLIN2 (white),DAPI (blue). Results were similar among 6 coverslips. c PLIN2 area covered, normalized to cell numbers, decreases with Etomoxir ( n = 6 coverslips shown as dots, from 3 independent experiments, bars indicate the mean value ± SEM, one-way ANOVA, Holm-Sidak correction, p -value < 0.0001 for 20 and 50 μM Etomoxir). d The total DAPI area covered significantly decreased with Etomoxir ( n = 6 coverslips shown as dots, from 3 independent experiments, bars indicate the mean value ± SEM, one-way ANOVA, Holm-Sidak correction, p -value = 0.066 for 20 μM and 0.0015 for 50 μM Etomoxir). e Representative images are maximum intensity projections of Ctrl -shRNA and Atgl -shRNA transfected proliferating NSPCs. DAPI (blue). Arrows indicate pH3+/RFP+cells. Results were similar among 6 coverslips per condition. f Atgl knockdown leads to a significant reduction in proliferating (pH3+) NSPCs compared to Ctrl -shRNA transfected NSPCs. ( n = 6 coverslips from 2 independent experiments, bars indicate the mean ± SEM, unpaired t -test, two-tailed, p -value = 0.0113). g Representative images of proliferating NSPCs treated with Atglistatin. PLIN2 (white), pH3 (green), DAPI (blue). h PLIN2 area covered, normalized to cell numbers, significantly increases with Atglistatin ( n = 9 coverslips shown as dots, from 3 independent experiments, bars indicate the mean ± SEM, one-way ANOVA, Holm-Sidak correction, p -value = 0.0018 for 5 μM and <0.0001 for 10 μM Atglistatin). i Proliferating pH3-positive NSPCs significantly decrease with Atglistatin ( n = 9 coverslips shown as dots, from 3 independent experiments, bars indicate the mean ± SEM, one-way ANOVA, Holm-Sidak correction, p -value = 0.0022 for 2 μM, 0.0059 for 5 μM and 0.0094 for 10 μM Atglistatin). j PLIN2 (white) is reduced in Fasn KO NSPCs (positive for Cre-GFP, green) compared to Fasn Ctrl NSPCs. Representative images are maximum intensity projections. DAPI-positive nuclei (blue). k LD numbers and total LD volume significantly decrease in Fasn KO NSPCs ( n = 3 coverslips, from 3 independent experiments with a total of 68 Fasn KO and 50 Fasn Ctrl cells analyzed, bars indicate the mean ± SEM, one-sample t -test on log2-transformed FC-values, two-tailed, p -value = 0.0972 and 0.026). Asterisks indicate the following p -value: + = 0.097. * < 0.05. ** < 0.01. *** < 0.001.

Article Snippet: Plasmids encoding for shRNAs against Atgl or control shRNA were bought from Origene (TF512624).

Techniques: shRNA, Transfection, Knockdown, Two Tailed Test, Transformation Assay