Journal: Cell reports

Article Title: RalA and PLD1 promote lipid droplet growth in response to nutrient withdrawal

doi: 10.1016/j.celrep.2021.109451

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-ADRP (B-6) (Perilipin2) , Santa Cruz Biotechnology , Cat# sc-377429.

Techniques: Recombinant, Software, Microscopy

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: Cannabinoid receptor 1 knockout alleviates hepatic steatosis by downregulating perilipin 2

doi: 10.1038/s41374-019-0327-5

Figure Lengend Snippet: CB1 knockout reduced hepatic PLIN2 in HBs transgenic mice. a Quantitative real-time PCR analysis of PLIN2 revealed reduced PLIN2 mRNA in CB1 −/− and HBS/CB1 −/− -mice. Data were normalized to β-actin as reference gene and shown as relative expression (ΔΔCt). b Western blot analysis of total protein lysates from the liver of 52-week-old mice demonstrates reduced PLIN2 protein levels in HBs/CB1 −/− mice. c Immunofluorescence analysis of paraffin-embedded liver sections from 52-week-old mice was performed using specific anti-PLIN2 antibody (red). Nuclei were stained with DAPI (blue). Original magnification ×1000, bar 40 µm

Article Snippet: Primary antibodies used for double staining; anti-rabbit LC3B, 1:100 (Proteintech, 18725-1-AP), and anti-rabbit PLIN2, 1:200 (Proteintech, 15294-1-AP), anti-mouse LAMP1, 1:100 (Genetex, GT25212), p62, 1:100 (Proteintech, 18420-1-AP).

Techniques: Knock-Out, Transgenic Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: Cannabinoid receptor 1 knockout alleviates hepatic steatosis by downregulating perilipin 2

doi: 10.1038/s41374-019-0327-5

Figure Lengend Snippet: Pharmacologic antagonization of CB1 reduced PLIN2 expression in cell culture a AML12 cells were treated with rimonabant (rimo) or methanadamide (meth-AEA) at 1 µM final concentration for 48 h in the absence or presence of oleic acid (200 µM). Western blot analysis demonstrated enhanced PLIN2 expression with meth-AEA and reduced PLIN2 expression with rimo-treatment. Equal protein loading was confirmed using anti-GAPDH antibody. A representative Western Blot is shown. b Immunofluorescence analysis of PLIN2 (red) in AML12 cells treated with rimonabant or meth-AEA. Nuclei were stained with DAPI (blue). Representative stainings are shown. Original magnification ×400, bar 100 µm. c Dose dependent decrease in PLIN2 expression in AML12 cells treated with rimonabant at different doses (0.1, 0.5, 1, and 2 µM), respectively. in the presence of oleic acid (200 µM). Cell lysates were analyzed by western blot using specific anti-PLIN2 antibodies. Equal protein loading was confirmed using an anti-GAPDH antibody

Article Snippet: Primary antibodies used for double staining; anti-rabbit LC3B, 1:100 (Proteintech, 18725-1-AP), and anti-rabbit PLIN2, 1:200 (Proteintech, 15294-1-AP), anti-mouse LAMP1, 1:100 (Genetex, GT25212), p62, 1:100 (Proteintech, 18420-1-AP).

Techniques: Expressing, Cell Culture, Concentration Assay, Western Blot, Immunofluorescence, Staining

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: Cannabinoid receptor 1 knockout alleviates hepatic steatosis by downregulating perilipin 2

doi: 10.1038/s41374-019-0327-5

Figure Lengend Snippet: a Double immunofluorescence analysis of paraffin-embedded liver sections of 52-week-old mice was performed using specific anti-LC3B (green) and LAMP1 (red) antibodies. Nuclei were stained with DAPI (blue). Colocalization of these two proteins appears in yellow. Magnification ×1000 Scale bar 40 µm. b Representative double immunofluorescence analysis of paraffin-embedded liver sections from 52-week-old mice was performed using anti-p62 (green) and anti-LAMP1 (red). Arrowheads indicate colocalization of these proteins shown in yellow color and p62 accumulation in nuclear inclusion. Original image magnification ×1000, bars 40 µm. c Representative immunohistochemical analysis of paraffin-embedded liver sections from 52-week-old mice was performed using specific anti-LAL (lysosomal acid lipase) antibody. Original magnification ×200 and ×1000, bar 100 µm. d Representative immunohistochemical analysis of PLIN2 (blue) and LAMP1 (red). Arrows show costained areas (purple color). Original magnification ×200 and ×1000, bar 100 µm

Article Snippet: Primary antibodies used for double staining; anti-rabbit LC3B, 1:100 (Proteintech, 18725-1-AP), and anti-rabbit PLIN2, 1:200 (Proteintech, 15294-1-AP), anti-mouse LAMP1, 1:100 (Genetex, GT25212), p62, 1:100 (Proteintech, 18420-1-AP).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining

Journal: bioRxiv

Article Title: Thrombolysis exacerbates cerebrovascular injury after ischemic stroke via a VEGF-B dependent effect on adipose lipolysis

doi: 10.1101/2024.10.11.617532

Figure Lengend Snippet: (A) Schematic representation of the in vitro experimental outline in HUVECs. (B) Glucose uptake in response to high long-chain FA ( hi FA; 0.1 mM, oleic/palmitic acid mix) and/or glucose ( hi Glc, 20 mM D-Glucose) levels, compared to cells cultured in control media (Ctrl) for the indicated timepoints. (C) Glucose uptake after 0.05 mM ( mod FA) or 0.5 mM ( hi FA) oleic acid. (D-E) GLUT1 intensity after hi FA exposure. Insets show higher magnification. (F) FA uptake after mod FA or hi FA exposure. (G-H) Mitochondria (G) and PLIN2+ LD (H) visualization after mod FA exposure (0.05 mM; oleic/palmitic acid mix). Insets show higher magnification. (I-J) Neutral lipid content after hi FA exposure (0.5 mM oleic acid). (K) Trans-endothelial electrical resistance after hi FA exposure. (L) ZO-1 intensity (arrowheads) after hi FA exposure. LDs stained with PLIN2. (M-N) Dex TMR uptake after hi FA exposure. (O-P) Glucose (O) and FA (P) uptake after VEGF-B stimulation in the absence or presence of hi FAs exposure. Data presented as mean ± s.e.m of 3-9 biological replicates per condition, normalized to control treated cells. Scale bars, 10 μm (D, G, H, L); 50 μm (I, M). Statistical evaluation using unpaired t -test with Welch’s correction (B, F, J), unpaired t -test (C, E, K, N), or two-way ANOVA followed by Tukeýs multiple comparisons test (O-P). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. See also Figure S1.

Article Snippet: After permeabilization for 30 min with PBS/0.2% BSA/0.1% saponin, cells were incubated with primary antibodies: guinea pig anti-adipophilin/PLIN2 (1:200; 20R-AP002, Fitzgerald Industries International), mouse anti-ZO-1 (1:500; 33-9100, Zymed) or rabbit anti-GLUT1 (1:200; 07-1401, Millipore) in PBS/0.2% BSA/0.1% saponin overnight at 4°C.

Techniques: In Vitro, Cell Culture, Control, Staining

Journal: bioRxiv

Article Title: Thrombolysis exacerbates cerebrovascular injury after ischemic stroke via a VEGF-B dependent effect on adipose lipolysis

doi: 10.1101/2024.10.11.617532

Figure Lengend Snippet: (A) Chow and HFD mice subjected to MCAO (50 mg/kg Rose Bengal) and sacrificed 72 h later. (B) Infarct volumes determined after TTC staining. (C) Petechial hemorrhages counted and summed up to generate an ICH score. Representative score 0, 1 and 4 to the right. (D) CBF measured from the MCA territory in mice undergoing a milder MCAO protocol (12.5 mg/kg Rose Bengal). (E) Schematic representation of experimental outline for investigation of thrombosis and BBB function 3 h post MCAO (50 mg/kg Rose Bengal). (F-G) Microthrombi amount (F) and localization (G) in leaky blood vessels. (H) Cerebrovascular LDs in leaky blood vessels. (I) Overall Plin2 intensity quantified from ipsilateral hemisphere. (J) Plin2 quantified in three discrete ipsilateral regions: Cortical ischemic core, cortical-(dorsal) and striatal (medio-ventral) penumbra, and the corresponding dorsal cortical region in the contralateral hemisphere. (K) Quantification of endothelial LDs. (L) TEM image showing an endothelial LD (arrow). (M) EC mitochondria area. (N) Dex TMR extravasation in whole-mount views (upper panels) and histological cross-sections (lower panels). (O) Dex TMR intensity quantified in histological cross-sections. Scale bars, 50 μm (G-H); 500 μm (N); 1 nm (L). Data presented as mean ± s.e.m of n=10-11 mice/group (B-C), n=5 mice/group (D, F, I, O) and n=3 mice/group (K, M). Statistical evaluation using unpaired t -test (B, D, F, J, O), unpaired t -test with Welch’s correction (C, I) or two-way ANOVA followed by Holm-Šídák’s multiple comparisons test (K, M). * P <0.05, ** P <0.001, *** P <0.001. See also Figure S3.

Article Snippet: After permeabilization for 30 min with PBS/0.2% BSA/0.1% saponin, cells were incubated with primary antibodies: guinea pig anti-adipophilin/PLIN2 (1:200; 20R-AP002, Fitzgerald Industries International), mouse anti-ZO-1 (1:500; 33-9100, Zymed) or rabbit anti-GLUT1 (1:200; 07-1401, Millipore) in PBS/0.2% BSA/0.1% saponin overnight at 4°C.

Techniques: Staining

Journal: bioRxiv

Article Title: Thrombolysis exacerbates cerebrovascular injury after ischemic stroke via a VEGF-B dependent effect on adipose lipolysis

doi: 10.1101/2024.10.11.617532

Figure Lengend Snippet: (A) Chow or HFD mice pre-treated for 1 week with αVEGF-B or isotype control IgG antibodies before MCAO (50 mg/kg Rose Bengal) and sacrificed 72 h later. (B) Infarct volumes. (C) ICH scores. (D) Schematic representation of experimental outline in mice sacrificed 3 h post MCAO. (E-F) Evaluation of CBF with laser speckle contrast imaging (E). Boxed areas in the penumbra region utilized for quantification (F). ( G-H) Dex TMR extravasation in whole-mount views (G) and quantification (H). (I-J) Cerebrovascular Plin2+ LDs. Contralateral hemisphere shown for reference (I). Scale bars, 5 mm (G);10 μm (I). Data are presented as mean ± s.e.m. based on n=23+9-10 (chow vs. HFD) mice/group (B-C), n=5-6 mice/group (F) and n=3 mice/group (H, J). Statistical evaluation using two-way ANOVA followed by Tukeýs multiple comparisons test or one-way ANOVA followed by Fisheŕs LSD multiple comparisons test (F). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. See also Figures S4 and S5.

Article Snippet: After permeabilization for 30 min with PBS/0.2% BSA/0.1% saponin, cells were incubated with primary antibodies: guinea pig anti-adipophilin/PLIN2 (1:200; 20R-AP002, Fitzgerald Industries International), mouse anti-ZO-1 (1:500; 33-9100, Zymed) or rabbit anti-GLUT1 (1:200; 07-1401, Millipore) in PBS/0.2% BSA/0.1% saponin overnight at 4°C.

Techniques: Control, Imaging

Journal: bioRxiv

Article Title: Thrombolysis exacerbates cerebrovascular injury after ischemic stroke via a VEGF-B dependent effect on adipose lipolysis

doi: 10.1101/2024.10.11.617532

Figure Lengend Snippet: (A) HFD mice subjected to MCAO (50 mg/kg Rose Bengal) and 1 h later given one i.p. injection with αVEGF-B or isotype control antibodies, followed by intravenous thrombolysis (IVT) with rtPA (alteplase, 10 mg/kg) 5 h post MCAO. Experimental endpoint at 72 h post MCAO. (B) Infarct volumes. ( C) ICH scores. (D) Hemorrhagic transformation in control, but not αVEGF-B treated mice. (E) Correlation between ICH events and infarct size. (F) Kaplan-Meier survival analysis. (G) Experimental endpoint at 5.5 h post MCAO including non-IVT control groups and sham mice as control for surgery and post-op procedures. (H-I) Plin2+ LD distribution (H) and quantification (I). (J-K) Plasma NEFAs (J) and plasma TGs (K). Scale bars, 50 μm. Data are presented as mean ± s.e.m. based on n=10+14 (Ctrl IgG vs. αVEGF-B) mice/group (B-C, E), n=14 mice/group (F) and n=6-7 mice/group (I-K). Statistical evaluation using unpaired t -test (B), unpaired t -test with Welch’s correction (C), linear regression with Pearson correlation (E), Log-rank (Mantel-Cox) test (F) and two-way ANOVA followed by Fisheŕs LSD test (I-K). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. See also Figure S6.

Article Snippet: After permeabilization for 30 min with PBS/0.2% BSA/0.1% saponin, cells were incubated with primary antibodies: guinea pig anti-adipophilin/PLIN2 (1:200; 20R-AP002, Fitzgerald Industries International), mouse anti-ZO-1 (1:500; 33-9100, Zymed) or rabbit anti-GLUT1 (1:200; 07-1401, Millipore) in PBS/0.2% BSA/0.1% saponin overnight at 4°C.

Techniques: Injection, Control, Transformation Assay

Journal: bioRxiv

Article Title: Thrombolysis exacerbates cerebrovascular injury after ischemic stroke via a VEGF-B dependent effect on adipose lipolysis

doi: 10.1101/2024.10.11.617532

Figure Lengend Snippet: (A) HFD mice subjected to MCAO (50 mg/kg Rose Bengal) and 1 h later given one i.p. injection with αVEGF-B or isotype control antibodies, followed by IVT with rtPA (alteplase, 10 mg/kg) or vehicle as control, at 5 h post MCAO. Experimental endpoint at 5.5 h post MCAO. Sham mice included as control for surgery and post-op procedures. (B-C) Activated (phosphorylated, B) and total (pan, C) hormone-sensitive lipase (HSL) in epididymal WAT (eWAT). (D) Phosphorylated/total HSL ratios. (E) Intravenous rtPA (alteplase, 10 mg/kg) or vehicle control injection of naïve HFD mice. Experimental endpoint 30 min post injection. (F-G) Phosphorylated/total HSL expression (F) and ratios (G) in eWAT. (H) Ex vivo lipolysis in eWAT (basal and isoproterenol stimulated). (I-J) Plasma NEFAs (I) and TGs (J). (K-L) Plin2+ LD distribution (K) and quantification (L) in brain. Scale bars, 50 μm. Data presented as mean ± s.e.m. based on n=6-7 mice/group (D, H-J) and n=3 mice/group (G, L). Statistical evaluation using Brown-Forsythe and Welch ANOVA followed by Dunnett’s T3 multiple comparisons test (D), two-way ANOVA followed by Tukeýs multiple comparisons test (H), unpaired t -test (I-J, L), or unpaired t -test with Welch’s correction (G). * P <0.05, *** P <0.001, **** P <0.0001. See also Figure S7.

Article Snippet: After permeabilization for 30 min with PBS/0.2% BSA/0.1% saponin, cells were incubated with primary antibodies: guinea pig anti-adipophilin/PLIN2 (1:200; 20R-AP002, Fitzgerald Industries International), mouse anti-ZO-1 (1:500; 33-9100, Zymed) or rabbit anti-GLUT1 (1:200; 07-1401, Millipore) in PBS/0.2% BSA/0.1% saponin overnight at 4°C.

Techniques: Injection, Control, Expressing, Ex Vivo