Journal: Stem Cells International

Article Title: Hydrogen Peroxide-Induced DNA Damage and Repair through the Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

doi: 10.1155/2018/1615497

Figure Lengend Snippet: hADMSC immunophenotype and characterization of adipocyte differentiation. Positive and negative surface markers of MSCs and adipocyte differentiation marker hFATP-1 in (a). Cell cycle and G1 arrest of adipocytes in (b). hADMSC representative morphology at 4x magnification in (c) and 40x magnification in (d). Adipocyte differentiation after 14 days evidenced by Oil Red O staining. (e) and (g) are representative images at 20x and 40x magnification, respectively. Light microscopy images at day 14 of differentiation are represented in (f) and (h), corresponding to 20x and 40x magnification, respectively. The increase in the relative lipid accumulation between hADMSCs and adipocytes was determined by Student's t -test, ∗ p < 0.05 (i).

Article Snippet: Anti-hFATP-1 (human fat acid transporter protein 1) (R&D Systems; cat. number IC3304P) was used as the surface marker of adipocyte differentiation [ ].

Techniques: Marker, Staining, Light Microscopy

Journal: Nature Communications

Article Title: Fatty acid metabolism in aggressive B-cell lymphoma is inhibited by tetraspanin CD37

doi: 10.1038/s41467-022-33138-7

Figure Lengend Snippet: Human lymphoma BJAB cells supplemented with fluorescent analogue for palmitate (Bodipy FL C16) ( n = 13, p = 0.00010) ( a ), lauric acid analogue (Bodipy FL C12) ( n = 10) ( c ) and glucose analogue (2-NBDG) ( n = 3) ( d ) to assess uptake potential with flow cytometry. BJAB CD37KO cells ( n = 3) were transfected or mock transfected with a CD37 construct and stained with Bodipy FL C16 ( b ) or 2-NBDG ( e ) to check uptake potential after CD37 rescue ( b : NT vs rescue; p = 0.0012, NT vs Mock; p = 0.0178, e NT vs rescue; p = 0.0017, NT vs Mock; p = 0.0042). Confocal microscopic analysis revealed colocalization of CD37 ( f , yellow, middle panels) and FATP1 ( f , magenta, bottom panels) in the membranes of WT human lymphoma cells ( n = 13) (blue: DAPI). Pearson’s R ( p < 0.0001) analysis indicates the likelihood of CD37 and FATP1, or CD37 and CD36, to reside in the same microdomain ( g ). Visualisation of in situ proximity ligation on cells stained with isotype controls ( n = 21) ( h , left panel), positive control ( n = 21) (two different primary antibodies against FATP1) ( h , middle panel) and CD37 and FATP1 ( n = 20) ( h , right panel). The dots per cell were quantified over three independent experiments and visualised for one representative repeat (all; p < 0.0001) ( i ) CD37KO lymphoma cells (BJAB) were transfected with empty vector or FATP1-FLAG construct and alfa-tagged (aTAG) CD37 construct and subjected to pulldown on alfa-tagged CD37. Blots were stained with anti-FLAG antibody to reveal specific interaction between CD37 and FATP1 (FATP1: 63 kDa, red square) ( j ), and anti-CD37 antibody (30–45 kDa (glycosylation)) ( k ). Two-way unpaired t -tests ( a , c , d , g ) or Two-way ANOVA with Tukey’s post hoc test ( b , e , i ) were performed to check for significant differences between the indicated groups, * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars represent mean ± SEM. Data in a–e and g was derived from three independent experiments, h–k were repeated at least three times, yielding similar results. gMFI geometric mean fluorescence intensity. Source data are provided as a Source Data file. See also Supplementary Fig. .

Article Snippet: Antibodies directed against FATP1 (308420, conjugated) (R&D Systems, Minneapolis, MN, USA, IC3304R), CD36 (877302, conjugated) (Novus Biologicals, Centennial, CO, USA, MAB19553AF647) and CD37 (HH1, unconjugated) (Santa Cruz Biotechnology, Dallas, TX, USA, sc-18881) were added as 10 μg/mL in PBS and stained for 30 min at RT in the dark.

Techniques: Flow Cytometry, Transfection, Construct, Staining, In Situ, Ligation, Positive Control, Plasmid Preparation, Glycoproteomics, Derivative Assay, Fluorescence

Journal: Nature Communications

Article Title: Fatty acid metabolism in aggressive B-cell lymphoma is inhibited by tetraspanin CD37

doi: 10.1038/s41467-022-33138-7

Figure Lengend Snippet: WT and CD37KO lymphoma cells (BJAB) were incubated in a nutrient-rich medium with or without FATP1 inhibitor (compound 5k (12.5 µM)) for 2 h. Cells were then transferred to a nutrient-restricted medium with ( n = 8) or without ( n = 8) compound 5k and subjected to an acute substrate injection of medium or palmitate (50 µM) ( a ) or oleic acid (50 µM) ( d ) at t = 30 min. Continuous oxygen consumption ratio (OCR) values are shown in response to FCCP (1 µM) at t = 70 min and Rotenone/AntimycinA (Rot/AA) (1 µM) at t = 100 min. Substrate-induced OCR ( b , e ) were calculated as the difference between baseline and acute substrate injection ( b : p = 0.0099). The spare respiratory capacity (SRC) ( c , f ) was calculated as the difference in OCR between baseline and FCCP ( c : WT vs KO; p = 0.0365, KO vs KO palmitate; p = 0.0188). Relative ATP production (counts per second, CPS) was assessed in response to 24-h FATP1 inhibition with compound 5k (12.5 µM) ( n = 3, WT− vs KO−; p = 0.0008, KO− vs KO+; p = 0.0003) ( g ). Decrease in palmitate uptake was measured after 2 h FATP1 inhibition with compound 5k ( n = 6, WT; p = 0.0147, KO; p = 0.0033) ( h ) and 12a ( n = 6, KO; p = 0.0355, p = 0.0315) ( i ) and compared to non-treated cells ( n = 6). Experiments were repeated three times, yielding similar results. Two-way ANOVA with Tukey’s post hoc test were performed to check for significant differences between the indicated groups, ns not significant * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001. Error bars represent mean ± SD. Source data are provided as a Source Data file. See also Supplementary Fig. .

Article Snippet: Antibodies directed against FATP1 (308420, conjugated) (R&D Systems, Minneapolis, MN, USA, IC3304R), CD36 (877302, conjugated) (Novus Biologicals, Centennial, CO, USA, MAB19553AF647) and CD37 (HH1, unconjugated) (Santa Cruz Biotechnology, Dallas, TX, USA, sc-18881) were added as 10 μg/mL in PBS and stained for 30 min at RT in the dark.

Techniques: Incubation, Injection, Inhibition

Journal: Nature Communications

Article Title: Fatty acid metabolism in aggressive B-cell lymphoma is inhibited by tetraspanin CD37

doi: 10.1038/s41467-022-33138-7

Figure Lengend Snippet: Human lymphoma BJAB cells supplemented with fluorescent analogue for palmitate (Bodipy FL C16) ( n = 13, p = 0.00010) ( a ), lauric acid analogue (Bodipy FL C12) ( n = 10) ( c ) and glucose analogue (2-NBDG) ( n = 3) ( d ) to assess uptake potential with flow cytometry. BJAB CD37KO cells ( n = 3) were transfected or mock transfected with a CD37 construct and stained with Bodipy FL C16 ( b ) or 2-NBDG ( e ) to check uptake potential after CD37 rescue ( b : NT vs rescue; p = 0.0012, NT vs Mock; p = 0.0178, e NT vs rescue; p = 0.0017, NT vs Mock; p = 0.0042). Confocal microscopic analysis revealed colocalization of CD37 ( f , yellow, middle panels) and FATP1 ( f , magenta, bottom panels) in the membranes of WT human lymphoma cells ( n = 13) (blue: DAPI). Pearson’s R ( p < 0.0001) analysis indicates the likelihood of CD37 and FATP1, or CD37 and CD36, to reside in the same microdomain ( g ). Visualisation of in situ proximity ligation on cells stained with isotype controls ( n = 21) ( h , left panel), positive control ( n = 21) (two different primary antibodies against FATP1) ( h , middle panel) and CD37 and FATP1 ( n = 20) ( h , right panel). The dots per cell were quantified over three independent experiments and visualised for one representative repeat (all; p < 0.0001) ( i ) CD37KO lymphoma cells (BJAB) were transfected with empty vector or FATP1-FLAG construct and alfa-tagged (aTAG) CD37 construct and subjected to pulldown on alfa-tagged CD37. Blots were stained with anti-FLAG antibody to reveal specific interaction between CD37 and FATP1 (FATP1: 63 kDa, red square) ( j ), and anti-CD37 antibody (30–45 kDa (glycosylation)) ( k ). Two-way unpaired t -tests ( a , c , d , g ) or Two-way ANOVA with Tukey’s post hoc test ( b , e , i ) were performed to check for significant differences between the indicated groups, * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars represent mean ± SEM. Data in a–e and g was derived from three independent experiments, h–k were repeated at least three times, yielding similar results. gMFI geometric mean fluorescence intensity. Source data are provided as a Source Data file. See also Supplementary Fig. .

Article Snippet: In addition, a second FATP1 antibody (polyclonal) (Novus Biologicals, Centennial, CO, USA, NBP2-69016) was used at 10 μg/mL as a positive control.

Techniques: Flow Cytometry, Transfection, Construct, Staining, In Situ, Ligation, Positive Control, Plasmid Preparation, Glycoproteomics, Derivative Assay, Fluorescence

Journal: Nature Communications

Article Title: Fatty acid metabolism in aggressive B-cell lymphoma is inhibited by tetraspanin CD37

doi: 10.1038/s41467-022-33138-7

Figure Lengend Snippet: WT and CD37KO lymphoma cells (BJAB) were incubated in a nutrient-rich medium with or without FATP1 inhibitor (compound 5k (12.5 µM)) for 2 h. Cells were then transferred to a nutrient-restricted medium with ( n = 8) or without ( n = 8) compound 5k and subjected to an acute substrate injection of medium or palmitate (50 µM) ( a ) or oleic acid (50 µM) ( d ) at t = 30 min. Continuous oxygen consumption ratio (OCR) values are shown in response to FCCP (1 µM) at t = 70 min and Rotenone/AntimycinA (Rot/AA) (1 µM) at t = 100 min. Substrate-induced OCR ( b , e ) were calculated as the difference between baseline and acute substrate injection ( b : p = 0.0099). The spare respiratory capacity (SRC) ( c , f ) was calculated as the difference in OCR between baseline and FCCP ( c : WT vs KO; p = 0.0365, KO vs KO palmitate; p = 0.0188). Relative ATP production (counts per second, CPS) was assessed in response to 24-h FATP1 inhibition with compound 5k (12.5 µM) ( n = 3, WT− vs KO−; p = 0.0008, KO− vs KO+; p = 0.0003) ( g ). Decrease in palmitate uptake was measured after 2 h FATP1 inhibition with compound 5k ( n = 6, WT; p = 0.0147, KO; p = 0.0033) ( h ) and 12a ( n = 6, KO; p = 0.0355, p = 0.0315) ( i ) and compared to non-treated cells ( n = 6). Experiments were repeated three times, yielding similar results. Two-way ANOVA with Tukey’s post hoc test were performed to check for significant differences between the indicated groups, ns not significant * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001. Error bars represent mean ± SD. Source data are provided as a Source Data file. See also Supplementary Fig. .

Article Snippet: In addition, a second FATP1 antibody (polyclonal) (Novus Biologicals, Centennial, CO, USA, NBP2-69016) was used at 10 μg/mL as a positive control.

Techniques: Incubation, Injection, Inhibition

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a , Overlay of the extracted ion chromatograms for EVT0185 reaction mixture at 0 and 30 min. b , Mass spectrum of EVT0185-CoA at 30-min. c , Fragment ions of EVT0185-CoA at 30-min sample showing the fragment ion 605 m/z produced following the characteristic neutral loss of 3’-phosphonucleoside diphosphate (507 Da). Western blot showing d , Dkk tag expression in empty vector control (pCMV), SLC27A1 , SLC27A2 , SLC27A4 , and SLC27A5 transfected HEK293 cells e , SLC27A2 (ACSVL1) protein expression in non-transfected cells (lane 1), pCMV controls (lanes 2–4) and SLC27A2 (lanes 5–7) transfected cells; β-actin as a loading control. For gel source data, see Supplementary Fig. . f , Extracted ion chromatogram from the MS1 and MS2 scans of HEK293 cells transfected with SLC27A2 and treated with 30 µM EVT0185. g , Mass spectrum from the MS1 and MS2 scans which shows the parent ion (1112 m/z) and the neutral loss of 3’-phosphonucleoside diphosphate (507 Da), respectively. h , EVT0185-CoA detected only in extracts from HEK293 cells overexpressing SLC27A2 (ACSVL1); mean ± SEM, n = 6 biologically independent samples/group. i , Proportion of human liver cells expressing SLC27A2 mRNA at different stages of disease progression. j , Human SLC27A2 protein expression in different cell types. k , Flow cytometry gating strategy: lymphocyte population identified via FSC vs. SSC, singlets via FSC-A vs. FSC-H, live cells via FSC vs. e780 viability dye, and CD19 + B cells and CD3 + T cells via CD19 vs. CD3 plot. l , PBMCs stimulated with R848 and IL-2 showing CD19 + B cell proliferation compared to negative control. m , Average proliferation index of R848-induced CD19 + B cells with EVT0185 or vehicle, n = 2 biologically independent PBMC donors. n , PBMCs stimulated with Concanavalin A (Con A) and IL-2 showing CD3 + T cell proliferation compared to negative control. o , Average proliferation index of Con A-induced CD3 + T cells with EVT0185 or vehicle, n = 2 biologically independent PBMC donors.

Article Snippet: HEK293 cells were transfected with expression plasmid for SLC27A1 ( NM_198580 , human tagged ORF clone; RC209285, OriGene), SLC27A2 ( NM_003645 , human tagged ORF clone; RC221033, OriGene), SLC27A4 ( NM_005094 , human tagged ORF clone; RC209557, OriGene), SLC27A5 ( NM_012254 , human tagged ORF clone; RC215758, Origene) or control vector (pCMV6-entry mammalian expression vector; PS100001, OriGene).

Techniques: Produced, Western Blot, Expressing, Plasmid Preparation, Control, Transfection, Biomarker Discovery, Flow Cytometry, Negative Control

Journal: Frontiers in molecular biosciences

Article Title: RabGAP AS160/TBC1D4 deficiency increases long-chain fatty acid transport but has little additional effect on obesity and metabolic syndrome in ADMSCs-derived adipocytes of morbidly obese women.

doi: 10.3389/fmolb.2023.1232159

Figure Lengend Snippet: FIGURE 3 Transcript level of fatty acid transporters, i.e., CD36/SR-B2 (A), FATP1 (B), and FATP4 (C) in AS160 knocked down mature adipocytes differentiated from ADMSCs of patients with different metabolic status. Data are presented as mean ± SD. Values are expressed in arbitrary units, i.e., lean non-targeting siRNA group was set at 1 [AU]. # Difference between the cells transfected with siRNA targeting AS160 and scrambled siRNA (negative control) within the metabolic status of the donor patient; p < 0.05; n = 4 for each study group (measurements taken in duplicate). Designations of the groups: NC, negative control containing non-targeting siRNA fragment; siAS160, knockdown of AS160; obese (−), obese without metabolic syndrome; and obese (+), obese with metabolic syndrome.

Article Snippet: The acquired ADMSCs were subjected to staining with monoclonal antibodies targeting anti-FATP1 (ACSVL5; mouse anti-human) (R&D Systems), anti-FATP4 (ACSVL4; rabbit anti-human), and anti-CD36/SR-B2 (CD36/SR-B2; rabbit antihuman) (Abcam).

Techniques: Transfection, Negative Control, Knockdown

Journal: Frontiers in molecular biosciences

Article Title: RabGAP AS160/TBC1D4 deficiency increases long-chain fatty acid transport but has little additional effect on obesity and metabolic syndrome in ADMSCs-derived adipocytes of morbidly obese women.

doi: 10.3389/fmolb.2023.1232159

Figure Lengend Snippet: FIGURE 4 Total protein abundance of fatty acid transporters, i.e., CD36/SR-B2 (A), FATP1 (B), and FATP4 (C) in AS160 knocked down mature adipocytes differentiated from ADMSCs of patients with different metabolic status. Data are presented as mean ± SD. # Difference between cells transfected with siRNA targeting AS160 and scrambled siRNA (negative control) within the metabolic status of the donor patient; p < 0.05; n = 4 for each study group (measurements taken in duplicate). Designations of the groups: NC, negative control containing non-targeting siRNA fragment; siAS160, knockdown of AS160; obese (−), obese without metabolic syndrome; and obese (+), obese with metabolic syndrome.

Article Snippet: The acquired ADMSCs were subjected to staining with monoclonal antibodies targeting anti-FATP1 (ACSVL5; mouse anti-human) (R&D Systems), anti-FATP4 (ACSVL4; rabbit anti-human), and anti-CD36/SR-B2 (CD36/SR-B2; rabbit antihuman) (Abcam).

Techniques: Quantitative Proteomics, Transfection, Negative Control, Knockdown

Journal: Frontiers in molecular biosciences

Article Title: RabGAP AS160/TBC1D4 deficiency increases long-chain fatty acid transport but has little additional effect on obesity and metabolic syndrome in ADMSCs-derived adipocytes of morbidly obese women.

doi: 10.3389/fmolb.2023.1232159

Figure Lengend Snippet: FIGURE 5 Plasma membrane abundance of fatty acid transporters, i.e., CD36/SR-B2 (A), FATP1 (B), and FATP4 (C) in AS160 knocked down mature adipocytes differentiated from ADMSCs of patients with different metabolic status evaluated by flow cytometry analysis. Data are presented as mean ± SD. # Difference between the cells transfected with siRNA targeting AS160 and scrambled siRNA (negative control) within the metabolic status of the donor patient; p < 0.05; n = 4 for each study group (measurements taken in duplicate). Designations of the groups: NC, negative control containing non- targeting siRNA fragment; siAS160, knockdown of AS160; obese (−), obese without metabolic syndrome; and obese (+), obese with metabolic syndrome.

Article Snippet: The acquired ADMSCs were subjected to staining with monoclonal antibodies targeting anti-FATP1 (ACSVL5; mouse anti-human) (R&D Systems), anti-FATP4 (ACSVL4; rabbit anti-human), and anti-CD36/SR-B2 (CD36/SR-B2; rabbit antihuman) (Abcam).

Techniques: Clinical Proteomics, Membrane, Cytometry, Transfection, Negative Control, Knockdown

Journal: Nature Communications

Article Title: MYCN-driven fatty acid uptake is a metabolic vulnerability in neuroblastoma

doi: 10.1038/s41467-022-31331-2

Figure Lengend Snippet: a FA transporters ( SLC27A1–6 , CD36 ) and ODC1 mRNA expression in SK-N-AS MYCN-ER ™ cells (1 µM 4-OHT for 0, 24, 48 h). Mean±SEM ( n = 3); two-way ANOVA with Dunnett’s multiple comparisons test. b FA transporters ( SLC27A1–6 , CD36 ) mRNA expression in Tet21/N cells (±2 µg/mL DOX for 24 h). Mean±SEM ( n = 3); two-sided unpaired t -test. MYCN protein expression in Tet21/N cells (−DOX, MYCN-ON and +DOX, MYCN-OFF). c MYCN ChIP-qPCR assays in TET21/N cells (±2 µg/mL DOX for 48 h). Input and MYCN ChIP samples were analyzed by qPCR using specific primers for SLC27A1–6 and CD36 . Mean±SD ( n = 3); two-way ANOVA with Dunnett’s multiple comparisons test. d Gene expression analysis in Cohort 1 ( GSE45547 ). Left, correlation matrix of transporter gene expression, MYCN expression/activity, and c-MYC expression. Correlations with p -values < 0.05 are represented in the heatmap. Red = positive; blue = negative correlation. Middle, SLC27A2 expression in MNA ( n = 93) and non-MNA patients ( n = 550). Two-sided unpaired Welch’s t -test. Right, SLC27A2 expression in stage 1–4 S patients (stage 1: n = 153; stage 2: n = 113; stage 3: n = 91; stage 4: n = 214; stage 4 s: n = 78). One-way ANOVA with Tukey’s multiple comparisons test. Box plots indicate median (middle line), 25th and 75th percentiles (box), as well as min and max (whisker). e , f Survival analyses in Cohort 1 ( GSE45547 ). e OS and EFS rates for stage 1–4 S and stage 3–4 patients with high (top third) or low (bottom third) SLC27A2 expression. f OS and EFS predictions for genes in the long-chain FA transport geneset (GO: 0015909); –log 10 ( p -value). Kaplan–Meier method was used to plot survival curves, and log-rank test was used for statistical analysis. Red=high expression has poor prognosis ( p < 0.05); blue = low expression has poor prognosis ( p < 0.05); gray = no significance. FC fold change, OS overall survival, EFS event-free survival. Source data are provided in the Source Data file.

Article Snippet: FATP1 or FATP2 overexpression: FATP1 (C-BASS) or FATP2 (OriGene) cDNAs were cloned into pINDUCER20, a Tet-inducible lentiviral vector for ORF expression.

Techniques: Expressing, Activity Assay, Whisker Assay