Journal: Nature Communications

Article Title: N 1 -methyladenosine methylation in tRNA drives liver tumourigenesis by regulating cholesterol metabolism

doi: 10.1038/s41467-021-26718-6

Figure Lengend Snippet: a Macroscopic appearance of livers from Trmt6 f/+ /Trmt61a f/+ ; Alb-Cre (WT) and Trmt6/Trmt61a DKO ( Trmt6 f/f /Trmt61a f/f ; Alb-Cre) mice under DEN-induced cancer or in Myc OE mouse model of HCC. Black asterisks indicate liver tumors. Scale bar, 1 cm. b Quantitation of liver tumor numbers of Trmt6 KO, Trmt61a KO, and Trmt6/Trmt61a DKO in DEN-treated and in Myc OE mice. Results are shown as means ± SD ( n = 10 biologically independent mice). Exact P values from left to right: 0.0075, 0.041, 0.032, 0.0037, 0.045, 0.014. c Representative liver H&E staining and immunohistochemistry images of m 1 A, Pparδ, and Hmgcs2 (brown) from Trmt6 f/ + /Trmt61a f/+ (WT) and Trmt6 −/− /Trmt61a −/− (DKO) in Myc OE and DEN-treated mice. Black circle indicates tumor region. Scale bar, 100 μm. n = 3 independent samples. d Levels of cholesterol biogenesis related proteins in 10-week-old male Trmt6/Trmt61a deleted (DKO) in Myc OE mice transduced with AAV vectors encoding GFP control (oeGFP) or Pparδ (oePparδ) for 8 days. WT: Trmt6 f/+ /Trmt61a f/+ ; Myc OE mice. n = 5 biologically independent mice. e Total serum cholesterol levels in 10-week-old normal-fed male Trmt6/Trmt61a deleted Myc OE (DKO) mice transduced with AAV vectors encoding GFP control (oeGFP) or Pparδ (oePparδ) for 8 days. WT: Trmt6 f/+ /Trmt61a f/+ ; Myc OE mice. Data are means ± SD. n = 5 mice per group. Exact P values: 0.021. These experiments were repeated three times. * P < 0.05; ** P < 0.01 by two-tailed Student’s t -test.

Article Snippet: Anti-HMGCS2 antibody (#20940S) was from Cell Signaling Technology, Inc. (USA).

Techniques: Quantitation Assay, Staining, Immunohistochemistry, Transduction, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A , Volcano plot of the DEGs of bezafibrate treatment (200µM, 16 hours) from RNA-seq analysis of CAOV3 cells ( n =3, adj. P =0.01). B , Venn diagram comparison of a list of oppositely expressed genes from ascites-treated vs. bezafibrate-treated cells. C-D , western blot analysis of HMGCS2 protein expression upon 10% ascites treatment from three patients with or without 200µM bezafibrate for 16 hours ( n =3). E-F , ferroptosis sensitivity was compared between CAOV3 wildtype (WT) and HMGCS2 KO cells with ( E ) erastin or ( F ) RSL3 treatment ( n =3, 24 hours). G , HMGCS2 expression was restored in HMGCS2 KO cells with hmgcs2 OE plasmid and ferroptosis sensitivity was compared with 10µM erastin or 125nM RSL3 treatment for 24 hours ( n =3). H-I , lipid peroxidation was measured via flow cytometry analysis of BODIPY TM 581/591 C11 staining in HMGCS2 KO cells that were treated with 5µM erastin for 20 hours ( n =3). J-K , HMGCS2 OE cells were treated with 2% ascites for 16 hours and lipid droplet levels were measured via flow cytometry analysis of BODIPY TM 493/503 staining ( n =3). L , cell viability was assessed via treatment with 10µM erastin in the presence or absence of 20µM baicalin with 2% ascites ( n =3, 24 hours). M-N , cells were treated with 10% ascites in the presence or absence of 25µM baicalin (24 hours) and lipid droplet levels were measured as described ( N ) ( n =3). O , cell viability was assessed in cells treated with 10µM erastin in the presence or absence of 100µM malonyl CoA lithium for 24 hours ( n =3). P-Q , cells were treated with 100µM malonyl CoA lithium (16 hours) and lipid droplet levels were measured as described ( Q ) ( n =3). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance for D-O was assessed using correlated-samples one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. Student’s t -test was performed for Q . All statistical tests were two-tailed where applicable.

Article Snippet: For generating a stable HMGCS2 overexpressing cell line, the human HMGCS2 ORF clone with a pCMV6-Entry backbone (Origene, RC208128) was cut and ligated into lentiviral vector (Origene, PS100069) according to the manufacturer’s guidelines.

Techniques: RNA Sequencing Assay, Comparison, Western Blot, Expressing, Plasmid Preparation, Flow Cytometry, Staining, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-B , Cell viability was measured in CAOV3 cells transfected with ( A ) 50 nM si HMGCS2 or ( B ) 50 nM si TFRC and treated with 10µM erastin for 24 hours ( n =3). C-D , ( C ) HMGCS2 and ( D ) TFRC silencing was validated via qRT-PCR ( n =3). E-F , ( E ) HMGCS2 and ( F ) TFRC downregulation upon 10% ascites exposure for 16 hours was validated via qRT-PCR ( n =3). G-H , knockout of HMGCS2 was validated in HMGCS2 KO cells via ( G ) qRT-PCR and ( H ) western blot analysis ( n =3). I , HMGCS2 overexpression was validated via western blot analysis ( n =3). J-K , cells overexpressing HMGCS2 were treated with 5µM erastin for 20 hours and ( J ) lipid peroxidation was visualized and ( K ) measured with flow cytometry analysis of BODIPY TM 581/591 C11 staining ( n =3). L , patient overall survival data was extracted from the TCGA database and analyzed via the GEPIA 2 survival analysis tool. All cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Excluding L , statistical significance was assessed using correlated-samples one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. All statistical tests were two-tailed where applicable.

Article Snippet: For generating a stable HMGCS2 overexpressing cell line, the human HMGCS2 ORF clone with a pCMV6-Entry backbone (Origene, RC208128) was cut and ligated into lentiviral vector (Origene, PS100069) according to the manufacturer’s guidelines.

Techniques: Transfection, Quantitative RT-PCR, Knock-Out, Western Blot, Over Expression, Flow Cytometry, Staining, Glo Assay, Two Tailed Test

Journal: Journal of Lipid Research

Article Title: Increased hepatic mitochondrial FA oxidation reduces plasma and liver TG levels and is associated with regulation of UCPs and APOC-III in rats

doi: 10.1194/jlr.M074849

Figure Lengend Snippet: Hepatic ketone body production and muscle and cardiac enzyme activities in Wistar male rats given 100 mg/kg 1-triple TTA for 3 weeks compared with controls. A: Mitochondrial HMG-CoA synthase activity in liver. B: Hepatic mRNA level of mitochondrial Hmgcs2 . C: NEFA plasma concentration. D: Plasma concentration of the ketone body, β-hydroxybutyrate. E: Ratio of β-hydroxybutyrate and NEFA. F: Oxalate CoA-transferase activity in muscle and heart tissue. G: CPT-II activity in muscle and heart tissue. Values are shown as means and SD (n = 5–8). Significant difference compared with controls was determined with Student’s t -test (* P ≤ 0.05, **** P ≤ 0.0001).

Article Snippet: RT-PCR was performed on Sarstedt 384-well Multiply-PCR plates (Sarstedt Inc., Newton, NC,) using an ABI Prism 7900HT sequence detection system from Applied Biosystems with the software, SDS 2.3. mRNA levels of genes of interest were detected in 1× Taqman buffer by using the following probes and primers listed in alphabetical order: acetyl-CoA carboxylase ( Acaca ) (Rn00573474_m1); medium-chain acyl-CoA dehydrogenase ( Acadm ) (Rn00566390_m1); long-chain acyl-CoA dehydrogenase ( Acadl ) (Rn00563121_m1); very-long-chain acyl-CoA dehydrogenase ( Acadvl ) (Rn00563649_m1); 4- N -trimethylaminobutyraldehyde dehydrogenase ( Aldh9a1 ) (Rn01491039_m1); ApoB (Rn01499054_m1); ApoC-II (Rn01764530_g1); ApoC-III (Rn00560743); ATP synthase H + transporting mitochondrial F 1 complex γ polypeptide 1 ( Atp5c1 ) (Rn01487287_m1); ATP synthase H + transporting mitochondrial F 0 complex subunit F2 ( Atp5j2 ) (Rn01409509_g1); γ-butyrobetaine hydroxylase ( Bbox1 ) (Rn00575255_m1); cluster of differentiation 36 ( Cd36 ) (Rn00580728_m1); Cpt1a (Rn00580702_m1); Cpt2 (Rn00563995_m1); carnitine O -acetyltransferase ( Crat ) (Rn01758585_m1); somatic cytochrome c ( Cycs ) (Rn00820639_g1); mitochondrial 2,4 dienoyl-CoA reductase 1 ( Decr1 ) (Rn00589420_m1); diglyceride acyltransferase ( Dgat ) 1 (Rn00584870_m1); Dgat2 (Rn01506787_m1); FA binding protein 1 ( Fabp1 ) (Rn00664587_m1); Fasn (Rn00569117_m1); mitochondrial glycerol-3-phosphate dehydrogenase 2 ( Gpd2 ) (Rn00562472_m1); hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein) α subunit ( Hadha ) (Rn00590828_m1); hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein) β subunit ( Hadhb ) (Rn00592435_m1); mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 ( Hmgcs2 ) (Rn00597339_m1); Lpl (Rn00561482_m1); microsomal TG transfer protein (large subunit) ( Mttp ) (Rn01522970_m1); NADH dehydrogenase (ubiquinone) 1α subcomplex 9 ( Ndufa9 ) (Rn01462923_m1); Pparα (Rn00566193_m1); Pparδ (Rn00565707_m1); Pparγ (Rn00440945_m1); PPARγ coactivator 1α ( Ppargc1a ) (Rn00580241_m1); protein kinase AMP-activated α1 catalytic subunit ( Prkaa1 ) (Rn00569558_m1); protein kinase AMP-activated α2 catalytic subunit ( Prkaa2 ) (Rn00576935_m1); Slc25a20 (Rn00588652_m1); solute carrier family 22 (organic cation/carnitine transporter) member 5 ( Slc22a5 ) (Rn01471177_m1); mitochondrial transcription factor A ( Tfam ) (Rn00580051_m1); N ε -trimethyllysine hydroxylase ( Tmlhe ) (Rn00591314_m1); uncoupling protein ( Ucp ) 2 (Rn01754856_m1); Ucp3 (Rn00565874_m1); VLDL receptor ( Vldlr ) (Rn00565784_m1).

Techniques: Activity Assay, Clinical Proteomics, Concentration Assay

Journal: Oncotarget

Article Title: hsa-miR-29c-3p regulates biological function of colorectal cancer by targeting SPARC

doi: 10.18632/oncotarget.22356

Figure Lengend Snippet: KEGG pathway analysis of differentially expressed genes associated with NSCLC

Article Snippet: Membranes were performed blockage whit 5% skim milk for 3 h and then incubation with primary antibodies for ACAA1 (ab110289, Abcam, 1:1000) antibody, ACOX1 (ab184032, Abcam, 1:1000) antibody, COL1A1 (PB0980, Boster, 1:500) antibody, COL1A2 (ab208638, Abcam, 1:500) antibody, COL4A1 (PB0126, Boster, 1:500) antibody, COL5A2 (A03869, Boster, 1:1000) antibody, COL12A1 (ab121304, Abcam, 1:1000) antibody, CPT2 (ab181114, Abcam, 1:4000) antibody, ETHE1 (ab174302, Abcam, 1:4000) antibody, HMGCS2 (ab137043, Abcam, 1:4000) antibody, SPARC (ab207743, Abcam, 1:1000) antibody, SQRDL (ab71978, Abcam, 1:500) antibody, TST (ab166625, Abcam, 1:4000) antibody and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, Abcam, 1:4000) antibody, and subsequent incubation whit horseradish peroxidase (HRP)-coupled goat anti-rabbit (ZDR-5306, Beijing ZSBio, 1:5000) or HRP-coupled goat anti-mouse (W420B, Promega, 1:5000) secondary antibody, respectively.

Techniques: Starch

Journal: Oncotarget

Article Title: hsa-miR-29c-3p regulates biological function of colorectal cancer by targeting SPARC

doi: 10.18632/oncotarget.22356

Figure Lengend Snippet: The expression levels of mRNA COL12A1 (G) , COL1A2 (D) , COL4A1 (E) , SPARC (K) , COL5A2 (F) and COL1A1 (C) was increased, while ACAA1 (A) , ACOX1 (B) , CPT2 (H) , SQRDL (L) , HMGCS2 (J) , ETHE1 (I) and TST (M) were decreased. * P < 0.05.

Article Snippet: Membranes were performed blockage whit 5% skim milk for 3 h and then incubation with primary antibodies for ACAA1 (ab110289, Abcam, 1:1000) antibody, ACOX1 (ab184032, Abcam, 1:1000) antibody, COL1A1 (PB0980, Boster, 1:500) antibody, COL1A2 (ab208638, Abcam, 1:500) antibody, COL4A1 (PB0126, Boster, 1:500) antibody, COL5A2 (A03869, Boster, 1:1000) antibody, COL12A1 (ab121304, Abcam, 1:1000) antibody, CPT2 (ab181114, Abcam, 1:4000) antibody, ETHE1 (ab174302, Abcam, 1:4000) antibody, HMGCS2 (ab137043, Abcam, 1:4000) antibody, SPARC (ab207743, Abcam, 1:1000) antibody, SQRDL (ab71978, Abcam, 1:500) antibody, TST (ab166625, Abcam, 1:4000) antibody and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, Abcam, 1:4000) antibody, and subsequent incubation whit horseradish peroxidase (HRP)-coupled goat anti-rabbit (ZDR-5306, Beijing ZSBio, 1:5000) or HRP-coupled goat anti-mouse (W420B, Promega, 1:5000) secondary antibody, respectively.

Techniques: Expressing

Journal: Oncotarget

Article Title: hsa-miR-29c-3p regulates biological function of colorectal cancer by targeting SPARC

doi: 10.18632/oncotarget.22356

Figure Lengend Snippet: The expression levels of protein COL12A1 (G) , COL1A2 (D) , COL4A1 (E) , SPARC (K) , COL5A2 (F) and COL1A1 (C) was increased, while ACAA1 (A) , ACOX1 (B) , CPT2 (H) , SQRDL (L) , HMGCS2 (J) , ETHE1 (I) and TST (M) were decreased. * P < 0.05.

Article Snippet: Membranes were performed blockage whit 5% skim milk for 3 h and then incubation with primary antibodies for ACAA1 (ab110289, Abcam, 1:1000) antibody, ACOX1 (ab184032, Abcam, 1:1000) antibody, COL1A1 (PB0980, Boster, 1:500) antibody, COL1A2 (ab208638, Abcam, 1:500) antibody, COL4A1 (PB0126, Boster, 1:500) antibody, COL5A2 (A03869, Boster, 1:1000) antibody, COL12A1 (ab121304, Abcam, 1:1000) antibody, CPT2 (ab181114, Abcam, 1:4000) antibody, ETHE1 (ab174302, Abcam, 1:4000) antibody, HMGCS2 (ab137043, Abcam, 1:4000) antibody, SPARC (ab207743, Abcam, 1:1000) antibody, SQRDL (ab71978, Abcam, 1:500) antibody, TST (ab166625, Abcam, 1:4000) antibody and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, Abcam, 1:4000) antibody, and subsequent incubation whit horseradish peroxidase (HRP)-coupled goat anti-rabbit (ZDR-5306, Beijing ZSBio, 1:5000) or HRP-coupled goat anti-mouse (W420B, Promega, 1:5000) secondary antibody, respectively.

Techniques: Expressing

Journal: Oncotarget

Article Title: hsa-miR-29c-3p regulates biological function of colorectal cancer by targeting SPARC

doi: 10.18632/oncotarget.22356

Figure Lengend Snippet: Primer pairs of different genes

Article Snippet: Membranes were performed blockage whit 5% skim milk for 3 h and then incubation with primary antibodies for ACAA1 (ab110289, Abcam, 1:1000) antibody, ACOX1 (ab184032, Abcam, 1:1000) antibody, COL1A1 (PB0980, Boster, 1:500) antibody, COL1A2 (ab208638, Abcam, 1:500) antibody, COL4A1 (PB0126, Boster, 1:500) antibody, COL5A2 (A03869, Boster, 1:1000) antibody, COL12A1 (ab121304, Abcam, 1:1000) antibody, CPT2 (ab181114, Abcam, 1:4000) antibody, ETHE1 (ab174302, Abcam, 1:4000) antibody, HMGCS2 (ab137043, Abcam, 1:4000) antibody, SPARC (ab207743, Abcam, 1:1000) antibody, SQRDL (ab71978, Abcam, 1:500) antibody, TST (ab166625, Abcam, 1:4000) antibody and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, Abcam, 1:4000) antibody, and subsequent incubation whit horseradish peroxidase (HRP)-coupled goat anti-rabbit (ZDR-5306, Beijing ZSBio, 1:5000) or HRP-coupled goat anti-mouse (W420B, Promega, 1:5000) secondary antibody, respectively.

Techniques: