Journal: Cancer & Metabolism

Article Title: Metabolic reprogramming induced by ketone bodies diminishes pancreatic cancer cachexia

doi: 10.1186/2049-3002-2-18

Figure Lengend Snippet: Ketone bodies inhibit tumor cell-conditioned medium-induced degradation of myofibers and adipolysis. Differentiated C2C12 cells were treated with S2-013 (A) and Capan1 (B) cell-conditioned medium with or without solvent control and 10 and 20 mM NaHB and LiAcAc for 72 h, and bright-field images were represented for individual treatments. (C) Differentiated C2C12 cells were cultured in Capan1 and S2-013 cell-conditioned medium with or without ketone body treatment for 24 h. Total RNA was isolated and relative mRNA levels of MuRF1 and Atrogin were determined by performing qRT-PCR. β-Actin was utilized as an internal control. Differentiated 3T3L1 cells were cultured in S2-013 (D) and Capan1 (E) cell-conditioned medium with or without ketone body treatment for 72 h and stained with nile red. Fluorescent and bright-field images for individual treatments are presented. (F) Differentiated 3T3L1 cells were cultured in Capan1 and S2-013 cell-conditioned medium with or without ketone body treatment for 24 h. Total RNA was isolated and relative mRNA levels of Zag and HSL were determined by qRT-PCR. β-Actin was utilized as an internal control. Values represented are mean ± SEM. All statistical analyses were conducted with one-way ANOVA with Dunnett’s post hoc test and CM as the reference group. * P < 0.05; ** P < 0.01.

Article Snippet: The human pancreatic cancer cell line Capan1, mouse myoblast C2C12, and mouse embryo fibroblast (preadipocyte) 3T3L1 were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Solvent, Control, Cell Culture, Isolation, Quantitative RT-PCR, Staining

Journal: Cancer & Metabolism

Article Title: Metabolic reprogramming induced by ketone bodies diminishes pancreatic cancer cachexia

doi: 10.1186/2049-3002-2-18

Figure Lengend Snippet: Pretreatment of tumor cells with ketone bodies or glycolytic inhibition diminishes their cachectic potential. S2-013 cells were treated with solvent control, 20 mM NaHB (NaHB-S2-013), 20 mM LiAcAc (LiAcAc-S2-013), and 10 μM 3-bromopyruvic acid (BPA-S2-013) for 24 h. The cells were then washed twice with phosphate-buffered saline and cultured in serum-free DMEM. After 24 h, the conditioned medium was collected. The conditioned medium was also prepared from GLUT1 knockdown S2-013 (S2-013-sh GLUT1 ) and control cells (S2-013-shScr). Differentiated myotubes from C2C12 cells were cultured in (A) control, S2-013-CM, NaHB-S2-013-CM, LiAcAc-S2-013-CM, and BPA-S2-013-CM or (B) control, S2-013-shScr-CM, and S2-013-sh GLUT1 -CM for 72 h, and bright-field images were represented for individual treatments. Differentiated 3T3L1 cells were cultured in (C) control, S2-013-CM, NaHB-S2-013-CM, LiAcAc-S2-013-CM, and BPA-S2-013-CM or (D) control, S2-013-shScr-CM, and S2-013-sh GLUT1 -CM for 72 h and stained with nile red, and images for individual treatments are represented. (E) Differentiated myotube form C2C12 cells were cultured in similar conditions for 24 h. Total RNA was isolated and relative mRNA levels of MuRF1 and Atrogin were determined by qRT-PCR. β-Actin was utilized as an internal control. (F) Differentiated 3T3L1 cells were cultured in the above-mentioned conditions for 24 h. Total RNA was isolated and relative mRNA levels of Zag and HSL were determined by qRT-PCR. β-Actin was utilized as an internal control. Values represented are mean ± SEM. All statistical analyses were conducted with one-way ANOVA with Dunnett’s post hoc test and S2-013-CM as the reference group.* P < 0.05; ** P < 0.01.

Article Snippet: The human pancreatic cancer cell line Capan1, mouse myoblast C2C12, and mouse embryo fibroblast (preadipocyte) 3T3L1 were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Inhibition, Solvent, Control, Saline, Cell Culture, Knockdown, Staining, Isolation, Quantitative RT-PCR

Journal: Current Issues in Molecular Biology

Article Title: Thymoquinone (TQ) Inhibits Inflammation and Migration of THP-1 Macrophages: Mechanistic Insights into the Prevention of Atherosclerosis Using In-Vitro and In-Silico Analysis

doi: 10.3390/cimb44040120

Figure Lengend Snippet: The effect of TQ on cholesterol efflux in THP-1 macrophages. Cholesterol efflux was assessed using cholesterol-loaded THP-1 cells treated with 5 or 10 µM of TQ for 24 h. Cholesterol efflux was calculated as a percent of media [3H]cholesterol per total cell and media [3H]cholesterol. Data were normalized to cells treated with DMSO as vehicle control. The data are presented as the mean ± SEM from two independent experiments.

Article Snippet: Cholesterol efflux was performed using a commercial cholesterol efflux assay kit (Cat No. ab196985) from (Abcam, Cambridge, UK), to investigate the effect of TQ on the progression of atherosclerosis in human THP-1 macrophages.

Techniques: Control

Journal: bioRxiv

Article Title: Tamoxifen Targets Wisp2 to Impair Subcutaneous Adipocyte Progenitor Self-Renewal and Adipogenic Differentiation

doi: 10.64898/2025.12.21.695772

Figure Lengend Snippet: (A) Mouse subcutaneous adipose stromal cells (primary ASCs) differentiate into lipid-laden adipocytes after 10 days in culture. Images are oil red O (ORO) stained cells at day 0 (D0) or day 10 (D10) of adipocyte differentiation. Graph shows quantification of ORO accumulation over time. (B-D) Expression of adipocyte progenitor markers Cd34 (b), Pdgfra (c), and Cd24a (d) in mouse primary ASCs during proliferation (pro) or through differentiation. (E-F) Expression of preadipocyte markers Pparg (e) and Fabp4 (f) in mouse primary ASCs during proliferation (pro) or through differentiation. (G-H) Expression of Esr1 gene (g) or ERα protein (h) in mouse immortalized APCs during proliferation (pro) or through differentiation. (I) undifferentiated mouse primary ASCs treated with EtOH (Veh), E 2 , 4-OH tamoxifen (TAM) or E 2 +TAM. Images represent cells at day 4 of treatment. Scale=200µm. Inset graph represents cell number over the 4-day proliferation assay. Two-way ANOVA testing for main effects of treatment and time; interaction p<0.001. (J) ORO accumulation after 10 days of primary ASC differentiation; cells treated as in (i). Graph represents ORO absorbance on day 10. Scale=200µm. (K) undifferentiated primary human subcutaneous adipose stromal cells treated with EtOH (Veh), E 2 , 4-OH tamoxifen (TAM) or E 2 +TAM for 7.5 days. Two-way ANOVA testing for main effects of treatment or time; interaction p<0.001. Inset graph is cell number on the final day of proliferation; E 2 versus Veh p=0.011; E 2 +TAM vs E 2 p<0.001 by t-test. Images represent cells at day 7.5 of treatment. (L) ORO in human primary adipose stromal cells. (M) Representative FACS plots of mouse primary ASCs treated with EtOH (Veh), E 2 , or E 2 +4-OH tamoxifen (E 2 +TAM) for 48 hours and stained for Sca1 and CD24. (N-O) Percent of cells positive for Sca1 and CD24 (progenitors; n) or Sca1-positive, CD24-negative (preadipocytes, o) after treated as described in (m). T-tests determined significance.

Article Snippet: Primary human subcutaneous preadipocytes (PCS-210-01, ATCC) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast low serum growth supplement (ATCC PCS-201-041).

Techniques: Staining, Expressing, Proliferation Assay

Journal: bioRxiv

Article Title: Tamoxifen Targets Wisp2 to Impair Subcutaneous Adipocyte Progenitor Self-Renewal and Adipogenic Differentiation

doi: 10.64898/2025.12.21.695772

Figure Lengend Snippet: (A-B) Primary mouse ASCs were treated with EtOH (Veh), E 2 , or E 2 +4-OH tamoxifen (E 2 +TAM) prior to and during adipogenic differentiation for 10 days. (a). Schematic of adipogenesis was created with BioRender https://BioRender.com/az4hymd . Cells were then analyzed for Sca1+/CD24+ progenitors or Sca1+/CD24-preadipocytes by FACS (b). (C) Primary mouse subcutaneous ASCs were isolated from 6 different adult females (ages 20-24 weeks), and plated in media containing EtOH (Veh), E 2 , or E 2 +4-OH tamoxifen (E 2 +TAM). Cells were grown under these conditions and passaged each time they reached 80% confluence. The number of passages was recorded. For each treatment group, the experiment ended when cells no longer reached 80% confluence after 21 days and did not adhere to the plate with the subsequent passage, having reached the end of their proliferative lifespan. The passage number at this point was recorded for each experiment. At each passage, an aliquot of cells was plated for adipogenic differentiation and stained with oil red O (ORO). The absorbance of ORO was measured in technical triplicate wells from cells under each treatment at each passage. (D) Final passage numbers of primary ASCs were recorded under each treatment condition. Mann-Whitney test determined significance. (E) Representative quantification of ORO absorbance from key passage numbers, representing the final passage achieved for each treatment condition (E 2 +TAM=P6; Vehicle=P8; E 2 =P10). Images are phase contrast and ORO-stained plates from the respective passages.

Article Snippet: Primary human subcutaneous preadipocytes (PCS-210-01, ATCC) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast low serum growth supplement (ATCC PCS-201-041).

Techniques: Isolation, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Tamoxifen Targets Wisp2 to Impair Subcutaneous Adipocyte Progenitor Self-Renewal and Adipogenic Differentiation

doi: 10.64898/2025.12.21.695772

Figure Lengend Snippet: (A) Venn diagram of genes that were higher in cells from obese versus lean female mice treated with E 2 , compared with genes that were lower after treatment of obese females with E 2 +tamoxifen (E 2 +TAM) or estrogen deprivation/withdrawal (EWD). Wisp2/Ccn5 was one of two genes in the overlap of all three gene lists. (B) UMAP showing the distribution and level of Wisp2/Ccn5 expression in primary mouse subcutaneous ASCs. (C) Violin plot showing the level of Wisp2 expression in each cluster, previously defined in Scalzo et al. (D) Bubble plot indicating the average expression level and percent positive cells for Wisp2 in each cluster and treatment group from primary mouse subcutaneous ASCs, as described in Scalzo, et al. (E-G) The percent of Wisp2 positive cells by treatment group in transitional progenitors (e), preadipocytes (f), and adipocyte regulatory cells (AREGs, g).

Article Snippet: Primary human subcutaneous preadipocytes (PCS-210-01, ATCC) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast low serum growth supplement (ATCC PCS-201-041).

Techniques: Expressing

Journal: bioRxiv

Article Title: Tamoxifen Targets Wisp2 to Impair Subcutaneous Adipocyte Progenitor Self-Renewal and Adipogenic Differentiation

doi: 10.64898/2025.12.21.695772

Figure Lengend Snippet: (A) Expression of Wisp2 gene (left) and protein (right) in mouse immortalized APCs expressing shRNA for GFP (Con) or Wisp2 (W2KO). (B-D) Expression of adipocyte progenitor markers Cd34 (b), Pdgfra (c), or Cd24a (d) in Con and W2KO immortalized APCs during proliferation in standard growth media. (E) Cell number over 4 days in Con or W2KO populations treated with EtOH (veh), E 2 , or E 2 +4-OH tamoxifen (E 2 +TAM). 3-way ANOVA tested for main effects of time, treatment, and Wisp2 modification. (F) Oil red O (ORO) staining of Con or W2KO cells at day 0 of differentiation. Representative images on the left, quantification of ORO absorbance on the right. (G) Heatmap of expression of progenitor and preadipocyte markers in Con and W2KO cells treated with EtOH (veh) or E 2 for 48 hours. Data are expressed as fold change versus vehicle for each gene, showing 3 replicates per group. (H-I) Quantification of percent Sca1+/CD24+ progenitors (h) and Sca1+/CD24-preadipocytes (i) in Con or W2KO cells treated with vehicle or E 2 . T-tests determined significance.

Article Snippet: Primary human subcutaneous preadipocytes (PCS-210-01, ATCC) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast low serum growth supplement (ATCC PCS-201-041).

Techniques: Expressing, shRNA, Modification, Staining

Journal: bioRxiv

Article Title: Tamoxifen Targets Wisp2 to Impair Subcutaneous Adipocyte Progenitor Self-Renewal and Adipogenic Differentiation

doi: 10.64898/2025.12.21.695772

Figure Lengend Snippet: (A) Proliferation of mouse primary ASCs during 3 days of treatment EtOH (Veh), E 2 , E 2 +4-OH tamoxifen (E 2 +TAM), exogenous Wisp2 protein (Wisp2), or E 2 +TAM+Wisp2. Two-way ANOVA tested for main effects of time and treatment; interaction p<0.001. Inset shows relative cell number at the final time point. T-test determined significance. (B) oil red O accumulation after 10 days of differentiation of cells treated as in (a). T-tests determined significance at day 10. (C-E) Representative FACS plots showing cells treated as in (a) for 48 hours and stained for Sca1 and CD24. Percent of Sca1+/CD24+ progenitors shown in (d); percent of Sca1+/CD24-preadipocytes shown in (e). T-tests determined significance. (F-K) Expression of Cd34 (f), Pdgfra (g), Sca1/Ly6a (h), Cd24a (i), Pparg (j), or Fabp4 (k) in mouse primary ASCs treated as in (a) and differentiated for 10 days. Cells were treated for the duration of the differentiation assay, replenishing media every 2 days.

Article Snippet: Primary human subcutaneous preadipocytes (PCS-210-01, ATCC) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast low serum growth supplement (ATCC PCS-201-041).

Techniques: Staining, Expressing, Differentiation Assay

Journal: bioRxiv

Article Title: Tamoxifen Targets Wisp2 to Impair Subcutaneous Adipocyte Progenitor Self-Renewal and Adipogenic Differentiation

doi: 10.64898/2025.12.21.695772

Figure Lengend Snippet: (A) Expression of Wisp2 gene (left) and protein (right) in primary mouse subcutaneous ASCs from wild type (Con) or Wisp2 transgenic females (W2OE) both treated with Cre adenovirus in vitro. (B) Oil red O (ORO) accumulation in control and W2OE primary ASCs differentiated for 10 days in vitro. Graph shows ORO absorbance. (C) Proliferation of control or W2OE primary ASCs over 5 days, during treatment with EtOH (Veh), E 2 , or E 2 +4-OH tamoxifen (E 2 +TAM). Three-way ANOVA tested for main effects of time, treatment, or cell genotype. Interaction p-value <0.001. (D) Quantification of cell number after 5 days of proliferation as shown in (c). Statistical analysis is two-way ANOVA testing for main effects of treatment or cell genotype, with post-hoc analyses. (E-F) Quantification of Sca1+/CD24+ progenitors (e) or Sca1+/CD24-preadipocytes (f) after 2 days of treatment with EtOH (Veh), E 2 , or E 2 +TAM measured by FACS. (G) ORO accumulation in CON or W2OE cells treated with EtOH (Veh), E 2 , or E 2 +TAM for 10 days during differentiation. Graph represents quantification of ORO absorbance at day 10. T-tests determined significance.

Article Snippet: Primary human subcutaneous preadipocytes (PCS-210-01, ATCC) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast low serum growth supplement (ATCC PCS-201-041).

Techniques: Expressing, Transgenic Assay, In Vitro, Control

Journal: Cells

Article Title: Chronic Fatty Acid Depletion Induces Uncoupling Protein 1 (UCP1) Expression to Coordinate Mitochondrial Inducible Proton Leak in a Human-Brown-Adipocyte Model

doi: 10.3390/cells11132038

Figure Lengend Snippet: Regulation of UCP1 expression by FAs in an immortalized human-brown-preadipocyte cell line (hTERT A41BAT-SVF). ( A ) The expression of UCP1 and FABP4 was quantified using qRT-PCR during differentiation of A41BAT-SVF preadipocytes using RoFB in the treatment with either FA BSA or FA-free BSA, from 1 to 4 weeks as indicated. ( B ) UCP1 protein levels were compared between ciBAs and A41BAT-SVF adipocytes. ( C ) The expression of UCP1 , CIDEA , and FABP4 was quantified using qRT-PCR in A41BAT-SVF adipocytes differentiated using carnitine or PA. ( D ) The UCP1 protein was immunoblotted in A41BAT-SVF adipocytes differentiated under various conditions. ( E ) Cellular triglyceride content and GPDH activity were measured in A41BAT-SVF adipocytes. ( F ) The expression of UCP1 and FABP4 was quantified using qRT-PCR in adipocytes differentiated from adipose-tissue-derived mesenchymal stem cells (AdMSCs) using carnitine or PA. ( G ) Cellular triglyceride content and GPDH activity were measured in AdMSC-derived adipocytes. Data represent mean ± SD ( n = 3). Student’s t -test: * p < 0.05, ** p < 0.01, *** p < 0.001, N.S.; not significant.

Article Snippet: The immortalized preadipocyte cell line (hTERT A41hBAT-SVF), isolated from human deep-neck fat tissue, was purchased from the American Type Culture Collection (CRL-3385; ATCC, VA, USA).

Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Derivative Assay

Journal: Hormones & Cancer

Article Title: CREB-Regulated Transcription Co-Activator Family Stimulates Promoter II-Driven Aromatase Expression in Preadipocytes

doi: 10.1007/s12672-013-0142-1

Figure Lengend Snippet: Role of CRTCs in aromatase PII activation. a Relative abundance of CRTC transcripts in primary breast preadipocytes with and without PGE2 treatment. b Confocal images of immunofluorescence on endogenous CRTC proteins (green) in primary breast preadipocytes with and without PGE2 treatment, lamin B1 + B2 nuclear stain in red. c Chromatin immunoprecipitation (ChIP) showing endogenous binding of CRTCs to aromatase PII in human breast preadipocytes with and without FSK/PMA treatment. d Reporter assays demonstrating the effect of CRTCs on aromatase PII activity with and without FSK/PMA treatment. vc Vehicle control; mean ± SEM, n = 3 for qPCR and reporter assays; confocal images are representative of the majority of cells examined; scale bar 20.0 μm; all experiments repeated twice

Article Snippet: Cell culture Primary human breast preadipocytes were isolated after collagenase treatment of whole subcutaneous adipose tissue obtained from women undergoing reduction mammoplasty and cultured in Waymouth’s medium (Invitrogen, USA), as previously described [ 19 ].

Techniques: Activation Assay, Immunofluorescence, Staining, Chromatin Immunoprecipitation, Binding Assay, Activity Assay, Control

Journal: Hormones & Cancer

Article Title: CREB-Regulated Transcription Co-Activator Family Stimulates Promoter II-Driven Aromatase Expression in Preadipocytes

doi: 10.1007/s12672-013-0142-1

Figure Lengend Snippet: Role of CRTCs in regulating aromatase expression and activity in primary human breast preadipocytes. Aromatase activity assays demonstrating the effect of silencing (a) and overexpressing (b) CRTCs on the FSK/PMA-mediated stimulation of aromatase activity. c Effect of CRTC overexpression on aromatase transcript expression. Mean ± SEM, n = 3, repeated twice

Article Snippet: Cell culture Primary human breast preadipocytes were isolated after collagenase treatment of whole subcutaneous adipose tissue obtained from women undergoing reduction mammoplasty and cultured in Waymouth’s medium (Invitrogen, USA), as previously described [ 19 ].

Techniques: Expressing, Activity Assay, Over Expression

Journal: Hormones & Cancer

Article Title: CREB-Regulated Transcription Co-Activator Family Stimulates Promoter II-Driven Aromatase Expression in Preadipocytes

doi: 10.1007/s12672-013-0142-1

Figure Lengend Snippet: Effect of phosphorylating CRTC2 at Ser171 on its subcellular localization and the activation of aromatase PII in preadipocytes. a Confocal images on overexpressed GFP-tagged wt, S171A and S171D of CRTC2 (green) and immunofluorescence on lamin B1 + B2 nuclear stain in red, with or without FSK/PMA or AICAR treatment. b Reporter assays demonstrating the effect of wt, S171A and S171D of CRTC2 on aromatase PII activity with or without FSK/PMA treatment. vc Vehicle control, RLU relative luciferase units, β-gal β-galactosidase activity. Mean ± SEM, n = 3 for reporter assays, confocal images are representative of the majority of cells examined, scale bar represents 50.0 μm, all experiments repeated twice

Article Snippet: Cell culture Primary human breast preadipocytes were isolated after collagenase treatment of whole subcutaneous adipose tissue obtained from women undergoing reduction mammoplasty and cultured in Waymouth’s medium (Invitrogen, USA), as previously described [ 19 ].

Techniques: Activation Assay, Immunofluorescence, Staining, Activity Assay, Control, Luciferase

Journal: Immune Network

Article Title: Differential Chemokine Signature between Human Preadipocytes and Adipocytes

doi: 10.4110/in.2016.16.3.189

Figure Lengend Snippet: Chemokine receptor signature in human preadipocytes and adipocytes. (A) Representative images of human adipocytes during adipogenesis. Human preadipocyte and differentiated adipocytes were visualized under a digital inverted microscope at 20 X magnification. Human preadipocytes show long, thin and flatted cells, while human adipocytes show differentiated fat cells in spherical form. (B) Comparison of chemokine ligands in preadipocytes vs. adipocytes. A PCR array for chemokine receptors was performed by using total RNA isolated from nondifferentiated (preadipocytes) and differentiated cells (adipocytes). Different colors indicate the average cycle threshold with expressions that ranged from >35 to <25. Expression levels of chemokine receptors were defined as absent (>35), low (30-35) and high (<30) on average threshold cycles. Chemokines with a >2-fold increase ( * ) or decrease ( # ) were recognized as the major differences between preadipocytes and adipocytes.

Article Snippet: The human preadipocytes and adipocytes were purchased from Cell Applications Inc. (San Diego, CA, USA).

Techniques: Inverted Microscopy, Comparison, Isolation, Expressing

Journal: Immune Network

Article Title: Differential Chemokine Signature between Human Preadipocytes and Adipocytes

doi: 10.4110/in.2016.16.3.189

Figure Lengend Snippet: Chemokine ligand signature in human preadipocytes and adipocytes. (A) Comparison of chemokine ligands in preadipocytes and adipocytes using PCR array containing complementary sequences for human chemokine ligand genes. Different colors indicate the average cycle threshold with expressions that ranged from >35 to <25. Expression levels of chemokines were defined as absent (>35), low (30-35) and high (<30) on average threshold cycles. Chemokines with a >2-fold increase ( * ) or decrease ( # ) were recognized as the major differences between preadipocytes and adipocytes. (B) Changes in mRNA expression of CCL2, CCL7, CCL8, CXCL6, CXCL10 and (C) CXCL14 were confirmed using specific PCR primer sets by qRT-PCR. Fold changes were calculated as a relative value after setting the first sample of adipocytes as a control group (1.0). Also, secreted CXCL14 in conditioned media from preadipocytes and adipocytes was assessed by ELISA. Experiments values for PCR array are the means from duplicate experiments, while experiments for qRT-PCR were performed at least in triplicate. All data value are presented as mean±SEM. * and # indicates significant increase or decrease (p≤0.05), respectively when tested with Student's t test.

Article Snippet: The human preadipocytes and adipocytes were purchased from Cell Applications Inc. (San Diego, CA, USA).

Techniques: Comparison, Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Oncology reports

Article Title: Adipocyte culture medium stimulates invasiveness of MDA-MB-231 cell via CCL20 production.

doi: 10.3892/or_00000593

Figure Lengend Snippet: Figure 1. CCL20 production is increased by adipocyte-CM. (A) MDA-MB-231 cells were treated with preadipocyte-CM or adipocyte-CM for 16 h, and supernatants were centrifuged to remove cells. Chemokines in supernatants were analyzed. (B) MDA-MB-231 cells were incubated with preadipocyte- CM or adipocyte-CM for 6 h. Total RNA was isolated and subjected to RT-PCR analysis, and band density was measured. The experiment was performed in triplicate. A representative experiment is shown. (C) MDA-MB-231 cells were incubated with adipocyte-CM for indicated times. CCL20 mRNA expression was upregulated after stimulation with adipocyte-CM. The experiment was performed in triplicate. A representative experiment is shown.

Article Snippet: The MDA-MB-231 human breast cancer cell line and the OP9 mouse preadipocyte cell line were purchased from the American Type Culture Collection (ATCC).

Techniques: Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Oncology reports

Article Title: Adipocyte culture medium stimulates invasiveness of MDA-MB-231 cell via CCL20 production.

doi: 10.3892/or_00000593

Figure Lengend Snippet: Figure 2. A protein factor in adipocyte-CM activates NF-κB with a resultant increase in CCL20 mRNA expression in MDA-MB-231 cells. (A) MDA- MB-231 cells were treated with preadipocyte-CM, adipocyte-CM, or pro- teinase K-treated adipocyte CM, for 6 h. Total RNA was isolated and subjected to RT-PCR analysis. (B) MDA-MB-231 cells were pretreated with 20 μM SP600125, 20 μM SB203580, 20 μM PD98059, or 1.5 μM parthenolide (PTN), 1 h before treatment with adipocyte-CM, and incubated for 6 h. Total RNA was isolated and subjected to RT-PCR analysis.

Article Snippet: The MDA-MB-231 human breast cancer cell line and the OP9 mouse preadipocyte cell line were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Incubation

Journal: Oncology reports

Article Title: Adipocyte culture medium stimulates invasiveness of MDA-MB-231 cell via CCL20 production.

doi: 10.3892/or_00000593

Figure Lengend Snippet: Figure 3. The TNF-· gene is not more highly expressed in adipocytes than in preadipocytes. (A) MDA-MB-231 cells were treated with TNF-· (10 ng/ml) or IL-1ß (15 ng/ml) for 6 h. The levels of CCL20 mRNA expression were examined by RT-PCR. (B) OP9 preadipocytes were permitted to differentiate into adipocytes. Cells were harvested at the indicated days during differentiation. The levels of TNF-· and IL-1ß gene expression were examined using RT-PCR analysis.

Article Snippet: The MDA-MB-231 human breast cancer cell line and the OP9 mouse preadipocyte cell line were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: Oncology reports

Article Title: Adipocyte culture medium stimulates invasiveness of MDA-MB-231 cell via CCL20 production.

doi: 10.3892/or_00000593

Figure Lengend Snippet: Figure 4. Anti-TNF-· neutralizing antibody blocks adipocyte-CM-induced CCL20 production in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with preadipocyte-CM, adipocyte-CM, or proteinase K-treated adipocyte-CM, for 6 h. The levels of TNF-· and IL-1ß gene expression were examined by RT- PCR. (B) MDA-MB-231 cells were treated with adipocyte-CM, and total RNA was isolated at the indicated times. The levels of TNF-· gene expression were examined by RT-PCR. (C) MDA-MB-231 cells were treated with adipocyte-CM preincubated with various concentrations of anti-TNF-· neutralizing antibody for 1 h. The level of CCL20 expression was examined by RT-PCR 6 h after incubation with adipocyte-CM containing anti-TNF-· neutralizing antibody. (D) Preadipocytes were treated with TNF-· siRNA and GFP siRNA as a control for 24 h and then differentiated into adipocytes. MDA-MB-231 cells were treated with TNF-·-deficient preadipocyte-CM and TNF-·-deficient adipocyte-CM for 6 h. The level of CCL20 expression was examined by RT-PCR. (E) MDA-MB-231 cells were treated with TNF-· receptor siRNA or GFP siRNA as a control for 24 h and then preadipocyte-CM or adipocyte- CM was treated for 6 h. The level of CCL20, TNF-·, and TNF-· receptor 1 expression was examined by RT-PCR. (F) MDA-MB-231 cells were treated with TNF-· receptor siRNA or GFP siRNA as a control for 24 h and then preadipocyte-CM or adipocyte-CM was treated for 30 min. Lysates were used to determine the level of phospho-IKK·/ß and phospho-IκB· using Western blot analysis.

Article Snippet: The MDA-MB-231 human breast cancer cell line and the OP9 mouse preadipocyte cell line were purchased from the American Type Culture Collection (ATCC).

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

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: ( A – C ) hTERT pre-adipocytes and differentiated adipocytes infected with SARS-CoV-2 (WA1/2020, MOI = 1). ( A , B ) Relative expression of viral ( A ) genomic RNA (nucleocapsid, *** p = 0.0002, ** p = 0.0018) and ( B ) sub-genomic RNA (ORF1ab, *** p = 0.0003, * p = 0.0263), normalized to β-actin. ( C ) Viral loads measured from supernatant using plaque assay. (**** p < 0.0001, ** p = 0.0024). Data are pooled from two independent experiments ( n = 8, biological replicates). Statistical analysis was performed using two-way ANOVA. ( D ) IL-6 levels in the supernatant of differentiated adipocytes with or without viral infection (MOI = 1), measured by ELISA. Data are pooled from three independent experiments ( n = 14, biological replicates, **** p < 0.0001). Statistical analysis was performed using one-way ANOVA. ( E – G ) Adipocytes were infected at 4, 9, 12, 16, and 20 days post-differentiation (MOI = 1), with measurements taken 48 h post-infection. ( E ) Viral loads measured by plaque assay; data represent two independent experiments ( n = 4, biological replicates, **** p < 0.0001). Statistical analysis was performed using one-way ANOVA. ( F ) Western blot of viral nucleocapsid, FABP4, and β-actin proteins levels in cell lysates. ( G ) Quantification of nucleocapsid and FABP4 band intensities normalized to β-actin, representative of two independent experiments ( n = 4, biological replicates, **** p < 0.0001, *** p = 0.0001 and 0.0006). Statistical analysis was performed using two-way ANOVA. ( H , I ) Percent area of lipid droplets in infected cells and neighboring cells, quantified by fluorescence neutral lipid staining (Bodipy). Infected cells identified by ( H ) nucleocapsid-positive staining and ( I ) dsRNA-positive staining. Data pooled from two independent experiments ( n = 6, biological replicates, **** p < 0.0001, *** p = 0.0001); statistical analysis was performed using a standard t-test. ( J ) Representative confocal images of infected differentiated adipocytes (MOI = 1), stained for dsRNA (red), lipid droplets (yellow), and nucleus (DAPI, blue). Scale bar = 50 μm; magnified regions = 10 μm ( n = 3). ( K ) Percent lipid droplet area relative to dsRNA-positive area per cell. Pearson correlation coefficient indicated as r. ( L , M ) Representative confocal images of control and infected adipocytes at 8, 12, 24, and 48 h post-infection, stained for dsRNA (red), lipid droplets (yellow), calnexin (blue), and FABP4 (gray). ( L ) Merged image of all stains (Scale bar = 50 μm); insets highlight overlap of FABP4, dsRNA, and calnexin (Scale bar = 5 μm). ( M ) Signal overlap between dsRNA, FABP4, and calnexin, and between FABP4 and calnexin individually (Scale bar = 5 μm). ( N – P ) Colocalization of target signals over time, represented as Pearson correlation R. Cells infected with MOI = 3 at 8 and 12 hpi; data pooled from two independent experiments ( n = 6, biological replicates, **** p < 0.0001). For 24 and 48 hpi, MOI = 1 was used ( n = 3) biological replicates. Statistical analysis was performed using one-way ANOVA. Data shown as mean ± s.e.m. .

Article Snippet: hTERT human pre-adipocytes. , ATCC , CRL-3386.

Techniques: Infection, Expressing, Plaque Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Fluorescence, Staining, Control

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: hTERT human pre-adipocytes. , ATCC , CRL-3386.

Techniques: Knock-Out, Sequencing, Recombinant, shRNA, Staining, Electron Microscopy, Plasmid Preparation, Software, Isolation, cDNA Synthesis, Luciferase, Enzyme-linked Immunosorbent Assay