Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: Expression of CD36 in preadipocytes is induced in adipose tissue of obese patients. General health survey data were collected from 28,035 patients who underwent health examinations at the Affiliated Hospital of CQMU. Body mass index (BMI), waistline and serum hs-CRP measurements were divided into quartiles. The ranges of the different quartiles of BMI were <21.55 kg/m 2 (1st quartile), 21.55–23.66 kg/m 2 (2nd quartile), 23.66-25.81 kg/m 2 (3rd quartile) and >25.81 kg/m 2 (4th quartile). The ranges of the different quartiles of waist circumference were <76 cm (1st quartile), 76–82 cm (2nd quartile), 82–88 cm (3rd quartile) and 88 cm (4th quartile). The ranges of the different quartiles of hs-CRP were 0-0.30 mg/L (1st quartile), 0.31–0.60 mg/L (2nd quartile), 0.61–1.25 mg/L (3rd quartile) and >1.25 mg/L (4th quartile). Human visceral adipose tissue samples were obtained from patients who underwent laparoscopic cholecystectomy. The subjects were subdivided into two groups: non-obese patients (18.5 kg/m 2

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Expressing, Staining, Immunohistochemical staining, Double Immunofluorescence Staining

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: Expression of CD36 in preadipocytes is induced in HFD-fed mice accompanied with lysosomal impairment. C57BL/6J mice were fed a HFD (5 males and 5 females) or normal diet (ND) (5 males and 5 females) for 14 weeks. (a) Double immunofluorescence staining for CD36 and Pref1 in sections of mouse adipose tissue. The arrow indicates the colocalization area. (b) Preadipocytes (CD146 − /CD34 + cells) in mouse adipose tissue were sorted by a FACScan flow cytometer. CD36 expression (c) and lysosome function (d-e) in CD146 − /CD34 + cells were detected by a FACScan flow cytomete. * p <0.05 compared with the C57-ND group.

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Expressing, Double Immunofluorescence Staining, Flow Cytometry

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: FFA upregulates CD36 expression and induces lysosomal impairment, lipid accumulation and inflammation in 3T3L1 preadipocytes. (a) Cell viability assay in 3T3L1 preadipocytes treated with FFA for 60 h. (b) The relative protein expression of CD36 in 3T3L1 preadipocytes after treatment with FFA (0.4 mM PA+0.2 mM OA) for 15 h, 30 h and 60 h was detected by western blotting ( n= 3). * p <0.05 compared with 0h. (c) CD36 expression in plasma membrane (PM) of 3T3L1 preadipocytes treated with FFA for 0h and 60 h was detected by a FACScan flow cytometer ( n= 3). (d) Lysosome function in 3T3L1 preadipocytes treated with FFA for 15 h and 60 h were detected by a FACScan flow cytometer ( n= 3). (e) BODIPY staining of 3T3L1 preadipocytes treated with or without FFA (FFA group or control (CTL) group) for 60 h ( n= 3). (f) Relative mRNA levels of MCP1, TNF α, IL-6 and IL-1β in 3T3L1 preadipocytes treated with FFA for 60 h ( n= 3). * p <0.05 compared with the CTL group.

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Expressing, Viability Assay, Western Blot, Membrane, Flow Cytometry, Staining

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: Forced CD36 upregulation induces lipid accumulation and inflammation in 3T3L1 preadipocytes were infected with a recombinant lentivirus containing CD36 cDNA (wtCD36 OE preadipocytes) or empty vector (NC preadipocytes) and these cells were selected with puromycin. (a) Western blot analysis of CD36, NF-κB p65, LaminB1, caspase-1, and IL-1β in NC and wtCD36 OE preadipocytes ( n= 3). (b) The quantitative data of protein expression in A. (c) Relative mRNA levels of MCP1, TNF α, IL-6 and IL-1β in NC and wtCD36 OE preadipocytes ( n= 3). (d) Migration assay. A migration assay was performed by using Transwell migration chambers (8-μm pore size) to assess THP-1 cell migration co-cultured with the 3T3L1 preadipocyte lines ( n= 3). (e) BODIPY staining and oil red O staining of NC and wtCD36 OE preadipocytes ( n= 3). * p< 0.05 compared with the NC group.

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Infection, Recombinant, Plasmid Preparation, Western Blot, Expressing, Migration, Pore Size, Cell Culture, Staining

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: Forced CD36 upregulation impairs lysosomal function and lipophagy in 3T3L1 preadipocytes. (a) Western blot analysis of P62 in NC and wtCD36 OE preadipocytes ( n= 3). (b) Double fluorescent staining of mRFP-GFP-LC3 fusion protein in NC and wtCD36 OE preadipocytes. (c) Lysosome function in NC and wtCD36 OE preadipocytes were detected by a FACScan flow cytometer ( n= 3). (d) Representative images showed double immunofluorescence staining for BODIPY and Lysotracker in wtCD36 OE and NC preadipocytes. The arrow indicates the colocalization area. * p< 0.05 compared with the NC group.

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Western Blot, Staining, Flow Cytometry, Double Immunofluorescence Staining

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: Forced CD36 upregulation promotes IP3R1-mediated Ca 2+ transport from the ER to lysosomes in preadipocytes. (a) Representative images showed double immunofluorescence staining for OG-BAPTA-5N and lysotracker in wtCD36 OE and NC preadipocytes. (b) Western blot analysis of IP3R1 and P-IP3R1 (Tyr353) in wtCD36 OE preadipocytes and NC preadipocytes ( n= 3). After wtCD36 OE preadipocytes were treated with or without 2APB (0.5 μM) for 1 h, lysosome function (c-d) were detected by a FACScan flow cytometer ( n= 3). (e) BODIPY staining ( n= 3). (f) Mitochondrial reactive oxygen species assay ( n= 3). (g) Relative mRNA levels of MCP1, TNF α, IL-6 and IL-1β. * p< 0.05 compared with the NC group ( n= 3). # p< 0.05 compared with the wtCD36 OE group.

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Double Immunofluorescence Staining, Western Blot, Flow Cytometry, Staining

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: CD36 coordinates with Fyn to phosphorylate IP3R1, inducing lysosomal Ca 2+ overload and inflammation in preadipocytes. We constructed a lentivirus containing CD36 with a palmitoylation site mutation and infected the 3T3L1 cells with lentivirus (mt∆CD36 OE preadipocytes). (a) Co-IP for CD36 and Fyn in wtCD36 OE preadipocytes and mt∆CD36 OE preadipocytes ( n= 3). (b) Western blot analysis of IP3R1 and P-IP3R1 (Tyr353) in wtCD36 OE preadipocytes and mt∆CD36 OE preadipocytes ( n= 3). (c) Cells were incubated for 30 min in the presence of 0.5 μM fluo-4 AM, a Ca 2+ indicator dye, to detect total intracellular Ca 2+ ( n= 3). (d) ER Ca 2+ release assay. Cells were incubated with fluo-4 AM (0.5 μM) for 30 min. Intracellular Ca 2+ was monitored prior to and following exposure to 1 μM thapsigargin ( n= 3). (e) Representative images showed double immunofluorescence staining for OG-BAPTA-5N and Lysotracker in NC preadipocytes, wtCD36 OE preadipocytes (treated with or without PP2) and mt∆CD36 OE preadipocytes. (f) Western blot analysis of NF-kB and IL-1β in wtCD36 OE preadipocytes and mt∆CD36 OE preadipocytes ( n= 3). * p< 0.05 compared with the NC group. # p< 0.05 compared with the wtCD36 OE group.

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Construct, Mutagenesis, Infection, Co-Immunoprecipitation Assay, Western Blot, Incubation, Release Assay, Double Immunofluorescence Staining

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).

doi: 10.33594/000000038

Figure Lengend Snippet: Fig. 2. Demonstration of (A) representative blots of TSP1, TSP2, CD36 and GAPDH by immunoblotting at different stages of CL development in the riverine buffalo. The relative molecular weight of each protein is shown along the right side of each blot. The luteal proteins were loaded @ 100 mg/ well and resolved in 10% SDS-PAGE followed by electrotransfer to a PVDF membrane. Protein specific antibodies were used @ 1:500 while secondary antibody @1:2000 dilutions. GAPDH was used as reference protein. Relative expression of TSP and its receptors was analysed by densitometry using image J software (n = 6/group). One-way ANOVA to determine if treatment groups were significantly different. Tukey HSD test was done to find the pair-wise mean differences. Each bar represents Mean ± SEM. Different superscripts denote statistical significance (p<0.05). Abbreviations: CL, Corpus Luteum; TSP, Thrombospondin; CD, Cluster of differentiation.

Article Snippet: Antibodies and Growth factor Immunoblotting and immunohistochemistry were performed using goat polyclonal GAPDH (sc-48166; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse monoclonal TSP1 (sc-393504; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal TSP2 (sc-12313; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal CD36 (sc-5522; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse anti-goat IgG-HRP (sc-2354; Santa Cruz Biotechnology, Inc., Dallas, TX), goat anti-mouse IgG-HRP (sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX) bovine anti-goat IgG-CFL-647 (sc-362284, Lot#B0312) and goat anti-mouse IgG-CFL-647 (sc362257; Santa Cruz Biotechnology, Inc.).

Techniques: Western Blot, Molecular Weight, SDS Page, Electrotransfer, Membrane, Expressing, Software

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).

doi: 10.33594/000000038

Figure Lengend Snippet: Fig. 5. Immunohistochemical localization of CD36 in the late CL stage of riverine buffalo. The 5 μm thick sections of CL were deparaffinised and rehydrated, followed by antigen retrieval. Primary TSP1 antibody was used at 1:200 while the FITC was used at 1:500. DAPI was used to counterstain nucleus. Fluorescent signals were captured by microscopy (Carl Zeiss Micro Imaging GmbH). Representative images from (A) bright field, (B) through (C) indicate intense immunoreactivity in late stages of CL which was localized predominantly in the cytoplasm of luteal cells. No primary antibody was used in the negative control (D). Scale bar = 50 μm. Abbreviations: LL, Large luteal cell; SL, Small luteal cell; FITC, Fluorescein isothiocyanate; DAPI, 40, 6-diamidino-2-phenylindole dihydrochloride.

Article Snippet: Antibodies and Growth factor Immunoblotting and immunohistochemistry were performed using goat polyclonal GAPDH (sc-48166; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse monoclonal TSP1 (sc-393504; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal TSP2 (sc-12313; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal CD36 (sc-5522; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse anti-goat IgG-HRP (sc-2354; Santa Cruz Biotechnology, Inc., Dallas, TX), goat anti-mouse IgG-HRP (sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX) bovine anti-goat IgG-CFL-647 (sc-362284, Lot#B0312) and goat anti-mouse IgG-CFL-647 (sc362257; Santa Cruz Biotechnology, Inc.).

Techniques: Immunohistochemical staining, Microscopy, Imaging, Negative Control

Journal: PLoS ONE

Article Title: De novo Generation of Cells within Human Nurse Macrophages and Consequences following HIV-1 Infection

doi: 10.1371/journal.pone.0040139

Figure Lengend Snippet: (A) Immunophenotype of nonadherent small monocytoid cells (SMC) harvested from an 18-day-old MDM culture. White arrow (top left plot) identifies the gated population shown in other plots. (B) Forward scatter (FSC) versus side scatter (SSC) plot demonstrates that SMC (CD3negCD4dimCD14+) are smaller than resting lymphocytes. Cells from 17-day-old MDM. Analysis performed using Paint-a-Gate software. (C) Confocal microscopy demonstrating that SMC adhere following their exit from nurse macrophages. Shown here is an 18-day-old primary MDM culture. An SMC adhering to a large macrophage (green arrow) and another adhering to the culture dish (pink arrow) can be seen. Others with elongated processes are also visible (white arrows) and viewable in greater detail in the panel on the right. The pink color of the nuclei reflects cdc6 colocalization with DNA as identified by TOTO-3 staining. SMC nuclei are typically cdc6+ during and shortly after budding from nurse macrophages, reflecting recent DNA synthesis. Size bar: 50 µm.

Article Snippet: The following secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc.: Rhodamine Red-X (RRX)-conjugated affinity purified goat anti-mouse IgG subclass 1 specific (used with anti-CD68 and anti-MHC II), Cy2-conjugated affinity purified goat anti-mouse IgG subclass 2a specific (used with the mouse monoclonal anti-CD3), Cy3-conjugated affinity purified goat anti-mouse IgG subclass 2a specific (used with anti-CD36 and anti-cdc6), Cy5-conjugated affinity purified goat anti-rabbit IgG (H + L) used with the polyclonal anti-CD3, and fluorescein (FITC)-conjugated affinity purified donkey anti-goat IgG (H + L) and normal donkey serum for use with the anti-pre-TCRα antibody.

Techniques: Software, Confocal Microscopy, Staining, DNA Synthesis

Journal: PLoS ONE

Article Title: De novo Generation of Cells within Human Nurse Macrophages and Consequences following HIV-1 Infection

doi: 10.1371/journal.pone.0040139

Figure Lengend Snippet: (A-D) DNA synthesis, as evidenced by BrdU labeling, is occurring in multiple nuclei within a macrophage with nurse cell morphology. The phase contrast view (panel A) illustrates the nurse-like morphology of the living cell, prior to fixation. The culture was exposed to BrdU for 48 hours. (E-I) Confocal microscopy images demonstrating BrdU labeling in multinucleated macrophages. Culture was exposed to BrdU for 48 hours. Note that quiescent nuclei (blue only) are also present within both cells actively replicating DNA. Also apparent in the upper, somewhat smaller cell, are BrdU-labeled globular structures of multiple sizes. These colocalize with the DNA staining, confirming that they are active sites of DNA replication and not artifact. Panel I shows an enlarged view of this cell. (J-M) Confocal microscopy images demonstrating colocalization of expression of cell division cycle 6 (cdc6) protein with globular structures of DNA. Panels K-M are enlargements of the cell shown in Panel J. A gallery view of this cell is presented in . (N-R) DNA configurations and compartmentalization in Wright’s- Giemsa stained cultured macrophages. Size bars: (A) 50 µm, (E) 20 µm, (K) 10 µm and (N) through (R) 25 µm.

Article Snippet: The following secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc.: Rhodamine Red-X (RRX)-conjugated affinity purified goat anti-mouse IgG subclass 1 specific (used with anti-CD68 and anti-MHC II), Cy2-conjugated affinity purified goat anti-mouse IgG subclass 2a specific (used with the mouse monoclonal anti-CD3), Cy3-conjugated affinity purified goat anti-mouse IgG subclass 2a specific (used with anti-CD36 and anti-cdc6), Cy5-conjugated affinity purified goat anti-rabbit IgG (H + L) used with the polyclonal anti-CD3, and fluorescein (FITC)-conjugated affinity purified donkey anti-goat IgG (H + L) and normal donkey serum for use with the anti-pre-TCRα antibody.

Techniques: DNA Synthesis, Labeling, Confocal Microscopy, Staining, Expressing, Cell Culture