Journal: Current Issues in Molecular Biology

Article Title: Losartan Attenuates Insulin Resistance and Regulates Browning Phenomenon of White Adipose Tissue in ob /ob Mice

doi: 10.3390/cimb43030128

Figure Lengend Snippet: Losartan attenuated HIF1α expression and LDs formation in EWAT. ( A ) Representative HE and HIF1α staining. Red arrow highlights the positive staining. Scale bar: 100 μm. Tissue staining, as quantified by either stain intensity, is represented on the left-hand vertical axis in each graph. ( B ) Representative PLIN1, PLIN2, CIDEA, and CIDEC staining of EWAT. Green pseudocolor represents visualization of lipofuscin’s autofluorescence at 450–490 nm. Red arrow highlights the positive staining. Scale bar: 100 μm. Tissue staining, as quantified by either stain intensity, is represented on the left-hand vertical axis in each graph. Quantification of ( C ) HIF1α protein levels by Western blot. Below graph indicate quantification relative to Histone. Quantification of ( D ) PLIN1, PLIN2, CIDEA, and CIDEC protein levels by Western blot of EWAT. Below graphs indicate quantification relative to β-actin. For each animal group, n = 5. All values represent the mean ± SEM. Data were analyzed by Student’s t test. * p ≤ 0.05, normal vs. ob/ob ; # p ≤ 0.05, ob /ob vs. ob /ob + Losartan. HIF1α: hypoxia-inducible factor 1α; LDs: lipid droplets; EWAT: epididymal white adipose tissue; HE: hematoxylin and eosin stain; PLIN1: perilipin 1; CIDEA: cell death inducing DFFA like effector A.

Article Snippet: Total protein (60–80 μg) was run on a 10% SDS-polyacrylamide gel and transferred onto 0.45-μm PVDF blotting membrane (GE Healthcare), and detected with a specific antibody against PLIN1 (ab3526, abcam, Cambridge, UK), PLIN2 (NB110-40877, Novus Biologicals, Centennial, CO, USA), CIDEA (NBP1-76950, Novus Biologicals, Centennial, CO, USA), CIDEC (ab198204, abcam, Cambridge, UK), SREBP-1c (SC-366, Santa Cruz Biotechnology, Dallas, TX, USA), CD36 (SC-70644, Santa Cruz Biotechnology, Dallas, TX, USA), SIRT1 (ab110304, abcam, Cambridge, UK), PGC1α (ab54481, abcam, Cambridge, UK), UCP1 (ab10983, abcam, Cambridge, UK), Tmem26 (PA5-23477, Thermo Fisher Scientific, Waltham, MA, USA), HIF1α (ab179483, abcam, Cambridge, UK), TGFβR2 (SC-17791, Santa Cruz Biotechnology, Dallas, TX, USA), VEGF (ab69479, abcam, Cambridge, UK), NF-κB (SC-8008, Santa Cruz Biotechnology, Dallas, TX, USA), p-STAT3 (05-485, Millipore, Burlington, MA, USA), STAT3 (06-596, Millipore, Burlington, MA, USA), CD11b (ab133357, abcam, Cambridge, UK), CD11c (ab52632, ab11029, abcam, Cambridge, UK), CCR7 (ab32527, abcam, Cambridge, UK), CD163 (ab182422, abcam, Cambridge, UK), CD206 (ab64693, abcam, Cambridge, UK), β-actin (MAB1501, Millipore, Burlington, MA, USA), Histone (SC-56616, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Staining, Western Blot, H&E Stain

Journal: Current Issues in Molecular Biology

Article Title: Losartan Attenuates Insulin Resistance and Regulates Browning Phenomenon of White Adipose Tissue in ob /ob Mice

doi: 10.3390/cimb43030128

Figure Lengend Snippet: Losartan reduced LDs accumulation and lipogenesis in liver. ( A ) Representative HE, Oil Red O, HIF1α, PLIN1, PLIN2, and CIDEA staining of liver. Red arrow highlights the positive staining. Scale bar: 100 μm. Tissue staining, as quantified by either stain intensity, is represented on the left-hand vertical axis in each graph. ( B ) Quantification of HIF1α, PLIN1, PLIN2, and CIDEA protein levels in homogenates of liver. Below graphs indicate quantification relative to Histone (for HIF1α) and β-actin (for PLIN1, PLIN2, and CIDEA). ( C ) Quantification of Srebp-1c , Fas and Scd1 , ( D ) Cd36 , Fatp , Cpt1 , and Cpt2 by qRT-PCR. For each animal group, n = 5. All values represent the mean ± SEM. Data were analyzed by Student’s t test. * p ≤ 0.05, normal vs. ob /ob ; # p ≤ 0.05, ob /ob vs. ob /ob + Losartan. HIF1: hypoxia-inducible factor 1; LDs: lipid droplets; HE: hematoxylin and eosin stain; PLIN1, perilipin 1; CIDEA, cell death inducing DFFA like effector A; SREBP: sterol response element-binding protein; Fas : fatty acid synthase; Scd : stearoyl-CoA desaturase; Cd36 : cluster of differentiation 36; Fatp : fatty acid transport protein; Cpt1 : carnitine palmitoyltransferase 1; qRT-PCR: quantitative real time polymerase chain reaction.

Article Snippet: Total protein (60–80 μg) was run on a 10% SDS-polyacrylamide gel and transferred onto 0.45-μm PVDF blotting membrane (GE Healthcare), and detected with a specific antibody against PLIN1 (ab3526, abcam, Cambridge, UK), PLIN2 (NB110-40877, Novus Biologicals, Centennial, CO, USA), CIDEA (NBP1-76950, Novus Biologicals, Centennial, CO, USA), CIDEC (ab198204, abcam, Cambridge, UK), SREBP-1c (SC-366, Santa Cruz Biotechnology, Dallas, TX, USA), CD36 (SC-70644, Santa Cruz Biotechnology, Dallas, TX, USA), SIRT1 (ab110304, abcam, Cambridge, UK), PGC1α (ab54481, abcam, Cambridge, UK), UCP1 (ab10983, abcam, Cambridge, UK), Tmem26 (PA5-23477, Thermo Fisher Scientific, Waltham, MA, USA), HIF1α (ab179483, abcam, Cambridge, UK), TGFβR2 (SC-17791, Santa Cruz Biotechnology, Dallas, TX, USA), VEGF (ab69479, abcam, Cambridge, UK), NF-κB (SC-8008, Santa Cruz Biotechnology, Dallas, TX, USA), p-STAT3 (05-485, Millipore, Burlington, MA, USA), STAT3 (06-596, Millipore, Burlington, MA, USA), CD11b (ab133357, abcam, Cambridge, UK), CD11c (ab52632, ab11029, abcam, Cambridge, UK), CCR7 (ab32527, abcam, Cambridge, UK), CD163 (ab182422, abcam, Cambridge, UK), CD206 (ab64693, abcam, Cambridge, UK), β-actin (MAB1501, Millipore, Burlington, MA, USA), Histone (SC-56616, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Staining, Quantitative RT-PCR, H&E Stain, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Lipid Research

Article Title: Dynamic and differential regulation of proteins that coat lipid droplets in fatty liver dystrophic mice

doi: 10.1194/jlr.m000976

Figure Lengend Snippet: Fig. 5. The expression of CIDE genes is markedly induced in liver of fl d mice. Graphs depict results of RT- PCR analyses to quantify mRNA levels of CideA, CideB, and Fsp27 using liver RNA isolated from WT and fl d mice at indicated postnatal days. Values are normalized (= 1.0) to P8 WT control expression levels. * P < 0.05 versus WT littermates. Representative Western blotting analyses using hepatic protein isolated from lipid droplet fractions from WT and fl d mice at P8 and antibodies indicated at left are shown.

Article Snippet: Antibodies to glycogen synthase (Proteintech Group, Chicago, IL), peroxisome proliferator-activated receptor (PPAR ) (Santa Cruz, San Diego, CA), SREBP-1 (Santa Cruz), CideA (Genway, San Diego, CA), Fsp27 (generous gift of Dr. Vishwajeet Puri), and actin (Sigma) were used according to the manufacturer’s instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Control, Western Blot

Journal: Journal of Lipid Research

Article Title: Dynamic and differential regulation of proteins that coat lipid droplets in fatty liver dystrophic mice

doi: 10.1194/jlr.m000976

Figure Lengend Snippet: Fig. 8. Forced-expression of SREBP-1 leads to increased expression of CideA. Hepatocytes isolated from adult WT mice were infected with adenovirus expressing a constitutively active form of SREBP-1a (Ad- caSREBP-1) or control adenovirus expressing green fl uorescent protein (GFP). The graph depicts the results of quantitative RT-PCR analyses of Cide and Plin family genes. * P < 0.05 versus GFP control.

Article Snippet: Antibodies to glycogen synthase (Proteintech Group, Chicago, IL), peroxisome proliferator-activated receptor (PPAR ) (Santa Cruz, San Diego, CA), SREBP-1 (Santa Cruz), CideA (Genway, San Diego, CA), Fsp27 (generous gift of Dr. Vishwajeet Puri), and actin (Sigma) were used according to the manufacturer’s instructions.

Techniques: Expressing, Isolation, Infection, Control, Quantitative RT-PCR

Journal: Journal of Lipid Research

Article Title: Dynamic and differential regulation of proteins that coat lipid droplets in fatty liver dystrophic mice

doi: 10.1194/jlr.m000976

Figure Lengend Snippet: Fig. 9. SREBP-1 directly activates Cidea gene transcription. Graphs represent mean (± SEM) luciferase ac- tivity in relative luciferase units (RLU) corrected for renilla luciferase activity and normalized (=1.0) to the value of empty expression vector-transfected cells. The results of studies using 293 cells cotransfected with a deletion series of Cidea promoter-luciferase reporter constructs and expression vectors driving expression of ca-SREBP-1 or empty vector control. Schematics of the various reporter constructs are shown at left. The location of canonical SREBP-1 response elements is denoted (SRE). TSS, transcriptional start site. * P <0.05 versus the value of empty vector control. ** P <0.05 versus caSREBP-1-stimulated pCID2 and pCID3. The images depict the results of chromatin immunoprecipitation (ChIP) studies using chromatin from hepato- cytes isolated from WT mice infected with adenovirus to overexpress caSREBP-1 (abbreviated “S”) and/or GFP (abbreviated “G”). Crosslinked proteins were immunoprecipitated with SREBP-1 antibody or IgG con- trol. Input represents 0.2% of the total chromatin used in the IP reactions. Primers specifi c for the Cidea promoter (The general annealing site of primers used is shown in A) or an exon of Acadm (negative control) were used to detect immunoprecipitated DNA.

Article Snippet: Antibodies to glycogen synthase (Proteintech Group, Chicago, IL), peroxisome proliferator-activated receptor (PPAR ) (Santa Cruz, San Diego, CA), SREBP-1 (Santa Cruz), CideA (Genway, San Diego, CA), Fsp27 (generous gift of Dr. Vishwajeet Puri), and actin (Sigma) were used according to the manufacturer’s instructions.

Techniques: Luciferase, Activity Assay, Expressing, Plasmid Preparation, Transfection, Construct, Control, Chromatin Immunoprecipitation, Isolation, Infection, Immunoprecipitation, Negative Control

Journal: Scientific Reports

Article Title: Development of CIDEA reporter mouse model and its application for screening thermogenic drugs

doi: 10.1038/s41598-021-97959-0

Figure Lengend Snippet: Specificity of fluorescence reporter expression in brown and beige adipose tissue of the CIDEA reporter mice. ( a ) Immunofluorescence staining of CIDEA and tdTomato in adipose tissue frozen sections of homozygous (HOMO) CIDEA reporter mice. Nuclei were counterstained with DAPI (Blue). Bar = 20 μm. ( b ) Comparison of adipose tissue frozen section (BAT) or whole mount (iWAT and gWAT) of HOMO CIDEA reporter (TG) and wild type (WT) mice. Mice were maintained in cold (4 °C for a week) or room temperature (22 °C) condition. Nuclei and lipid droplets were counterstained with DAPI and HCS LipidTox deep red. Bar = 50 μm. ( c ) Ex vivo fluorescence imaging of tissues freshly isolated from WT, Heterozygous (HET), and HOMO CIDEA reporter mice. Quantification of fluorescence signal was normalized to each tissue weight. Data were analyzed by an unpaired, two-tailed t test (mean ± SEM; n = 3 *P < 0.05).

Article Snippet: CIDEA antibody (Rabbit, 1:100, Novus Biologicals, RRID: AB_11012002) was used for immunofluorescence detection.

Techniques: Fluorescence, Expressing, Immunofluorescence, Staining, Comparison, Ex Vivo, Imaging, Isolation, Two Tailed Test

Journal: Scientific Reports

Article Title: Development of CIDEA reporter mouse model and its application for screening thermogenic drugs

doi: 10.1038/s41598-021-97959-0

Figure Lengend Snippet: Establishment of in vitro CIDEA reporter primary culture system. Stromal vascular fractions of adipose tissue from homozygous CIDEA reporter mice were isolated, cultured, and differentiated. ( a ) In vitro bioluminescence imaging of preadipocyte (Pre) and fully differentiated adipocytes (AC) from BAT. Cells were treated with growth medium containing d -luciferin (150 μg/ml). ( b ) Luciferase assay of cell lysate from ( a ). ( c ) Immunofluorescence staining image of differentiated adipocytes from BAT and iWAT. CIDEA (Green) and tdT (Red) double positive cells (CIDEA + tdT + ) were indicated with a dashed yellow line. Nuclei were counterstained with DAPI (Blue). Bar = 10 μm. ( d ) Quantification of the number of CIDEA + tdT + cells per field. ( e ) High magnification images of CIDEA + tdT + adipocytes. Nuclei and lipid were counterstained with DAPI and HCS LipidTOX. Bar = 5 μm. Data were analyzed by an unpaired, two-tailed t test in ( b , d ) (mean ± SEM; n = 3–4 *P < 0.05, ***P < 0.001).

Article Snippet: CIDEA antibody (Rabbit, 1:100, Novus Biologicals, RRID: AB_11012002) was used for immunofluorescence detection.

Techniques: In Vitro, Isolation, Cell Culture, Imaging, Luciferase, Immunofluorescence, Staining, Two Tailed Test

Journal: JNCI Journal of the National Cancer Institute

Article Title: Immunotherapeutic Potential of Anti-Human Endogenous Retrovirus-K Envelope Protein Antibodies in Targeting Breast Tumors

doi: 10.1093/jnci/djr540

Figure Lengend Snippet: Effect of 6H5 monoclonal antibody (mAb) on apoptosis of breast cancer cells. A) Cells were treated with 6H5 mAb (red line) or control mIgG (gray line) (10 μg/mL of each antibody) for 16 hours, stained with annexin V–allophycocyanin and 7-AAD-phycoerythrin-cyanide 7, and analyzed by flow cytometry. B) Effect of 6H5 mAb treatment on cell death–inducing DFFA-like effector A (CIDEA) protein expression. Breast cancer cell lines were treated with 6H5 mAb or mIgG (10 μg/mL of each antibody) for 24 hours and analyzed for changes in protein expression by immunoblot using a mouse anti-human CIDEA antibody. ACTB was used as the protein loading control. Results are representative of two independent assays. C) The effect of 6H5 mAb treatment on expression of TP53 and TP53AIP1 proteins. MCF-7 and MDA-MB-231 breast cancer cell lines were treated with 6H5 mAb or mIgG (10 μg/mL of each antibody) for 24 hours, and an immunoblot assay was done using mouse anti-human TP53 and rabbit anti-human TP53AIP1 antibodies. ACTB was used as the protein loading control. Results are representative of two independent assays. D) Expression of active caspases 3 and 9 was assessed by immunoblot assay in ZR-75-1 and MDA-MB-231 breast cancer cells treated with 6H5 mAb or 6E11 mAb, or with mIgG (10 μg/mL of each antibody) for 24 hours using rabbit anti-human caspase 3 and mouse anti-human caspase 9 antibodies. ACTB was used as the protein loading control (top panel). Expression of active caspase 8 was assessed by immunoblot assay in MDA-MB-453 and MCF-7 breast cancer cells treated with 6H5 mAb (10 , 25, or 50 μg/mL), or with mIgG (10 μg/mL) for 24 hours using mouse anti-human caspase 8 antibody (bottom panel). Results are representative of at least two independent assays. E) Immunofluorescence assay to assess the expression of caspase proteins in MDA-MB-231 cells treated with 6H5 mAb or mIgG (10 μg/mL of each antibody) for 24 hours using rabbit anti-human caspase 3, mouse anti-human caspase 8, and mouse anti-human caspase 9 antibodies. Results are representative of two independent assays. Scale bar = 10 μm. F) Immunofluorescence assay to assess the expression of CDK5 and CDKN1A proteins. MDA-MB-231 cells were treated with 6H5 mAb or mIgG (10 μg/mL of each antibody) using mouse anti-human CDK5 and mouse anti-human CDKN1A antibodies. Results are representative of at least two independent assays. Scale bar = 10 μm.

Article Snippet: Mouse anti-human cell death–inducing DFFA-like effector A (CIDEA) antibody (Abnova, Taipei, Taiwan; 1:1000 dilution) was used for detection of CIDEA expression in cells treated with 6H5 mAb or mIgG.

Techniques: Control, Staining, Flow Cytometry, Expressing, Western Blot, Immunofluorescence

Journal: JNCI Journal of the National Cancer Institute

Article Title: Immunotherapeutic Potential of Anti-Human Endogenous Retrovirus-K Envelope Protein Antibodies in Targeting Breast Tumors

doi: 10.1093/jnci/djr540

Figure Lengend Snippet: Effect of anti-HERV-K antibody treatment on protein expression in breast cell lines, analyzed by flow cytometry *

Article Snippet: Mouse anti-human cell death–inducing DFFA-like effector A (CIDEA) antibody (Abnova, Taipei, Taiwan; 1:1000 dilution) was used for detection of CIDEA expression in cells treated with 6H5 mAb or mIgG.

Techniques: Expressing, Flow Cytometry