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: siRNA Duplex Sequences

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: Sequencing

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: Nature communications

Article Title: Grasp55 -/- mice display impaired fat absorption and resistance to high-fat diet-induced obesity.

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: Fig. 5 GRASP55 participates in the Golgi localization and LD targeting of ATGL and MGL. a Subcellular localization of ATGL and MGL in the intestinal epithelial cells of Grasp55+/+ and Grasp55−/−mice was determined using LD floating ultracentrifugation (for Fraction 1), followed by subcellular fractionation assay (for Fractions 2–9; using pooled jejunum tissue from four male mice 4 h after olive oil bolus [olive oil, 10 μl g−1 of body weight]) as described in Methods. RAB18/ADRP, Aldolase A, Giantin, ERGIC53, and calreticulin were used as organelle markers of the LDs, cytosol, Golgi, the ER-Golgi intermediate compartment (ERGIC), and the ER, respectively. Immunoblotting results with a full list of proteins involved in lipid metabolism are shown in the Supplementary Fig. 8. The Golgi localizations of ATGL and MGL were reduced by GRASP55 deficiency in mouse intestinal cells (red boxes). Three independent experiments showed similar results. b, c The protein expressions of ATGL, phospho-HSL (pHSL), HSL, MTP, and ADRP were analyzed by immunoblotting. Jejunum tissues were prepared from Grasp55+/+ and Grasp55−/−mice fasted for 16 or 4 h after olive oil bolus (olive oil, 10 μl g−1 body weight). Representative immunoblots are shown in (b). Densitometric analysis of ATGL are shown in (c, n = 5). A summary of pHSL, HSL, MTP, and ADRP is shown in Supplementary Fig. 11a–d. The level of β-actin was monitored as a cytosolic protein loading control. d, e Measurements of ATGL in the lipid droplet (LD) fraction of intestinal epithelial cells (using pooled jejunum tissue from four male mice 4 h after olive oil bolus [olive oil, 10 μl g−1 of body weight]). Representative immunoblots are shown in (d) and a summary of multiple experiments is shown in (e, n = 3). TM, total membrane. f, g Intracellular localization of ATGL and ADRP, a marker protein of LD, in jejunal epithelia of mice 4 h after olive oil bolus. Representative images are shown in (f) and quantitative analyses of colocalization between ATGL and ADRP are presented in (g, n = 5). MCC, Manders’ colocalization coefficient. Unprocessed blots can be found in Supplementary Fig. 22. Data are shown as mean ± SEM. Scale bars: 20 μm. **p < 0.01. P values were calculated by unpaired (c, g) or paired (e) two-tailed Student’s t tests. Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55-specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone (GRASP55: #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Fractionation, Western Blot, Control, Membrane, Marker, Two Tailed Test

Journal: Nature communications

Article Title: Grasp55 -/- mice display impaired fat absorption and resistance to high-fat diet-induced obesity.

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: Fig. 6 GRASP55 depletion reduces LD targeting of ATGL and transepithelial lipid absorption in Caco-2 cells. a, b Measurements of ATGL in the lipid droplet (LD) fraction of Caco-2 cells after treatment with 400 μM oleic acid for 16 h. Representative immunoblots are shown in (a) and a summary of multiple experiments is shown in (b, n = 3). c Presence of ATGL on the LD surface was examined by co-staining with anti-ATGL antibodies and BODIPY 493/503 (an LD marker) in Caco-2 cells, in which endogenous GRASP55 was depleted (shGRASP55, shG55) and/or exogenously supplemented (GRASP55- Myc, G55-Myc). Cells were treated with 400 μM oleic acid for 4 h before immunostaining. Arrows indicate the location of ATGL, which is targeted to the peripheral regions of LDs. Four independent experiments showed similar results. d, e ATGL protein stability was examined in Caco-2 cells, in which GRASP55 was depleted (shGRASP55, open arrowhead) and/or exogenously supplemented (GRASP55-Myc, filled arrowhead). Cytosolic proteins were collected 0, 2, and 4 h after treatment with cycloheximide (0.1 mg ml−1), an inhibitor of protein biosynthesis. Aldolase A was used as a cytosolic protein loading control. Representative immunoblot is shown in (d), and the results of multiple experiments are summarized in (e, n = 3). f Immunoblot results of Caco-2 monolayers, in which GRASP55 was depleted (shGRASP55) and exogenous ATGL (ATGL-Myc) was supplemented. Levels of endogenous ATGL (open arrowhead) and exogenous ATGL (filled arrowhead) are shown. A high level of ATGL-Myc supplementation restored LD-associated ATGL levels in GRASP55-depleted cells, although the LD transport efficiency was much lower (compare endogenous and exogenous ATGL levels in lanes 3 and 4). g Transepithelial lipid transport of Caco-2 monolayers were determined using 14C-labeled oleic acid. The basolateral (B) to apical (A) transport of oleic acid (left, B to A) was much smaller than that of apical to basolateral transport (middle, A to B), and it was not affected by GRASP55 depletion or ATGL overexpression. Transepithelial lipid absorption was determined by subtracting values of (B to A) from those of (A to B) in each paired experiment (right, n = 4). ATGL overexpression rescued lipid absorption in GRASP55-deficient Caco-2 Cells. Unprocessed blots are presented in Supplementary Fig. 22. CPM, counts per min. TM, total membrane. Data are shown as mean ± SEM. Scale bars: 10 μm. n.s.: not significant, *p < 0.05. P values were caluculated by paired (b), unpaired (e) two-tailed Student’s t tests or ANOVA followed by Tukey’s multiple comparison tests (g). Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55-specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone (GRASP55: #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Western Blot, Staining, Marker, Immunostaining, Control, Labeling, Over Expression, Membrane, Two Tailed Test, Comparison

Journal: Nature communications

Article Title: Grasp55 -/- mice display impaired fat absorption and resistance to high-fat diet-induced obesity.

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: Fig. 7 GRASP55-PDZ1 interacts with ATGL-patatin. a–d Coimmunoprecipitation experiments with anti-Myc antibodies were performed in Caco-2 cells. For the induction of ATGL protein expression, some cells were treated with 400 μM oleic acid for 16 h. A representative coimmunoprecipitation assay using GRASP55 with a COOH-terminal Myc-tag (GRASP55-Myc) is shown in (a), and the results of multiple experiments are summarized in (b, n = 3). A representative coimmunoprecipitation assay using ATGL with a COOH-terminal Myc-tag (ATGL-Myc) is shown in (c), and the results of multiple experiments are summarized in (d) (n = 3). Aldolase A was used as a cytosolic protein loading control. e, f Pull-down assays were performed with GRASP55 and ATGL fragments. The domain structures of His6-tagged GRASP55 and GST-tagged ATGL constructs used in this study are shown in (e), and a representative result from the pull-down assay is shown in (f). Expression of control GST and each GST-fusion protein is visualized by Ponceau S staining (f lowermost panel). An asterisk indicates the band of each GST-tagged protein. One microgram of each His6-tagged protein was loaded as an input control. The PDZ1 domain of GRASP55 interacted strongly with the ATGL-patatin domain. Three independent experiments showed similar results. Unprocessed blots can be found in Supplementary Fig. 22. Data are shown as mean ± SEM. n.s.: not significant, *p < 0.05, **p < 0.01. All p values were calculated by paired two-tailed Student’s t tests. Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55-specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone (GRASP55: #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Expressing, Co-Immunoprecipitation Assay, Control, Construct, Pull Down Assay, Staining, Two Tailed Test

Journal: Nature communications

Article Title: Grasp55 -/- mice display impaired fat absorption and resistance to high-fat diet-induced obesity.

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: Fig. 8 Decreased ATGL levels in LDs by GRASP55 depletion are not reversed by proteasome inhibition. a–d Immunoblot analysis of ATGL levels in Caco-2 cells, in which GRASP55 was depleted (shGRASP55) and/or exogenously supplemented (GRASP55-Myc). Cytosolic proteins were collected in the absence or presence of the proteasomal inhibitor MG132 (0.5 μM, 16 h). For the induction of ATGL protein expression, cells were treated with 400 μM oleic acid for 16 h. Representative immunoblots of cytosols and LDs are shown in (a) and (c), and the results of multiple experiments are summarized in (b) and (d), respectively (n = 3 each). Aldolase A and RAB18/ADRP were used as loading controls for cytosolic and LD proteins, respectively. MG132 reversed the decreased cytosolic levels of ATGL by GRASP55 depletion, but did not significantly affect the ATGL levels in LDs. In contrast, the add-back of GRASP55 restored ATGL levels in both the cytosol and LDs. e, f Coimmunoprecipitation (IP) experiments with anti-GRASP55 antibodies were performed in Caco-2 cells. The ER-to-Golgi trafficking was blocked by dominant-inhibitory forms of SAR1 (SAR1-T39N) and ARF1 (ARF1-T31N). For the induction of ATGL protein expression, cells were treated with 400 μM oleic acid for 16 h. A representative IP assay is shown in (e), and the results of multiple experiments are summarized in (f, n = 3). Aldolase A was used as a cytosolic protein loading control. MG132 does not rescue the dissociation of GRASP55 and ATGL induced by SAR1-T39N and ARF1-T31N. Unprocessed blots can be found in Supplementary Fig. 22. Data are shown as mean ± SEM. **p < 0.01, difference from control without MG132. ##p < 0.01, difference from control. All p values were calculated by ANOVA followed by Tukey’s multiple comparison tests. Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55-specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone (GRASP55: #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Inhibition, Western Blot, Expressing, Control, Comparison

Journal: Nature communications

Article Title: Grasp55 -/- mice display impaired fat absorption and resistance to high-fat diet-induced obesity.

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: Fig. 9 GRASP55 deficiency-induced lipid storage defects are rescued by supplementation of GRASP55 in mice. Wild-type (Grasp55+/+) and Grasp55−/−mice were crossed with transgenic mice overexpressing GRASP55-Myc (TgGrasp55). a, b Photographs (a) and a summary of body weight (b, n = 6) of 12-week-old male mice. c, d Photographs (c) and a summary of weight (d, n = 6) of epididymal white adipose tissues (EWAT, right side). e, f Representative light microscopic images (H&E) of EWAT are shown in (e). Quantitative analyses of the H&E images, that inversely correlate with fat contents of EWAT by counting the number of adipocyte nuclei in a light microscopic field (450 × 340 μm), are presented in (f, n = 6). g H&E and Oil Red O staining of mouse jejunum 4 h after olive oil bolus (10 μl g−1 body weight). TgGrasp55(+) reduced the postprandial lipid accumulation of jejunal epithelia in Grasp55−/−mice. Arrows indicate supersized LDs. h, i The protein amount ATGL of mouse jejunum was analyzed by immunoblotting. Representative immunoblots are shown in (h). In some experiments, jejunum tissues were prepared 4 h after olive oil bolus (10 μl g−1 body weight). The filled arrowhead and open arrowhead indicate levels of the exogenous GRASP55-Myc and endogenous GRASP55, respectively. A blot with a longer exposure time of GRASP55 is shown in the left four lanes to better visualize the presence and absence of endogenous GRASP55. The results of multiple experiments (n = 4) are summarized in (i). TgGrasp55(+) increases the protein levels of ATGL in jejunal epithelia of Grasp55−/−mice. Unprocessed blots can be found in Supplementary Fig. 22. Data are shown as mean ± SEM. Scale bars: 50 μm. n.s.: not significant, **p < 0.01. P values were calculated by unpaired two-tailed Student’s t tests (b) or ANOVA followed by Tukey’s multiple comparison tests (d, f, i). Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55-specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone (GRASP55: #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Transgenic Assay, Staining, Western Blot, Two Tailed Test, 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: 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