hbg bc2  (Inserm Transfert)

Journal: bioRxiv

Article Title: PPARγ, a key modulator of metabolic reprogramming, stemness and chemoresistance associated with retrodifferentiation in human hepatocellular carcinomas

doi: 10.1101/2024.09.02.610533

Figure Lengend Snippet: (A) Aldolase B (left y-axis) and CD44 (right y-axis) mRNA expression throughout the culture of HepaRG, BC2, Huh7, HepG2 and Huh6 cell lines: Sph=spheres, SP=side population, Prog=progenitors, Conf=committed/confluent, Diff=differentiated and Pro=proliferative. Results are expressed relative to progenitors for HepaRG or proliferative cells for BC2, Huh7, HepG2 and Huh6 (n≥3). Comparison with spheres *p<0.05, **p<0.01, ***p<0.001; comparison with SP $ p<0.05, $$ p<0.01, $$$ p<0.001 (B) Glycolysis, glycolytic capacity, basal and maximal respiration assessed with Seahorse analyzer for HepaRG, BC2, Huh7, HepG2 and Huh6 (n≥3). (C) Lactate and β-hydroxybutyrate assessed in culture supernatants by absorption spectrophotometry (n≥3). FAO and de novo lipogenesis assessed by quantifying the radioactivity subsequent to 14 C-palmitate and 14 C-acetate incorporation, respectively (n≥3). FAT/CD36 mRNA expression relative to progenitors for HepaRG and proliferative cells for BC2, Huh7, HepG2 and Huh6 (n>=3). Neutral lipid content assessed by nile red staining (n≥3). (D) Quantification by gas chromatography-mass spectrometry of fatty acids (FA) from cell total lipids (total FAs), triglycerides, phospholipids, cholesterol esters and free FAs in HepaRG and BC2 at different stages of differentiation. The saturated/unsaturated FA ratio is calculated from data obtained with the total lipid pool. Results are normalized by the number of cells (n≥3). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: We used 2 human HCC cell lines established in our laboratory, HBG-BC2( ) (Inserm UMR 1317, ex Inserm U 49) and HepaRG( ) (Inserm UMR 1317, ex Inserm UMR 552, Patent number US7456018).

Techniques: Expressing, Comparison, Spectrophotometry, Radioactivity, Staining, Gas Chromatography, Mass Spectrometry

Journal: bioRxiv

Article Title: PPARγ, a key modulator of metabolic reprogramming, stemness and chemoresistance associated with retrodifferentiation in human hepatocellular carcinomas

doi: 10.1101/2024.09.02.610533

Figure Lengend Snippet: (A) Upper left panel: PPAR mRNA expression throughout the differentiation process of HepaRG and BC2 cells: Sph=spheres, SP=side population, Prog=progenitors, Conf=committed/confluent, Diff=differentiated, Pro=proliferative, Hep=freshly isolated human hepatocytes. Results are expressed relative to HepaRG-progenitors or BC2-proliferative cells (n≥3). *, $ and # for PPARG , PPARD and PPARA , respectively; $ p<0.05; **, $$, ##p<0,01; ***, $$$, ###p<0,001; all compared with spheres. Upper right panel: Western Blot of PPARγ, PPARα and HSC70 in HepaRG and BC2 cells. Lower left panel: PPARG mRNA expression in HepaRG progenitors treated with SC-79 or LY294002 during 24, 48 or 72h. Lower right panel: Western Blot and densitometry of AKT and phosphoAKT (pAKT) in HepaRG progenitors treated with SC-79 or LY294002 during 24 or 72h. *p<0.05; **p<0,01; ***p<0,001. (B) Overall survival according to PPARG and/or PPARA expression in the TCGA-LIHC cohort. (C) Correlation between PPARG and PPARA expression and AFP level in the TCGA-LIHC cohort. (D) PPARG and PPARA expression in the Désert’s HCC subclasses. Results are expressed relative to PPARG and PPARA levels in the periportal subclass. ***p<0.001. (E) Immunostaining of PPARγ in human HCC. Both the tumor (left) and a tumor nodule invading adjacent tissue (right) show nuclear and cytoplasmic signal. Black arrows indicate a nuclear localization of PPARγ.

Article Snippet: We used 2 human HCC cell lines established in our laboratory, HBG-BC2( ) (Inserm UMR 1317, ex Inserm U 49) and HepaRG( ) (Inserm UMR 1317, ex Inserm UMR 552, Patent number US7456018).

Techniques: Expressing, Isolation, Western Blot, Immunostaining

Journal: bioRxiv

Article Title: PPARγ, a key modulator of metabolic reprogramming, stemness and chemoresistance associated with retrodifferentiation in human hepatocellular carcinomas

doi: 10.1101/2024.09.02.610533

Figure Lengend Snippet: (A) PPARG mRNA expression 48h after transfection of HepaRG-spheres with siNon-targeting (siNT) and siPPARG (n=3). Sphere number and ATP viability assay 48h after seeding of transfected HepaRG cells in sphere culture medium (n=3). ATP viability assay of HepaRG- and -spheres after treatment with 50µM or 100µM rosiglitazone during 72h (n=4). (B) ATP viability assay of HepaRG-progenitors and BC2-proliferative cells after treatment with 50µM or 100µM rosiglitazone during 72h (n=3). HepaRG and BC2 proliferation assessed by quantifying empty areas using ImageJ, three days after seeding. (n=3). (C) mRNA expression of E-cadherin ( CDH1 ), fatty acid transporter FAT/CD36 , PDK4 , perilipin2 ( PLIN2 ), EMT-inducing transcription factor SNAIL and stem-related marker KLF4 in HepaRG-progenitors and BC2- proliferative cells after treatment with rosiglitazone (50 or 100µM) during 72h (n=3). (D) Confocal microscopy of mitochondrial protein TOM22 immunostaining in HepaRG-progenitors 48h after treatment with 50µM rosiglitazone or transfection with siPPARG. Bar=10µm. Mitochondrial network branching and length analyzed using ImageJ (n=3). Mitochondrial DNA assessed by RT-qPCR, 24h, 48h and 72h after transfection of HepaRG-progenitors with siPPARG (n=4). (E) Basal respiration, maximal respiration, spare respiratory capacity and respiration linked to ATP production assessed with Seahorse analyzer in HepaRG-progenitors treated by rosiglitazone 50µM during 48h (n=3). Results are expressed relative to untreated cells or siNT. *p<0.05, **p<0.01, ***p<0.001

Article Snippet: We used 2 human HCC cell lines established in our laboratory, HBG-BC2( ) (Inserm UMR 1317, ex Inserm U 49) and HepaRG( ) (Inserm UMR 1317, ex Inserm UMR 552, Patent number US7456018).

Techniques: Expressing, Transfection, Viability Assay, Marker, Confocal Microscopy, Immunostaining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: PPARγ, a key modulator of metabolic reprogramming, stemness and chemoresistance associated with retrodifferentiation in human hepatocellular carcinomas

doi: 10.1101/2024.09.02.610533

Figure Lengend Snippet: (A) Phase-contrast microscopy of HepaRG-progenitors and BC2-proliferative cells 48h after transfection with siPPARG. Bar=100µm. (B) mRNA expression of PPARG , PLIN2 and PDK4 , 48h and 72h after transfection of siPPARG in HepaRG-progenitors and BC2-proliferative cells (n=3). (C) Basal respiration, maximal respiration, spare respiratory capacity, proton leak, respiration linked to ATP production and non-mitochondrial oxygen consumption, assessed with Seahorse analyzer in HepaRG-progenitors, 48h after transfection of siPPARG (n=3). Results are expressed relative to siNT. (D) Basal respiration, maximal respiration and proton leak assessed with Seahorse analyzer in HepaRG-spheres treated by DCA (50mM) during 4h. Results are expressed relative to untreated HepaRG-spheres (n>=3). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: We used 2 human HCC cell lines established in our laboratory, HBG-BC2( ) (Inserm UMR 1317, ex Inserm U 49) and HepaRG( ) (Inserm UMR 1317, ex Inserm UMR 552, Patent number US7456018).

Techniques: Microscopy, Transfection, Expressing

Journal: bioRxiv

Article Title: PPARγ, a key modulator of metabolic reprogramming, stemness and chemoresistance associated with retrodifferentiation in human hepatocellular carcinomas

doi: 10.1101/2024.09.02.610533

Figure Lengend Snippet: (A) PPARG and PPARA expression in HepaRG progenitors after treatment during 72h with cisplatin (10µl/ml) or sorafenib (2.5µM) (n=4). **p<0,01 (B) Superoxide anion production (ROS) assessed by Mitosox® in HepaRG-spheres after 50 µM dicloroacetate (DCA) treatment during 12h, 24h, 48h and 72h (n=4) or in HepaRG progenitors after 12h, 24h, 48h and 72h transfection with siPPARG or siNT (n=4). *p<0.05; **p<0,01 (C) Viability of HepaRG- progenitors and BC2-proliferative cells transfected with siPPARG or siNT and treated with cisplatin (10µg/ml for HepaRG; 20µg/ml for BC2) or sorafenib (2.5µM for HepaRG; 15µM for BC2) or 50mM DCA during 48h. Results are expressed relative to untransfected/untreated cells (n=4 for HepaRG and n=3 for BC2). *, #, $ in comparison to siNT, untreated siNT and untreated siPPARG, respectively. (D) Viability of HepaRG-progenitors pre-incubated 24h or not with N-acetyl-cysteine (NAC, 1mM) and treated during 48h with cisplatin (10µg/ml) or sorafenib (2.5µM) combined or not with T0070907 (10µM). Results are expressed relative to untreated cells (n=3). *, T0070907 vs untreated; #, cisplatin or sorafenib vs untreated; $, cisplatin or sorafenib vs cisplatin or sorafenib combined with T0070907. (E) Viability, superoxide anion production, lipid peroxidation assessed by malondialdehyde formation in HepaRG transfected with siPPARG or siNT, pre-incubated 48h or not with NAC (1mM) and treated during 48h with cisplatin (10µg/ml) or sorafenib (2.5µM). *, NAC vs without NAC; #, cisplatin or sorafenib vs untreated; $, siPPARG vs siNT. (F) Viability of HepaRG- and BC2-spheres pre-treated 24h with clofibrate (100 or 500µM) and then treated with sorafenib (2.5µM for HepaRG; 15µM for BC2) and/or DCA (50mM) during 72h. Results are expressed relative to untreated cells (n=4 for HepaRG and n=3 for BC2). *, #, $ p<0.05; **, $$ p<0.01; ***, ###, $$$ p<0.001. ****, #### p<0.0001.

Article Snippet: We used 2 human HCC cell lines established in our laboratory, HBG-BC2( ) (Inserm UMR 1317, ex Inserm U 49) and HepaRG( ) (Inserm UMR 1317, ex Inserm UMR 552, Patent number US7456018).

Techniques: Expressing, Transfection, Comparison, Incubation