Journal: PLoS ONE

Article Title: N-substituted phenylbenzamides of the niclosamide chemotype attenuate obesity related changes in high fat diet fed mice

doi: 10.1371/journal.pone.0204605

Figure Lengend Snippet: Glucose tolerance test in DIO mice. 2g/kg of glucose was injected IP and at 15, 30 and 60 min, blood glucose levels were measured. (A) Averaged glucose tolerance for 7-day sham cohort and 5 mg/Kg Niclosamide treatment group (n = 3 each, P = 0.05). (B) Comparison of pre and post-7 day treatment with 5 mg/kg Niclosamide (n = 3, P = 0.034). C) Western blot analyses of phosphorylated AMPKα T172, Fatty acid synthase (FAS) and carbonic anhydrase III (CAIII) protein expression in liver tissue samples from male C57BL/6J mice fed HFD treated with 10mg/kg Niclosamide or D) 500200, 500199 or 600453 for 12 days relative to Sham HFD alone (control).

Article Snippet: Proteins (50 μg) were then boiled in loading buffer and subjected to SDS/PAGE (Invitrogen), followed by Western analyses using the following primary antibodies: phospho-AMP activated protein kinase (AMPK) (Thr 172; Cell Signaling #2535), total AMPK (Cell Signaling #2603), Fatty acid synthase (FAS; Cell Signaling #3180) and Carbonic Anhydrase 3 (CAR3; Santa Cruz Biotechnology #sc-50715).

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

Journal: Cell Discovery

Article Title: Luminal hormone-responsive cells tune the regenerative remodeling of mammary glands in large mammals

doi: 10.1038/s41421-025-00848-3

Figure Lengend Snippet: a UMAP plot displaying 14 identified cell types within the goat mammary glands. Cells are annotated and colored by type. Subsets of cell types including luminal, basal, fibroblast, immune, endothelial cell are labeled. b The percentages of −4W (red) and +1 W (blue) cells in each cell type are shown in a UMAP plot. c UMAP plots showing the expression of selected marker genes in four luminal subtypes. d Pseudotemporal trajectory analysis of scRNA-seq data of luminal secretory cells is shown in a UMAP plot. e Changes in the proportion of LumSecP and LumSec cells in luminal cell types were identified by scRNA-seq data at −4W and +1 W. n = 3 goats per group. f Representative images of tissue immunofluorescence staining for ALDH1A3 (red), KRT18 (green), and DAPI at −4W and +1 W. Scale bars, 50 μm. g Bar plots exhibiting the percentage of ALDH1A3-positive cells in luminal cells (labeled by KRT18) in f . n = 15 sections per group. h Representative images of tissue immunofluorescence staining for FABP3 (red), KRT18 (green), and DAPI at −4W and +1 W. Scale bars, 50 μm. i Bar plots exhibiting the percentage of FABP3-positive cells in luminal cells (labeled by KRT18) in h . n = 20 sections per group. The data are presented as the mean ± SEM. The P values of two-sided Student’s t -tests are shown in e , g , i .

Article Snippet: For immunofluorescence staining, goat mammary tissues were stained with primary antibodies against ALDH1A3 (#25167-1-AP, Proteintech), FABP3 (#10676-1-AP, Proteintech), KRT17 (#17516-1-AP, Proteintech), KRT14 (#SC65-06, HUABIO), KRT18 (conjugated with iFluorTM 488, #SZ80-07, HUABIO), KRT8 (#10384-1-AP, ProteinTech), PR (#25871-1-AP, ProteinTech), ER (#21244-1-AP, ProteinTech), and PCNA (#ab29, Abcam).

Techniques: Labeling, Expressing, Marker, Immunofluorescence, Staining

Journal: Cell Discovery

Article Title: Luminal hormone-responsive cells tune the regenerative remodeling of mammary glands in large mammals

doi: 10.1038/s41421-025-00848-3

Figure Lengend Snippet: a Relative proportions of LumHR cells in total luminal cells identified by scRNA-seq data at −4W and +1 W. n = 3 goats per group. b , c Bar plots exhibiting the percentage of PGR - and ESR1 -positive cells within luminal cells in scRNA-seq data. n = 3 goats per group. d Representative images of immunofluorescence staining for PR (red), KRT18 (green), and DAPI (blue). Scale bars, 50 μm. e Bar plots exhibiting the percentage of PR-positive cells in luminal cells (labeled by KRT18) in ( d ). n = 5 goats per group. f Representative images of immunofluorescence staining for ER (red), KRT18 (green), and DAPI (blue). Scale bars, 50 μm. g Bar plots exhibiting the percentage of ER-positive cells in luminal cells (labeled by KRT18) in ( f ). n = 5 goats. h Violin plot showing the specific expression of PRLR in LumHR cells by scRNA-seq. i Heatmap displaying the transcriptional level of indicated genes related to milk protein and luminal differentiation in the goat mammary organoids ( n = 3 biological replicates) treated with or without prolactin and in the mammary tissue at −4W (non-lactation) and +1 W (lactation). n = 3 goats for tissues. j Proportions of luminal cell types in goat mammary organoids incubated with or without prolactin predicted by CIBERSORTx deconvolution. n = 3 biological replicates. k Representative images of immunofluorescence staining for ALDH1A3 (red), KRT18 (green), and DAPI in mammary organoids incubated with or without prolactin. Scale bars, 10 μm. l Bar plots exhibiting the percentage of ALDH1A3-positive cells in luminal cells (labeled by KRT18) in ( k ). n = 14 domes in the control group and n = 10 domes in the prolactin treated group. m Representative images of immunofluorescence staining for FABP3 (red), KRT18 (green) and DAPI in mammary organoids incubated with or without prolactin. Scale bars, 10 μm. n Bar plots exhibiting the percentage of FABP3-positive cells in luminal cells (labeled by KRT18) in ( m ). n = 6 domes in the control group and n = 7 domes in the prolactin-treated group. The data are presented as the mean ± SEM. The P values of two-sided Student’s t -tests are shown in ( a – c , e , g , j , l , n ).

Article Snippet: For immunofluorescence staining, goat mammary tissues were stained with primary antibodies against ALDH1A3 (#25167-1-AP, Proteintech), FABP3 (#10676-1-AP, Proteintech), KRT17 (#17516-1-AP, Proteintech), KRT14 (#SC65-06, HUABIO), KRT18 (conjugated with iFluorTM 488, #SZ80-07, HUABIO), KRT8 (#10384-1-AP, ProteinTech), PR (#25871-1-AP, ProteinTech), ER (#21244-1-AP, ProteinTech), and PCNA (#ab29, Abcam).

Techniques: Immunofluorescence, Staining, Labeling, Expressing, Incubation, Control

Journal: Cell Discovery

Article Title: Luminal hormone-responsive cells tune the regenerative remodeling of mammary glands in large mammals

doi: 10.1038/s41421-025-00848-3

Figure Lengend Snippet: a Schematic illustration of targeted ablation of LumHR cells using the prlr -promoter to drive expression of DTA. b Experimental setup used in AAV intraductally injected mammary gland of ROSA-DTA +/ − mice under RR. c Whole-mount staining with carmine alum of mammary glands from ROSA-DTA +/ − mice (lactation day 2) intraductally injected with AAV-pPrlr-Cre or AAV-Control . Scale bars, 4 mm (top) and 500 μm (bottom). d , e Immunohistochemical staining and quantification of ER-positive luminal cells in mammary glands intraductally injected with AAV-pPrlr-Cre or AAV-Control. n = 3 mice per group. Scale bars, 50 μm. f , g Immunohistochemical staining and quantification of PR-positive luminal cells in mammary glands intraductally injected with AAV-pPrlr-Cre or AAV-Control . n = 3 mice per group. Scale bars, 50 μm. h , i Immunohistochemical staining and quantification of β-casein-positive alveoli number per mm 2 in mammary glands intraductally injected with AAV-pPrlr-Cre or AAV-Control . n = 3 mice per group. Scale bars, 50 μm. j Representative images of immunofluorescence staining for FABP3 (red), KRT18 (green), and DAPI in mammary glands intraductally injected with AAV-pPrlr-Cre or AAV-Control . Scale bars, 50 μm. k Bar plots exhibiting the percentage of FABP3-positive cells in luminal cells (labeled by KRT18) in j . n = 3 mice per group. l Representative images of immunofluorescence staining for ALDH1A3 (red), KRT18 (green), and DAPI in mammary glands intraductally injected with AAV-pPrlr-Cre or AAV-Contro l. Scale bars, 50 μm. m Bar plots exhibiting the percentage of FABP3-positive cells in luminal cells (labeled by KRT18) in ( l ). n = 3 mice per group. The data are presented as the mean ± SEM. The P values of two-sided Student’s t -tests are shown in d , g , i , l , m .

Article Snippet: For immunofluorescence staining, goat mammary tissues were stained with primary antibodies against ALDH1A3 (#25167-1-AP, Proteintech), FABP3 (#10676-1-AP, Proteintech), KRT17 (#17516-1-AP, Proteintech), KRT14 (#SC65-06, HUABIO), KRT18 (conjugated with iFluorTM 488, #SZ80-07, HUABIO), KRT8 (#10384-1-AP, ProteinTech), PR (#25871-1-AP, ProteinTech), ER (#21244-1-AP, ProteinTech), and PCNA (#ab29, Abcam).

Techniques: Expressing, Injection, Staining, Control, Immunohistochemical staining, Immunofluorescence, Labeling

Journal: Nature Communications

Article Title: Histone demethylase JMJD1C is phosphorylated by mTOR to activate de novo lipogenesis

doi: 10.1038/s41467-020-14617-1

Figure Lengend Snippet: a Immunoblotting of lysates from HepG2 cells infected with JMJD1C adenovirus (left). RT-qPCR for mRNA levels (middle) and nascent RNA levels (right) of lipogenic genes in HepG2 cells with or without insulin treatment. n = 4 wells of cells per group. Experiment was repeated three times. b Immunoblotting of lysates from HepG2 cells infected with sh-JMJD1C adenovirus (left). RT-qPCR for mRNA levels of lipogenic genes (middle) and mRNA levels of lipogenic genes in HepG2 cells with or without 10 µM Methylstat treatment (right). n = 4 wells of cells per group. c Immunoblotting for JMJD1C protein and RT-qPCR for mRNA levels (left) in livers of mice 10 days after tail-vein injection of 2 × 10 8 pfu JMJD1C adenovirus in PBS ( n = 5). Mice were fasted for 16 h and then refed with HCD for 8 h before tissue harvesting. mRNA levels of lipogenic genes (middle). Immunoblotting of lysates from livers for Fas and Srebp-1c (right). d De novo lipogenesis, e Oil red O staining of liver tissue sections (left), and liver triglyceride levels (right) n = 5 per group. f Jmjd1c protein and mRNA levels (left two) in livers of mice 10 days after tail-vein injection of sh-JMJD1C adenovirus ( n = 5). mRNA levels of lipogenic genes (right). g De novo lipogenesis, h Oil red O staining of liver tissue sections (left) and liver triglyceride levels (right). Data are expressed as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, determined by two-tailed t -test. Scale bar = 100 μm. Source data are provided as a source data file.

Article Snippet: The following commercial antibodies were used: JMJD1C (sc-101073, 1:500), GAPDH (sc-32233, 1:2000), USF-1 (sc-229, 1:1000), HDAC1 (sc-7872, 1:1000), Fatty acid synthase (sc-55580, 1:1000), and SREBP-1c (sc-366, 1:1000) from SCBT.

Techniques: Western Blot, Infection, Quantitative RT-PCR, Injection, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Histone demethylase JMJD1C is phosphorylated by mTOR to activate de novo lipogenesis

doi: 10.1038/s41467-020-14617-1

Figure Lengend Snippet: a Schematics of conditional knockout allele of JMJD1C (left). RT-qPCR for Jmjd1c mRNA levels in tissues, n = 5 mice (middle) and immunoblotting of liver tissue lysates with JMJD1C antibody (right). b RT-qPCR for mRNA (left), immunoblotting of liver tissue lysates with Fas and Srebp-1c antibodies (middle), and nascent RNA (right). c Oil red O staining of liver tissue sections and liver triglyceride level. d Serum triglyceride and FFA levels in 12-week-old mice on chow diet. e Periodic acid–Schiff (PAS) staining for glycogen of liver tissues and glycogen levels in liver. a – e n = 8 mice per group. f – n JMJD1C-LKO mice and their WT littermates were fed a High CHO (70 kcal% carb) for 4 months after weaning, n = 6 mice per group. f mRNA (left) and nascent RNA (right), g de novo lipogenesis, h liver triglycerides, i Oil red O staining of liver tissue sections, j serum triglyceride levels, k liver glycogen levels, l and tissue weights. m GTT and ITT for high-CHO diet, and n serum insulin level. Data are expressed as means ± SEM. * P < 0.05, ** P < 0.01, determined by two-tailed t -test. Scale bar = 100 μm. Source data are provided as a source data file.

Article Snippet: The following commercial antibodies were used: JMJD1C (sc-101073, 1:500), GAPDH (sc-32233, 1:2000), USF-1 (sc-229, 1:1000), HDAC1 (sc-7872, 1:1000), Fatty acid synthase (sc-55580, 1:1000), and SREBP-1c (sc-366, 1:1000) from SCBT.

Techniques: Knock-Out, Quantitative RT-PCR, Western Blot, Staining, Two Tailed Test

Journal: Molecular Metabolism

Article Title: NF-κB signaling in tanycytes mediates inflammation-induced anorexia

doi: 10.1016/j.molmet.2020.101022

Figure Lengend Snippet: IL-1β stimulates the release of PGE 2 from tanycytes by activating the NF-κB pathway. (A) After treating primary tanycytes with IL-1β (0.25 μg/mL) or PBS for 24 h, Il1r1 (172 bp) and Gapdh (122 bp) mRNA were detected by RT-PCR (representative images of the agarose gels). (B–D) Expression of Il1b (B), Vcam1 (C), and Ptgs 2 (D) over time after IL-1β stimulation of primary tanycytes. Two-way ANOVA for treatment: Il1b , F (1,20) = 21.33, P = 0.0002; Vcam1 , F (1,20) = 12.09, P = 0.0024; Ptgs2 , F (1,19) = 6.11, P = 0.023. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 (Bonferroni posttest, n = 3 independent primary cell cultures of 3 wells/experiment). (E, F) Western blots of COX-2 after IL-1β (0.25 μg/mL) stimulation of primary tanyctes. GAPDH served as a loading control. Quantification of COX-2 protein levels at various time points after IL-1β stimulation (F). Two-way ANOVA for treatment, F (1,20) = 41.57, P < 0.0001. (Bonferroni posttest, n = 3 independent primary cell cultures of 3 wells/experiment). (G, H) Pretreatment with the IKK inhibitor BMS-345541 (BMS, 25 μM) blocked the induction of COX-2 by IL-1β (0.25 μg/mL) in primary tanycytes. Quantification of COX-2 protein levels (H). Two-way ANOVA; Bonferroni posttest, n = 2 independent primary cell cultures of 3 wells/experiment. (I) Quantification of PGE 2 levels in the medium showed that IL-1β stimulated PGE 2 secretion in an IKK-dependent manner. For IL-1β treatment, F (1,14) = 8.745, P = 0.0104; for BMS treatment, F (1,14) = 15.48, P = 0.0015; interaction, F (1,14) = 14.08, P = 0.0021, posttest: ∗ P = 0.017 (two-way ANOVA; Bonferroni posttest, n = 3 independent primary cell cultures of 3 wells/experiment).

Article Snippet: Real-time PCR was carried out on Applied Biosystems 7900 HT Fast Real-Time PCR System using the following exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystems): Il1r1 , Mm00434237_m1; Il1b , Mm00434228_m1; Ikbkg , Mm00494927_m1; Nfkb1 , Mm00476361_m1; Ptgs2 , Mm00478374_m1; Vcam1 , Mm01320970_m1; Vimentin , Mm01333430_m1; Darpp32 ( Ppp1r1b ), Mm00454892_m1; Gpr50 , Mm00439147_m1; Gfap , Mm01253033_m1; Npy , Mm03048253_m1; Pomc , Mm00435874_m1; r18S , Mm03928990_g1; Actb , Mm00607939.

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

Journal: Molecular Metabolism

Article Title: NF-κB signaling in tanycytes mediates inflammation-induced anorexia

doi: 10.1016/j.molmet.2020.101022

Figure Lengend Snippet: Glial deletion of Nemo ( Nemo gliaKO ) interferes with the induction of NF-κB target genes by IL-1β. (A–I) Immunohistochemistry showed that IL-1β (20 μg/kg, i.v.) upregulated the NF-κB target gene VCAM1 in α-tanycytes of Nemo FL but not of Nem o gliaKO mice. Representative immunostainings of VCAM1 in coronal mediobasal hypothalamus (MBH) sections of Nemo FL (A–C) and Nemo gliaKO (D–F) mice 8 h after IL-1β treatment and PBS-treated Nemo FL mice (G–I). (B), (C); (E), (F); and (H), (I) are higher magnifications of the boxed areas in (A), (D), and (G), respectively. Scale bar, 100 μm. (J) Quantification of VCAM1 staining in α-tanycytes projecting mainly into the VMH at various time points after IL-1β administration. Values are means ± SEM. Two-way ANOVA for genotype, F (1, 29) = 4.46, P = 0.044. ∗ P < 0.05 (Bonferroni posttest, n = 7–8 animals per group). (K) Nissl-stained coronal section of the MBH showing microdissected areas of the tanycytic layer and arcuate nucleus (ARC). ME, median eminence; 3 V, 3rd ventricle; scale bar, 100 μm. (L) Representative agarose gels after RT-PCR for Nemo and β-actin in microdissected tanycytes from Nemo FL and Nem o gliaKO mice. (M, N) Ptgs2 mRNA was reduced in tanycytes of Nem o gliaKO mice in comparison to Nemo FL controls 4 h after IL-1β treatment (M). In contrast, there was no difference in the ARC (N). ∗ P < 0.05 (Mann–Whitney test, n = 6–8 mice/group). (O) Pomc mRNA was reduced in tanycytes of Nem o gliaKO mice in comparison to Nemo FL controls 4 h after IL-1β treatment (Mann–Whitney test, n = 6–8 mice/group). (P, Q) Plasma concentrations of IL-6 (P) and IL-1β (Q) did not differ between Nemo FL and Nemo gliaKO animals after intravenous IL-1β injection. Values are means ± SEM (n = 6–8 mice/group).

Article Snippet: Real-time PCR was carried out on Applied Biosystems 7900 HT Fast Real-Time PCR System using the following exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystems): Il1r1 , Mm00434237_m1; Il1b , Mm00434228_m1; Ikbkg , Mm00494927_m1; Nfkb1 , Mm00476361_m1; Ptgs2 , Mm00478374_m1; Vcam1 , Mm01320970_m1; Vimentin , Mm01333430_m1; Darpp32 ( Ppp1r1b ), Mm00454892_m1; Gpr50 , Mm00439147_m1; Gfap , Mm01253033_m1; Npy , Mm03048253_m1; Pomc , Mm00435874_m1; r18S , Mm03928990_g1; Actb , Mm00607939.

Techniques: Immunohistochemistry, Staining, Reverse Transcription Polymerase Chain Reaction, Comparison, MANN-WHITNEY, Clinical Proteomics, Injection

Journal: Pharmaceuticals

Article Title: Computational Approach Reveals Pronociceptive Potential of Cannabidiol in Osteoarthritis: Role of Transient Receptor Potential Channels

doi: 10.3390/ph14100964

Figure Lengend Snippet: Molecular changes in inflammatory factor expression in rat cartilage and subchondral bone. Gene expression of Il6 ( A ), Ccl2 ( B ), Fabp3 ( C ), Comp ( D ), Ptgs2 ( E ) and Alox12 ( F ) was evaluated in cartilage and subchondral bone tissue at Day 28 post MIA and 5 h after drug administration. CBD was administered at a dose of 50 mg/kg at both Days 21 and 28 post MIA injection, either alone (CBD) or in combination with AP-18 (0.2 mg/kg) at Day 21 and HC-067047 (10 mg/kg) at Day 28 post MIA (HC). ANOVA revealed significant changes in expression of Il6 , Ccl2 , Fabp3 , Comp with p < 0.005. Individual data points are shown in boxes presenting the mean ± SEM of fold change normalized to the reference gene beta-2 microglobulin ( B2m ). ● denotes samples in control group; ■ denotes samples in MIA group; ▲ denotes samples in MIA + CBD group; ▼ denotes samples in MIA + CBD + AP-18 followed by HC-067047 group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test with a p < 0.05 confidence interval. Each experimental group includes N = 4–6 rats. * Denotes significant differences vs. control; # Denotes significant differences vs. MIA; $ Denotes significant differences vs. CBD.

Article Snippet: The following assays (TaqMan Gene Expression Assays, Life Technologies, Carlsbad, USA) were used in the experiment: Rn00560865_m1 ( B2m ), Rn00563255_m1 ( Comp ), Rn00580555_m1 ( Ccl2 ), Rn01410330_m1 ( Il6 ), Rn00577366_m1 ( Fabp3 ), Rn04342831_s1 ( Cb2 ), Rn00583117_m1 (Trpv1), Rn00577086_m1 ( Faah ), Rn00440861_m1 (Prkcg), Rn01432300_g1 ( Prkaca ), Rn00578842_m1 ( Mapk14 ), Rn00820922_g1 ( Mapk3 ), Rn01483828_m1 ( Ptgs2 ), Rn01461082_mL ( Alox12 ), Rn01473803_m1 ( Trpa1 ), Rn00576745_m1 ( Trpv4 ), Rn00592665_m1 ( Trpm8 ), Rn01482210_m1 ( Htr3a ), Rn00568803_m1 ( Gabra5 ), and Rn00440945_m1 ( Pparg ), Rn03037213_s1 ( Gpr55 ).

Techniques: Expressing, Gene Expression, Injection, Control

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: CD36 is overexpressed in human CRC. (A,B) Immunoreactivity score of CD36 expression was analyzed in matched normal colon mucosa and tumor tissues from patients diagnosed with Stage I–IV CRC (TMA: n = 56, * p < 0.001 vs. normal tissue). (C) CD36 staining in matched normal colon mucosa, primary CRC, and CRC metastasis to liver and lung [representative images are shown; liver ( n = 12) and lung metastasis ( n = 5)]. (D) Correlations between FASN and CD36 was determined based on RNASeq data of CRC patient samples ( n = 22 of normal tissues and n = 215 of tumors) from The Cancer Genome Atlas. (E) Expression of FASN and CD36 in human normal colon mucosa and tumor tissues. N, normal mucosa; T, primary tumor; L, normal liver tissue; M, liver metastasis.

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Expressing, Staining

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: Expression of CD36 is selectively regulated by the level of de novo fatty acid synthesis in CRC. (A) shRNA-mediated knockdown of FASN leads to upregulation of CD36 mRNA expression in HCT116 cells (* p < 0.05). (B) TVB-3664 treatment of CRC tissue slices (18 h) selectively upregulates CD36 mRNA expression (* p < 0.05). (C) TVB-3664 treatment of Pt 93 and Pt 130 primary CRC cells increases CD36 mRNA and protein expression. (D) shRNA mediated knockdown of FASN increases CD36 protein expression in HCT116 and HT29 cells. (E) Relative mRNA expression of FASN and CD36 in intestinal tumors collected from APC/Cre and FASN +/− /APC/Cre mice. (F) FASN and CD36 protein expression in intestinal mucosa collected from Apc/Cre and Apc/Cre mice with hetero- and homo-zygous deletion of FASN. (G,H) Inhibition of FASN increases membrane-associated expression of CD36. (G) Confocal images of FASN and CD36 in control and 0.2 μM TVB-3664 treated (6 days) Pt 93 primary CRC cells. (H) Flow cytometry analysis of Pt 93 and Pt 130 primary CRC cells treated with 0.2 μM TVB-3664 (6 days) in normal and serum free media conditions. Mean fluorescence for CD36 is shown for representative data from three different experiments (** p < 0.01, * p < 0.05). (I) FA uptake in HCT116, NTC, and FASN shRNA. Cells were pre-treated with anti-CD36 antibody or vehicle for 24 h and then treated with BODIPY FL for 10 min.

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Expressing, shRNA, Knockdown, Inhibition, Membrane, Control, Flow Cytometry, Fluorescence

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: Inhibition of CD36 is associated with decreased cellular proliferation. (A) Primary Pt 93 and Pt 130 CRC cells treated with 100 μM SSO for 6 days. Cellular proliferation assays were performed via cell count. Representative data from three experiments is shown (* p < 0.05). (B) Confocal images of FASN and CD36 in Pt 93 cells in normal and serum free media (6 days). (C) Expression of proteins associated with apoptosis and survival in HCT116 transfected with CD36 shRNAs and analyzed via western blot. (D) Cellular proliferation assay with HCT116, NTC, and CD36 shRNA (* p < 0.05 for normal medium, # p < for SFM). (E) qRT-PCR confirmation of CD36 knockdown using CD36 shRNA #2 (73%) and CD36 shRNA #4 (67%).

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Inhibition, Cell Counting, Expressing, Transfection, Western Blot, Proliferation Assay, shRNA, Quantitative RT-PCR, Knockdown

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: SSO treatment and CD36 knockdown inhibit tumor growth in vivo . (A) Tumor volume, tumor weight, and mouse weight of control and SSO treated (20 mg/kg) mice are shown. SSO was dissolved in 10% PEG and administered in 200 μl dosages via oral gavage daily. 1.0 × 10 6 cells were injected into NU/NU mice. Treatment was initiated when tumors reached ~100 mm 3 (day 0). (B) RT-PCR analysis of HCT116 tumors showing the effect of SSO treatment on CD36, FASN, and survivin mRNA expression. (C) Tumor volume of HCT116 NTC and CD36 shRNA #2 and #4 xenografts is shown. 1.0 × 10 6 cells were injected into NU/NU mice and tumor growth was measured every 3 days. (D) H&E and Ki67 staining of HCT116 NTC and CD36 shRNA tumors. (E) Tumor volume and tumor weight of HT29 LuM3 NTC and CD36 shRNA #4 xenografts are shown. (F) mRNA expression of survivin in HT29 LuM3 xenografts (analysis of tumors from 2 mice per group is show).

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Knockdown, In Vivo, Control, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, shRNA, Staining

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: High expression of CD36 is associated with an increase in pAkt and survivin in CRC. (A) Diagram of Pt 2402 propagation after flow cytometry sorting for CD36 high and CD36 low cells. (B) Numbers of CD36 high and CD36 low Pt 2402 cells for first and second flow cytometry sorts. (C) Protein expression levels of FASN, CD36, and survivin in CD36 high and CD36 low Pt 2402 primary cells from first flow cytometry sort. (D) IHC staining for Ki67 in Pt 2402 CD36 high and CD36 low tumors. (E) Protein expression levels of FASN, CD36, pAkt, cleaved PARP, and survivin in HCT116 CRC cells, control, and CD36 overexpression.

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Expressing, Flow Cytometry, Immunohistochemistry, Control, Over Expression

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: Inhibition of CD36 and FASN have a synergetic effect in reducing cell proliferation. (A) Pt 93, Pt 130, and HCT116 cells were treated with SSO and TVB-3664 alone or in combination for 6 days and cell number was counted. Representative data from three experiments is shown (* p < 0.05). (B) Western blot analysis of cells treated with TVB-3664, SSO, or TVB-3664 and SSO in combination.

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Inhibition, Western Blot

Journal: Nature

Article Title: IL-11 is a crucial determinant of cardiovascular fibrosis

doi: 10.1038/nature24676

Figure Lengend Snippet: 84 patients undergoing coronary artery bypass grafting donated right atrial biopsies

Article Snippet: The mouse plasma levels of urea and creatinine were quantified using urea assay kit (ab83362, Abcam) and creatinine assay kit (ab65340, Abcam), respectively.

Techniques:

Journal: Nature

Article Title: IL-11 is a crucial determinant of cardiovascular fibrosis

doi: 10.1038/nature24676

Figure Lengend Snippet: a, Representative histological images of the epicardium with Masson’s trichrome staining and ACTA2 immunostaining in mice with myocardial infarction (MI) treated with rmIL-11 or PBS. b, c, Epicardial thickness of regions distal (b) or proximal (c) to the region of myocardial infarction. a–c, Sham, n = 5; myocardial infarction, n = 6; myocardial infarction + rmIl-11, n = 8 biologically independent samples. d, e, Echocardiography show a decrease in ejection fraction (d) and increase in end-systolic volume (ESV) (e) after myocardial infarction and rmIl-11 treatment (sham, n = 3; myocardial infarction, myocardial infarction + rmIl-11, n = 5 biologically independent samples). b–e, two-tailed, Holm–Sidak-corrected Student’s t-test; Data are mean ± s.d. f, Representative histological images (chosen from control, n = 3; rmIl-11 injection, n = 4 biologically independent samples) of tissues from a Col1a1–GFP-reporter mouse after rmIL-11 injection (100 μg kg−1 per day, three weeks). g, Serum urea and creatinine levels after rmIL-11 injection (control urea, n = 8; control creatinine, n = 7; rmIl-11, n = 12 biologically independent samples). h, Reduced ejection fraction (echocardiography) in rmIl-11-treated mice. i, Hydroxyproline assay (HPA) quantifies cardiac and renal collagen content after rmIL-11 treatment. h, i, Control, n = 8; rmIl-11, n = 11 biologically independent samples. j, Representative histological images of Masson’s trichrome staining and ACTA2 immunostaining in the epicardium Il-11-Tg mice. k, HPA indicates cardiac and renal collagen content in Il-11-Tg mice. j, k, Control, n = 12; Il-11-Tg, n = 4 biologically independent samples. i, k, Two-tailed Student’s t-test; box-and-whisker plots show median (middle line), 25th–75th percentiles (box) and 10th–90th percentiles (whiskers). l, Reduction in ejection fraction (echocardiography) in Il-11-Tg mice (control, n = 6; Il-11-Tg, n = 4 biologically independent samples). g, h, l, Two-tailed Student’s t-test; data are mean ± s.d.; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The mouse plasma levels of urea and creatinine were quantified using urea assay kit (ab83362, Abcam) and creatinine assay kit (ab65340, Abcam), respectively.

Techniques: Staining, Immunostaining, Two Tailed Test, Control, Injection, Hydroxyproline Assay, Whisker Assay

Journal: Diabetes

Article Title: Genetic Disruption of Myostatin Reduces the Development of Proatherogenic Dyslipidemia and Atherogenic Lesions In Ldlr Null Mice

doi: 10.2337/db09-0349

Figure Lengend Snippet: Effects of Mstn deletion on liver of Ldlr −/− mice. A : Hematoxylin and eosin (H&E) and Oil Red O staining of the liver of Mstn +/+ /Ldlr −/− and Mstn −/− /Ldlr −/− mice after 12 weeks of HFD (HF-diet). B and C : mRNA expression of Srebf1 ( B ) and fatty acid synthase ( Fasn ) ( C ) in the liver of mice before and after 12 weeks of HFD. Values are expressed with respect to Mstn +/+ /Ldlr −/− controls. D : Protein expression of fatty acid synthase. Averages were taken from three different gels. E : Plasma apoB100 at 0 and 180 min after injection of Triton WR1339, a lipoprotein lipase inhibitor. VLDL secretion is determined as percent increase of apoB100 from baseline. Averages were taken from three different gels. F : Akt serine-473 and GSKα/β serine-21/9 phosphorylation in the liver of mice after 12 weeks of HFD. The graphs demonstrate the quantification of phosphorylation of each molecule. ++/−−, Mstn +/+ /Ldlr −/− . −−/−− , Mstn −/− /Ldlr −/− . AU, arbitrary units. Data are shown as the means ± SE ( n = 7–10). * P < 0.05, ** P < 0.01. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: All primary, except anti–fatty acid synthase antibodies (Cell Signaling Technology, Danvers, MA), and secondary antibodies were purchased from Santa Cruz Biotechnnology.

Techniques: Staining, Expressing, Clinical Proteomics, Injection, Phospho-proteomics

Journal: Nature Communications

Article Title: Light-responsive adipose-hypothalamus axis controls metabolic regulation

doi: 10.1038/s41467-024-50866-0

Figure Lengend Snippet: a–l Studies in 12-15-week-old C57BL/6 J male mice. a Schematic showing µLED device implantation in mouse scWAT (NO, RED, and BLUE light groups). b % BW change over 8 days (NO: n = 10, RED: n = 8, BLUE: n = 7). c Average food intake (NO: n = 7, RED: n = 4, BLUE: n = 4). d Tissue weight relative to BW (NO: n = 10, RED: n = 6, BLUE: n = 7). e Treated and untreated scWAT weight relative to BW (NO: n = 10, RED: n = 6, BLUE: n = 7). f Change in energy expenditure (ΔHeat) after norepinephrine (NE) injection (NO: n = 8, RED: n = 6, BLUE : n = 5). * P < 0.05, vs NO light group. g AUC quantification of ΔHeat (NO: n = 8, RED: n = 6, BLUE: n = 5). h Glucose levels during intraperitoneal glucose tolerance test (IPGTT) (NO, RED, BLUE: n = 6). * P < 0.05, vs NO light group, # P < 0.05, vs RED light group. (i) AUC of IPGTT (NO, RED, BLUE: n = 6). j–l Fasting plasma insulin (j ) (NO: n = 8, RED: n = 6, BLUE: n = 5), HOMA-IR ( k ) (NO: n = 7, RED: n = 5, BLUE : n = 5), and plasma leptin levels (l ) (NO: n = 9, RED: n = 7, BLUE: n = 6). m–u Studies in 20-25-week-old C57BL/6 J male DIO mice. ( m ) % BW change over 8 days (NO: n = 6, RED: n = 5, BLUE: n = 6). ( n ) Average food intake (NO, RED, BLUE: n = 5). o % changes in fat and lean mass over 8 days (DEXA scan) (NO: n = 6, RED: n = 5, BLUE: n = 6). p Treated and untreated scWAT weight relative to BW (NO: n = 6, RED: n = 5, BLUE: n = 5). (q) ΔHeat after NE injection (NO: n = 5, RED: n = 4, BLUE: n = 5). * P < 0.05 vs NO light group. r AUC quantification of ΔHeat (NO: n = 5, RED: n = 4, BLUE: n = 5). s Glucose levels during IPGTT (NO, RED, BLUE: n = 5). t AUC of IPGTT (NO, RED, BLUE: n = 5). u Fasting plasma insulin level (NO: n = 4, RED: n = 4, BLUE: n = 5). Statistics were performed by two-tailed paired Student’s t-tests ( e and p ), one-way ANOVA followed by Tukey’s post hoc test ( b–d, g, i–o, r, t, and u ), and two-way ANOVA followed by Tukey’s post hoc test ( f, h, q , and s ). n.s. indicates no significant difference. Data are represented as mean ± SEM. The diagram in a was created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: Plasma leptin, FA, and NE were determined by ELISA (Mouse/Rat Leptin Quantikine ELISA kit, cat. no. MOB00B, R&D systems; Free fatty acid assay kit, cat. no. ab65341, Abcam; and Norepinephrine ELISA kit, cat. no. MBS760375, MyBioSource), respectively.

Techniques: Injection, Clinical Proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: Light-responsive adipose-hypothalamus axis controls metabolic regulation

doi: 10.1038/s41467-024-50866-0

Figure Lengend Snippet: a–d Principal components analysis (PCA) plot ( a ), metabolite set enrichment analysis (MSEA) of altered pathways in circulation in blue lighted groups compared with non-lightened group (b ) or red lightened group ( c ), and variable importance in projection (VIP) ( d ) from metabolomics analysis of plasma from 12-15-week-old C57BL/6 J male mice exposed to scWAT by different light wavelengths for 8 days (NO light: n = 7, RED light: n = 6, and BLUE light: n = 6). The colored boxes on the right ( d ) indicate the relative concentrations of the corresponding metabolite in each group under study. e The relative abundance of circulating histidine and carnosine within the histidine metabolism pathway, employing metabolomics analysis (NO light: n = 7, RED light: n = 6, and BLUE light: n = 6). ( f and g ) Circulating histidine levels in C57BL/6 J male mice (NO light: n = 6, RED light: n = 6, and BLUE light: n = 6) ( f ) and Opn3-GKO male mice (NO light: n = 5, and BLUE light: n = 4) ( g ) with/without light treatment for 8 days, measured by ELISA. h and i Clustered heatmap from metabolomics analysis ( h ) and the relative abundance of histidine and carnosine ( i ) in treated scWAT with 8 days of blue light exposure relative to no light, employing metabolomics analysis (NO light: n = 6, and BLUE light: n = 5). ( j ) A positive correlation between the relative abundance of circulating histidine and the histidine in treated scWAT ( n = 11). k–m Relative mRNA expression of histidine metabolism genes, Hdc and Carns1, in treated scWAT ( n = 8 per group) ( k ), murine white adipocytes (n = 4 per condition, three biological replicates) ( l ), and human white adipocytes ( n = 3 per condition, three biological replicates) ( m ) exposed to 8 days of blue light relative to dark condition. Statistics were performed by MSEA ( b and c ), VIP score ( d ) using MetaboAnalyst, two-tailed unpaired Student’s t-tests ( g, i, and k–m ), one-way ANOVA followed by Tukey’s post hoc test ( e and f ), and Spearman’s Rank correlation test (two-tailed) ( j ). n.s. indicates no significant difference. n.d. indicates no determined. Data are represented as mean ± SEM.

Article Snippet: Plasma leptin, FA, and NE were determined by ELISA (Mouse/Rat Leptin Quantikine ELISA kit, cat. no. MOB00B, R&D systems; Free fatty acid assay kit, cat. no. ab65341, Abcam; and Norepinephrine ELISA kit, cat. no. MBS760375, MyBioSource), respectively.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test

Journal: Journal of Translational Medicine

Article Title: TC2N inhibits distant metastasis and stemness of breast cancer via blocking fatty acid synthesis

doi: 10.1186/s12967-023-04721-3

Figure Lengend Snippet: TC2N expression is related to tumor metastasis and differentiation status of BC patients. A The protein expression of TC2N was detected by IHC staining in BC tissues. High TC2N expression group contains the ≥ 3 score patients. Low TC2N expression group contains the < 3 score patients. Scale bars represent 20 μm. B TC2N expression was significantly decreased in advanced-stage BC patients than that in early-stage BC patients. The P value was measured with Student’s t-tests. *P < 0.05; **P < 0.01; ***P < 0.001. C TC2N expression was obviously decreased in BC patients with lymph node metastasis (N 1–3 ) than that without lymph node metastasis (N 0 ). The P value was measured with Student’s t-tests. ***P < 0.001. D TC2N expression was significantly decreased in BC patients with distant-metastasis (M 1 ) than that without metastasis (M 0 ). The P value was measured with Student’s t-tests. ***P < 0.001. Scale bars represent 20 μm. E The expression of TC2N in different metastatic sites. The P value was measured with Student’s t-tests. ns, no significance. F TC2N expression in BC tissues at different differentiating degree. The P value was measured with Student’s t-tests. ns, no significance, *P < 0.05. G TCGA BC database identified the association between TC2N expression and metastasis and differentiation-related processes. H CPTAC BC protein dataset identified the association between TC2N expression and metastasis and differentiation-related processes. I Kaplan–Meier analysis of the correlation between TC2N expression and overall survival time in 212 BC patients. J Cox-regression analysis of the correlation between TC2N expression and overall survival time in 212 BC patients

Article Snippet: Briefly, the protein expression of TC2N and FASN were detected using their primary antibodies: 1:200 diluted rabbit polyclonal antibody (Cat. #: HPA027549; Sigma) for TC2N; 1:500 diluted rabbit polyclonal antibody (Cat. #: 10624-2-AP; Proteintech) for FASN.

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Translational Medicine

Article Title: TC2N inhibits distant metastasis and stemness of breast cancer via blocking fatty acid synthesis

doi: 10.1186/s12967-023-04721-3

Figure Lengend Snippet: TC2N inhibits metastatic phenotype of BC cells in vitro and in vivo. A Overexpression of TC2N in M231, MCF7 and SKBR3 cells were examined by WB. B Effects of TC2N overexpression on migration and invasion of M231, MCF7 and SKBR3 cells were detected by transwell assays. Mean ± SEM (n = 3). The P value was measured with Student’s t-tests. **P < 0.01, ***P < 0.001. C Silencing of TC2N in M231-TC2N, MCF7-TC2N and SKBR3-TC2N stable transfectants were examined by WB. D Effects of TC2N knockdown on migration and invasion of M231-TC2N, MCF7-TC2N and SKBR3-TC2N cells were detected by transwell assays. Mean ± SEM (n = 3). The P value was measured with Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001. E Bioluminescent images of brain and heart tissues from female nude mice 50 days post intracardiac injection of SKBR3 cell line with TC2N ectopic expression. F Tissues were exfoliated from mice (n = 4) that injected with stable transfected SKBR3 cells. Organs invaded by BC cells were evaluated by H&E staining (Left). Scale bar represent 100 μm. The number of metastatic nodules in each organ were showed (Right). The P value was measured with Student’s t-tests. *P < 0.05

Article Snippet: Briefly, the protein expression of TC2N and FASN were detected using their primary antibodies: 1:200 diluted rabbit polyclonal antibody (Cat. #: HPA027549; Sigma) for TC2N; 1:500 diluted rabbit polyclonal antibody (Cat. #: 10624-2-AP; Proteintech) for FASN.

Techniques: In Vitro, In Vivo, Over Expression, Migration, Knockdown, Injection, Expressing, Transfection, Staining

Journal: Journal of Translational Medicine

Article Title: TC2N inhibits distant metastasis and stemness of breast cancer via blocking fatty acid synthesis

doi: 10.1186/s12967-023-04721-3

Figure Lengend Snippet: TC2N interferes with the CSC-like characteristics of BC cells. A WB analysis of NANOG and SOX2 in stable M231, MCF7 and SKBR3 cells. B Sphere formation ability of BC cells with ectopic expression of TC2N. Mean ± SEM (n = 3). The P value was measured with Student’s t-tests. **P < 0.01. C Fractions of CD44 + CD24 − BC cell-spheres with TC2N ectopic expression were determined by flow cytometry. Mean ± SEM (n = 3). The P value was measured with Student’s t-tests. ***P < 0.001. D Fractions of ADLH + BC cells with TC2N ectopic expression were determined by flow cytometry. Mean ± SEM (n = 3). The P value was measured with Student’s t-tests. **P < 0.01, ***P < 0.001. E The tumor growth curves of M231 spheres (n = 5). *P < 0.05. F The image of nude mice subcutaneously injected with M231 spheres. G Tumor weight from the Vc and TC2N group. **P < 0.01. H IHC evaluation of CD133 in xenograft tumor. Scale bar represent 100 μm

Article Snippet: Briefly, the protein expression of TC2N and FASN were detected using their primary antibodies: 1:200 diluted rabbit polyclonal antibody (Cat. #: HPA027549; Sigma) for TC2N; 1:500 diluted rabbit polyclonal antibody (Cat. #: 10624-2-AP; Proteintech) for FASN.

Techniques: Expressing, Flow Cytometry, Injection

Journal: Journal of Translational Medicine

Article Title: TC2N inhibits distant metastasis and stemness of breast cancer via blocking fatty acid synthesis

doi: 10.1186/s12967-023-04721-3

Figure Lengend Snippet: TC2N decreases the fatty acid content in BC. A CPTAC BC protein dataset identified the association between TC2N expression and lipid metabolism. B Effects of TC2N overexpression on total free fatty acids levels of M231, MCF7 and SKBR3 cells. The P value was measured with Student’s t-tests. **P < 0.01, ***P < 0.001. C Lipid metabolism was detected by LC–MS/MS in stable transfected M231 cells. Heatmap was performed and the significant differences were analyzed using Student’s t-test, n = 4. D The growth of E0771 cells stably expressing control lentivirus or TC2N overexpression lentivirus in nude mice. Block arrows indicate the tumor masses. E The fatty deposit of E0771 tumor masses were detected by oil red O staining. Scale bar represent 100 μm. F Immunofluorescence analysis of the localization of TC2N and FASN in MCF7-TC2N cells. DAPI serves as a nuclear counterstain. Scale bars represent 25 μm. G Immunofluorescence revealed that TC2N decreases the protein levels of FASN in MCF7 cells. Scale bars represent 25 μm. H qRT-PCR revealed that TC2N do not change the mRNA expression of FASN in M231 and MCF7 cells. The P value was measured with Student’s t-tests. ns, no significance. I WB revealed that TC2N decreases the protein expression of FASN in M231 and MCF7 cells

Article Snippet: Briefly, the protein expression of TC2N and FASN were detected using their primary antibodies: 1:200 diluted rabbit polyclonal antibody (Cat. #: HPA027549; Sigma) for TC2N; 1:500 diluted rabbit polyclonal antibody (Cat. #: 10624-2-AP; Proteintech) for FASN.

Techniques: Expressing, Over Expression, Liquid Chromatography with Mass Spectroscopy, Transfection, Stable Transfection, Control, Blocking Assay, Staining, Immunofluorescence, Quantitative RT-PCR

Journal: Journal of Translational Medicine

Article Title: TC2N inhibits distant metastasis and stemness of breast cancer via blocking fatty acid synthesis

doi: 10.1186/s12967-023-04721-3

Figure Lengend Snippet: TC2N promotes the degradation of FASN via blocking the interaction between PTEN and FASN. A The extracts from M231 cells with control lentivirus or TC2N overexpression lentivirus treated with 20 µM cycloheximide (CHX) for the indicated times were subjected to WB (Upper). Relative FASN protein levels were quantified by ImageJ software (Lower). B M231 cells with control lentivirus or TC2N overexpression lentivirus were transfected with 2 µg of ubiquitin-expressing plasmids. At 24 h after the transfection, cells were treated with 20 µM MG132 for 24 h. Cell extracts were subjected to IP with anti-FASN antibody and further analyzed by WB with anti-Ubiquitin and anti- Tyrosine antibody. Normal IgG was used as a negative control. Whole-cell lysates were used as a positive control (Input). C The extracts from BC cells were subjected to IP with anti-Flag antibody and further analyzed by WB with TC2N, FASN, TRIM21 and PTEN antibodies. Normal IgG was used as a negative control. Whole-cell lysates were used as a positive control (Input). D and E BC cells were treated with 20 µM MG132 for 24 h, and then cell extracts were subjected to IP with anti-FASN or anti-TRIM21 antibodies and further analyzed by WB with indicated antibodies. Normal IgG was used as a negative control. Whole-cell lysates were used as a positive control (Input). F The cell extracts from BC cells were subjected to IP with anti-FASN antibody and further analyzed by WB with indicated antibodies. Normal IgG was used as a negative control. Whole-cell lysates were used as a positive control (Input). G The extracts from M231 cells stably expressing FLAG-Vc or FLAG-TC2N (full-length and truncation) were subjected to IP with anti-Flag antibody. Elutes were resolved using SDS-PAGE and silver-stained. Block arrows indicate the bands of TC2N. H The extracts from M231 cells stably expressing FLAG-Vc or FLAG-TC2N (full-length and truncation) were subjected to IP with anti-FASN antibody and further analyzed by WB with indicated antibodies. Normal IgG was used as a negative control. Whole-cell lysates were used as a positive control (Input)

Article Snippet: Briefly, the protein expression of TC2N and FASN were detected using their primary antibodies: 1:200 diluted rabbit polyclonal antibody (Cat. #: HPA027549; Sigma) for TC2N; 1:500 diluted rabbit polyclonal antibody (Cat. #: 10624-2-AP; Proteintech) for FASN.

Techniques: Blocking Assay, Control, Over Expression, Software, Transfection, Ubiquitin Proteomics, Expressing, Negative Control, Positive Control, Stable Transfection, SDS Page, Staining

Journal: Journal of Translational Medicine

Article Title: TC2N inhibits distant metastasis and stemness of breast cancer via blocking fatty acid synthesis

doi: 10.1186/s12967-023-04721-3

Figure Lengend Snippet: TC2N decreases the levels of PTEN neddylation by preventing the nuclear import of PTEN. A M231 cell extracts were subjected to IP with anti-PTEN antibody and further analyzed by WB with anti-NEDD8 antibody. Normal IgG was used as a negative control. Whole-cell lysates were used as a positive control (Input). B Immunofluorescence revealed that TC2N decreases the nuclear localization of PTEN in MCF7 cells. Scale bars represent 25 μm. C PTEN protein level in nucleus and cytoplasm were analyzed by WB. D The extracts from BC cells were subjected to IP with anti-Flag antibody and further analyzed by WB with anti-importinβ and anti-IPO5 antibodies. Normal IgG was used as a negative control. Whole-cell lysates were used as a positive control (Input). E The cell extracts from BC cells were subjected to IP with importinβ antibody and further analyzed by WB with indicated antibodies. Normal IgG was used as a negative control. Whole-cell lysates were used as a positive control (Input)

Article Snippet: Briefly, the protein expression of TC2N and FASN were detected using their primary antibodies: 1:200 diluted rabbit polyclonal antibody (Cat. #: HPA027549; Sigma) for TC2N; 1:500 diluted rabbit polyclonal antibody (Cat. #: 10624-2-AP; Proteintech) for FASN.

Techniques: Negative Control, Positive Control, Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: TC2N inhibits distant metastasis and stemness of breast cancer via blocking fatty acid synthesis

doi: 10.1186/s12967-023-04721-3

Figure Lengend Snippet: TC2N represses FASN-driven tumor progression and its high expression in BC indicate low fatty deposition and good prognosis. A WB analysis of TC2N and FASN expression in stable M231, MCF7 and SKBR3 cells with 0.2 μM TVB-3166 treatment (Upper). Transwell assays were used to examine the effect of FASN ablation on cell migration and invasion of BC stable cells (Under). The P value was measured with Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001. B CD133 + analysis was used to examine the effect of FASN ablation on stemness of BC stable cell spheres. Mean ± SEM (n = 3). The P value was measured with Student’s t-tests. ***P < 0.001. C ALDH + analysis was used to examine the effect of FASN ablation on stemness of BC stable cells. Mean ± SEM (n = 3). The P value was measured with Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001. D The fatty deposits were detected by oil red O staining in tumor tissues of BC patients. Scale bar represent 100 μm. E and F Survivorship curves showed that the overall survival rates of BC patients with high or low levels of FASN and of those with low expression of TC2N and high expression of FASN or high expression of TC2N and low expression of FASN. The high or low levels of TC2N and FASN were defined by their median levels in the BC

Article Snippet: Briefly, the protein expression of TC2N and FASN were detected using their primary antibodies: 1:200 diluted rabbit polyclonal antibody (Cat. #: HPA027549; Sigma) for TC2N; 1:500 diluted rabbit polyclonal antibody (Cat. #: 10624-2-AP; Proteintech) for FASN.

Techniques: Expressing, Migration, Stable Transfection, Staining

Journal: Journal of Translational Medicine

Article Title: TC2N inhibits distant metastasis and stemness of breast cancer via blocking fatty acid synthesis

doi: 10.1186/s12967-023-04721-3

Figure Lengend Snippet: A schematic diagram reveals the regulatory mechanism by which TC2N induces FASN degradation

Article Snippet: Briefly, the protein expression of TC2N and FASN were detected using their primary antibodies: 1:200 diluted rabbit polyclonal antibody (Cat. #: HPA027549; Sigma) for TC2N; 1:500 diluted rabbit polyclonal antibody (Cat. #: 10624-2-AP; Proteintech) for FASN.

Techniques:

Journal: Journal of Translational Medicine

Article Title: TC2N inhibits distant metastasis and stemness of breast cancer via blocking fatty acid synthesis

doi: 10.1186/s12967-023-04721-3

Figure Lengend Snippet: Association of TC2N expression with BC clinicopathological characteristics

Article Snippet: Briefly, the protein expression of TC2N and FASN were detected using their primary antibodies: 1:200 diluted rabbit polyclonal antibody (Cat. #: HPA027549; Sigma) for TC2N; 1:500 diluted rabbit polyclonal antibody (Cat. #: 10624-2-AP; Proteintech) for FASN.

Techniques: Expressing

Journal: PLOS Pathogens

Article Title: Retinol binding protein 4 enhances cellular cholesterol uptake to facilitate influenza A virus infection

doi: 10.1371/journal.ppat.1013623

Figure Lengend Snippet: (A, B) Quantification of total cholesterol levels in multiple WT and RBP4-deficient cell types (A) and in serum from WT and RBP4-deficient mice (B). (C) Heatmap representation of RNA sequencing analysis showing differentially expressed genes related to cholesterol metabolism in WT and RBP4-deficient BMDMs. (D, E) qPCR analysis of CD36 mRNA expression in WT and RBP4-deficient BMDMs (D, left) and HEK293T cells (D, right), or in lung tissues from WT and RBP4-deficient mice (E). (F, G) Immunoblotting analysis of CD36 protein levels in multiple WT and RBP4-decficent cells as indicated (F) or in lung tissues from WT and RBP4-deficient mice (G). (H to J) Immunoblotting analysis of protein levels of CD36, total JNK, phosphorylated (p)-JNK, STAT1, and p-STAT1 as indicated in PK-15 or 3D4/21 cells pretreated with SP600125 (SP, 10 μM) or Resatorvid (TAK, 1 μM) for 2 hours, followed by treatment with rpRBP4 for 24 hours. (K) Immunoblotting analysis of protein expression of CD36, NP and M1 in PK-15 cells treated as described in (H), followed by infection with IAV WSN strain (MOI = 0.1) for 24 hours. (L) Immunoblotting analysis of CD36, p-STAT1, total STAT1 and RBP4 protein levels in HEK293T cells transfected with RBP4 expression plasmid or control empty vector, followed by treatment with Ruxolitinib (Ruxo, 5 μM) for 2 hours. (M) Immunoblotting analysis of CD36, p-STAT1, STAT1, STRA6 and RBP4 protein levels in HEK293T cells co-transfected with RBP4 expression plasmid and siRNA for STRA6 or negative control for 36 hours. (N) Immunoblotting analysis of CD36, p-JAK2, JAK2, p-STAT1, STAT1 and RBP4 protein levels in HEK293T cells transfected with RBP4 expression plasmid or control empty vector, followed by treatment with Ruxo (5 μM) for 2 hours or transfected with siRNA for STRA6 or negative control for 36 hours. Data are pooled from three independent experiments (A, B, D, E, mean ± SD) or representative of two independent experiments (F-N). * p < 0.05, ** p < 0.01 (Student’s t -tes t ).

Article Snippet: The primary antibodies used in this study were sourced from commercial suppliers as follows: Rabbit polyclonal antibodies against RBP4 (1 : 2,000, 11774–1-AP), CD36 (1 : 2,000, 18836–1-AP), STRA6 (1 : 3,000, 22001–1-AP), p65 (1 : 1,000, 10745–1-AP) and β-actin (1 : 20,000, 66009–1-lg) were purchased from Proteintech Group Inc. Rabbit polyclonal antibodies against CD36 (1 : 1,000, CY5796) and FLAG (1 : 10,000, AB0030) were purchased from Abways.

Techniques: RNA Sequencing, Expressing, Western Blot, Infection, Transfection, Plasmid Preparation, Control, Negative Control

Journal: PLOS Pathogens

Article Title: Retinol binding protein 4 enhances cellular cholesterol uptake to facilitate influenza A virus infection

doi: 10.1371/journal.ppat.1013623

Figure Lengend Snippet: (A to D) Confocal microscopy (A and B) or flow cytometry (C and D) analysis of cholesterol uptake by Dil-OxLDL in WT and RBP4-deficient HEK293T cells or BMDMs. Scale bars, 100 μm. Statistics of mean fluorescence intensity was calculated by Image J software (A and B, right) or Flow Jo software (C and D, right). (E, F) Flow cytometry analysis of cholesterol uptake by Dil-OxLDL in WT, RBP4-deficient and CD36 overexpressing RBP4-deficient HEK293T cells (E) and BMDMs (F). Mean fluorescence intensities were quantified with Flow Jo software. (G, H) Flow cytometry analysis of SA expression in HEK293T cells (G, upper) transfected with hCD36 plasmid or in stable CD36-overexpressing iBMDMs (H, upper). Cells were left untreated or incubated with cholesterol for 1 hour at 37°C, followed by staining with SNA or MAL. Mean fluorescence intensities were quantified by Flow Jo software (lower panels). (I) Confocal microscopy analysis of SA expression and distribution in WT and CD36-overexpressing iBMDMs. Cells were left untreated or incubated with cholesterol for 1 hour at 37°C, followed by staining with SNA or MAL (green). Cell membranes were stained with Dil dye (red), and nuclei were counterstained with DAPI (blue). Data are representative (A-F, left, G, H, upper and I) or pooled from at least three independent experiments (A-F, right, G and H, lower, mean ± SD). * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t -test).

Article Snippet: The primary antibodies used in this study were sourced from commercial suppliers as follows: Rabbit polyclonal antibodies against RBP4 (1 : 2,000, 11774–1-AP), CD36 (1 : 2,000, 18836–1-AP), STRA6 (1 : 3,000, 22001–1-AP), p65 (1 : 1,000, 10745–1-AP) and β-actin (1 : 20,000, 66009–1-lg) were purchased from Proteintech Group Inc. Rabbit polyclonal antibodies against CD36 (1 : 1,000, CY5796) and FLAG (1 : 10,000, AB0030) were purchased from Abways.

Techniques: Confocal Microscopy, Flow Cytometry, Fluorescence, Software, Expressing, Transfection, Plasmid Preparation, Incubation, Staining

Journal: PLOS Pathogens

Article Title: Retinol binding protein 4 enhances cellular cholesterol uptake to facilitate influenza A virus infection

doi: 10.1371/journal.ppat.1013623

Figure Lengend Snippet: (A, B) Flow cytometry analysis of α-2,3-linked SA or α-2,6-linked SA in WT, RBP4-deficient and CD36 overexpressing RBP4-deficient HEK293T cells, as wells as murine BMDMs transduced with CD36 retrovirus. Mean fluorescence intensities were quantified with FlowJo software. (C, D) qPCR analysis of vRNA levels of the NP gene or NP and M1 mRNA expression in HEK293T cells (C) or BMDMs (D) infected with IAV WSN strain (MOI = 5) during the viral attachment stage. (E) Flow cytometry analysis of α-2,3-linked SA or α-2,6-linked SA in WT and stable CD36-overexpressing iBMDMs cultured in serum-free medium. Cells were cultured with serum-free medium for 6 hours and subsequently stained with Lectins. Mean fluorescence intensities were quantified with FlowJo software. Data are pooled from three independent experiments (A-E, mean ± SD). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t -test).

Article Snippet: The primary antibodies used in this study were sourced from commercial suppliers as follows: Rabbit polyclonal antibodies against RBP4 (1 : 2,000, 11774–1-AP), CD36 (1 : 2,000, 18836–1-AP), STRA6 (1 : 3,000, 22001–1-AP), p65 (1 : 1,000, 10745–1-AP) and β-actin (1 : 20,000, 66009–1-lg) were purchased from Proteintech Group Inc. Rabbit polyclonal antibodies against CD36 (1 : 1,000, CY5796) and FLAG (1 : 10,000, AB0030) were purchased from Abways.

Techniques: Flow Cytometry, Transduction, Fluorescence, Software, Expressing, Infection, Cell Culture, Staining

Journal: PLOS Pathogens

Article Title: Retinol binding protein 4 enhances cellular cholesterol uptake to facilitate influenza A virus infection

doi: 10.1371/journal.ppat.1013623

Figure Lengend Snippet: (A) Experimental scheme for lentiviral transduction and influenza virus infection in mice. WT and RBP4-deficient mice were intravenously injected with lentivirus (10 9 PFU in 200 μL per mouse) followed by infection with 10 4 PFU of IAV (WSN strain) 5 days later. Lungs were collected 3 days post-IAV infection. (B) qPCR analysis of CD36 mRNA levels in lung tissues from WT, RBP4-deficient, and CD36-overexpressing RBP4-deficient mice as treated in (A). (C) Body weight changes of indicated mouse genotypes at day 3 post-IAV infection. (D) Plaque assay analysis of virus titer in lung tissues from mice described in (A). (E, F) Immunofluorescence images (E, left) and quantitative analysis of NP protein intensity (E, right) or immunoblotting analysis of M1, NP and CD36 protein expression (F) in lung tissues from the mice described in (A). Mean fluorescence intensity was determined by Image J software (E, right). Scale bars, 100 μm. (G, H) Representative H&E-stained lung sections (G) and corresponding histopathological scores (H) from indicated groups. Each dot in (B-D, H) represents an individual mouse. (I) Proposed model of RBP4-mediated promotion of influenza virus infection. Created in BioRender. Huang, L. (2025) https://BioRender.com/rll2ll1 . Data are representative of two independent experiments (E-G). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t -test).

Article Snippet: The primary antibodies used in this study were sourced from commercial suppliers as follows: Rabbit polyclonal antibodies against RBP4 (1 : 2,000, 11774–1-AP), CD36 (1 : 2,000, 18836–1-AP), STRA6 (1 : 3,000, 22001–1-AP), p65 (1 : 1,000, 10745–1-AP) and β-actin (1 : 20,000, 66009–1-lg) were purchased from Proteintech Group Inc. Rabbit polyclonal antibodies against CD36 (1 : 1,000, CY5796) and FLAG (1 : 10,000, AB0030) were purchased from Abways.

Techniques: Transduction, Virus, Infection, Injection, Plaque Assay, Immunofluorescence, Western Blot, Expressing, Fluorescence, Software, Staining