Journal: bioRxiv

Article Title: The hepatokine FGL1 regulates hepcidin and iron metabolism during the recovery from hemorrhage-induced anemia in mice

doi: 10.1101/2023.04.06.535920

Figure Lengend Snippet: Relative HAMP expression in Hep3B and HepG2 cells in response to BMP6 (25 ng/ml, 6h) (A) or BMP6 + recombinant FGL1 (10µg/ml) (B). Relative ID1 expression in hepatoma cell lines treated with BMP6 and FGL1 (C). HAMP (D) and ID1 (E) expression in Hep3B cells and mouse primary hepatocytes treated for 6h with BMP6 and either Fc, full length FGL1 and the N-terminal or globular domain of FGL1. Hepatic Hamp RNA expression (F), serum hepcidin concentration (G) and liver Id1 (H) and Smad7 (I) mRNA expression in mice treated for 6 hours with saline, Fc or recombinant FGL1 (10 mg/kg) (n=5). Data shown are means ± s.e.m of three independent experiments (A- E) or treated mice and were compared for each condition to untreated cells or control mice by Student t-test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05.

Article Snippet: Mouse FGL1 cDNA sequences (full length, N-terminal domain, globular domain) were cloned into pFUSEN-hG2Fc plasmid (InvivoGen) with the following modifications: vector signal sequence (from Interleukin-2) was used instead of the native, followed by the Fc fragment of human IgG2.

Techniques: Expressing, Recombinant, RNA Expression, Concentration Assay, Saline, Control

Journal: bioRxiv

Article Title: The hepatokine FGL1 regulates hepcidin and iron metabolism during the recovery from hemorrhage-induced anemia in mice

doi: 10.1101/2023.04.06.535920

Figure Lengend Snippet: Time course of Fgl1 mRNA expression in the liver (A) and the bone marrow (B) 1 to 6 days after phlebotomy in WT and Erfe-/- mice (n=5-8). (C) Hamp and Fgl1 mRNA expression in the liver of 7 week-old mice at t=0 to 20h after a single intra-peritoneal injection of EPO (200u) (n=5). (D) Fgl1 mRNA expression in liver of 8 week-old WT, Th3/+ and Erfe-/-; Th3/+ mice (n=7-9). (F) Fold change of Fgl1 mRNA expression in mouse primary hepatocytes in serum free or serum-containing (5% FBS) media and incubated for 15 hours in low oxygen condition (2%) or in presence of prolyl hydroxylases inhibitor DMOG compared to untreated cells (dashed line). Relative Fgl1 mRNA expression in the liver of Vhl -deficient mice (G), and in the liver of mice treated with prolyl hydroxylase inhibitor vadadustat (H). Data shown are means ± s.e.m and were compared for each time point to values for control WT at t = 0 by Two-way ANOVA (A, B, C) or to WT or vehicle treated mice by Student t-test (D, F, G). Data shown for experiment in primary hepatocytes are means of three independent experiments and were compared to control cells by Student t-test (E). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05.

Article Snippet: Mouse FGL1 cDNA sequences (full length, N-terminal domain, globular domain) were cloned into pFUSEN-hG2Fc plasmid (InvivoGen) with the following modifications: vector signal sequence (from Interleukin-2) was used instead of the native, followed by the Fc fragment of human IgG2.

Techniques: Expressing, Injection, Incubation, Control

Journal: bioRxiv

Article Title: The hepatokine FGL1 regulates hepcidin and iron metabolism during the recovery from hemorrhage-induced anemia in mice

doi: 10.1101/2023.04.06.535920

Figure Lengend Snippet: Fgl1 mRNA expression in the liver of male and female WT mice 36 hours after bleeding compared to control mice (A). Red blood cell count (RBC) (B) and hemoglobin (Hb) (C), marrow (D) and spleen (E) Erfe mRNA expression, liver Hamp (F), Id1 (G), Smad7 (H) mRNA expression in WT and Fgl1-/- mice at t=0 or 36 hours after phlebotomy. Data shown are means ± s.e.m (n= 5-11) and were compared between each group by Two-way ANOVA and corrected for multiple comparisons by Holm-Šidák method. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05.

Article Snippet: Mouse FGL1 cDNA sequences (full length, N-terminal domain, globular domain) were cloned into pFUSEN-hG2Fc plasmid (InvivoGen) with the following modifications: vector signal sequence (from Interleukin-2) was used instead of the native, followed by the Fc fragment of human IgG2.

Techniques: Expressing, Control, Cell Counting

Journal: bioRxiv

Article Title: The hepatokine FGL1 regulates hepcidin and iron metabolism during the recovery from hemorrhage-induced anemia in mice

doi: 10.1101/2023.04.06.535920

Figure Lengend Snippet: Relative expression of Hamp (A), Id1 (B), Smad7 (C) mRNA expression in mouse primary hepatocytes treated with BMP ligand (10ng/ml) and human Fc IgG2 (10µg/ml) or Fc-FGL1 (10µg/ml) for 6 hours. Data shown are means ± s.e.m of three independent experiments and were compared for each BMP between Fc or FGL1 treated cells and control cells by Two-way ANOVA. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05. (D) Western blotting of Hep3B cells treated for 6 hours with BMP6 (10 ng/ml), ERFE (1µg/ml), or FGL1 (10µg/ml) for P-SMAD5, SMAD5 and GAPDH. (E) Western blotting of pull down assay of BMP6 and FGL1 (FL, glob, Nter) for human Fc IgG2 and BMP6.

Article Snippet: Mouse FGL1 cDNA sequences (full length, N-terminal domain, globular domain) were cloned into pFUSEN-hG2Fc plasmid (InvivoGen) with the following modifications: vector signal sequence (from Interleukin-2) was used instead of the native, followed by the Fc fragment of human IgG2.

Techniques: Expressing, Control, Western Blot, Pull Down Assay

Journal: Cell Reports Medicine

Article Title: Single-cell spatial transcriptomics reveals a dynamic control of metabolic zonation and liver regeneration by endothelial cell Wnt2 and Wnt9b

doi: 10.1016/j.xcrm.2022.100754

Figure Lengend Snippet: Single-cell spatial transcriptomics profiling by Molecular Cartography reveals dynamic zonation changes in EC-Wnt2-9b-DKO mice (A) UMAP plots of single-cell transcriptomic analysis of Molecular Cartography data showing genetic clusters of hepatocytes from control and EC-Wnt2-9b-DKO livers based on expression of 16 zonated genes. Seurat clustering identifies C1-C6 clusters with C1 and C2 clusters representing hepatocytes expressing pericentral (zone 3) genes, C3 representing hepatocytes expressing midzonal genes, and C4-C6 representing hepatocytes expressing periportal genes. An enrichment of C4-C6 hepatocytes is observed in the EC-Wnt2-9b-DKO cluster at the expense of C1 and C2 clusters. (B) UMAP plots using spatial location of cells in Molecular Cartography analysis based on nine arbitrary but equivalent segments manually drawn between portal vein and central vein shows various clusters recognized in (A) to represent zonal location validating the accuracy of this technique in addressing single-cell spatial transcriptomics. (C) Molecular Cartography comparing spatial gene expression of Glul , Lect2 , Axin2 , Cldn2 , Cyp1a2 , Oat , Cyp2e1 , and Rgn (zone 3), and Fbp1 , G6pc , Pck1 , Gls2 , Arg1 , Ass1 , Cps1 , and Cyp2f2 (zone 1), and Sox9 (mostly in cholangiocytes) in control and EC-Wnt2-9b-DKO liver showing loss of zone 3 genes and overall “periportalization” of the DKO liver. CV, central vein; PV, portal vein. (D) Line plots showing impaired pericentral expression of Wnt target genes (shown here are Glul , Oat , Cyp2e1 , and Axin2) in DKOs compared with control. PC, pericentral; Mid, midzonal; PP, periportal. (E) Line plots showing dynamic changes in the expression of periportal genes (shown here are Fads1 , Gls2 , Cps1 , Elovl2 , and Ass1) in DKOs compared with control. Red arrow, upregulation; green arrow, downregulation; negative, no change. Color stands for the extent of up- or downregulation. (F) Molecular Cartography depicting altered spatial expression of Ccnd1 and Igfbp2 in EC-Wnt2-9b-DKO versus control liver. While the expression of both genes is midzonal with some overlap, both genes are located pericentrally in DKOs with Ccnd1 being more restricted to hepatocytes next to the central vein. CV, central vein; PV, portal vein. (G) Line plots of three representative midzonal genes ( Ccnd1 , Pon1 , and Igfbp2 ) showing altered location to pericentral hepatocytes in DKOs compared with control. The hue around line plots represents SEM.

Article Snippet: Rabbit monoclonal anti-Cyclin D1 , Abcam , Cat#ab134175; RRID: AB_2750906.

Techniques: Expressing

Journal: Cell Reports Medicine

Article Title: Single-cell spatial transcriptomics reveals a dynamic control of metabolic zonation and liver regeneration by endothelial cell Wnt2 and Wnt9b

doi: 10.1016/j.xcrm.2022.100754

Figure Lengend Snippet: Hepatic EC-derived Wnt2 and Wnt9b contribute to normal liver regeneration after partial hepatectomy (A) A representative WB showing notably lower cyclin D1 level at 40 h post PH, especially in males in single KO of endothelial cell Wnt2 and Wnt9b, but a more profound decrease is evident in DKOs in both sexes. (B) IHC for cyclin D1 shows decreased staining at 40 h post PH in male EC-Wnt2-KO, EC-Wnt9b-KO, and EC-Wnt2-9b-DKO mice. Staining for markers of proliferation, such as Ki67 and BrdU, were concurrently decreased in EC-Wnt2-KO and EC-Wnt9b-KO, and almost absent in EC-Wnt2-9b-DKO. In females, cyclin D1 was not changed at 40 h in single KO but was notably decreased in DKOs. Despite no difference in cyclin D1, both Ki67 and BrdU were decreased in single and almost absent in DKOs showing impairment of proliferation. Scale bars, 100 μm. (C) Quantification of BrdU-positive hepatocytes per field (200×) at 40 h. Both males and females were included. The bars represent means ± SEM, ∗∗∗∗p < 0.0001. n = 6, 4, 6, and 7 mice.

Article Snippet: Rabbit monoclonal anti-Cyclin D1 , Abcam , Cat#ab134175; RRID: AB_2750906.

Techniques: Derivative Assay, Staining

Journal: Cell Reports Medicine

Article Title: Single-cell spatial transcriptomics reveals a dynamic control of metabolic zonation and liver regeneration by endothelial cell Wnt2 and Wnt9b

doi: 10.1016/j.xcrm.2022.100754

Figure Lengend Snippet: FL6.13 rescues liver zonation and liver regeneration in mice lacking Wnt secretion, specifically Wnt2 and Wnt9b from the endothelial cells, but not in mice lacking Wnt co-receptors LRP5-6 from hepatocytes (A) Study design showing dosing schedule of pan-Frizzled agonist FL6.13 or isotype control IgG administration to various mouse groups. (B) Representative IHC for β-catenin pericentral targets GS and CYP2E1, midzonal target cyclin D1, and indicator of cell proliferation BrdU, in baseline livers after four treatments of control mice with control IgG and four treatments of EC-Wnt2-9b-DKO, EC-Wls-KO, and LRP5-6-LDKO mice with FL6.13. Re-appearance of these markers to almost control levels is seen in EC-Wnt2-9b-DKO and EC-Wls-KO mice but not in LRP5-6-LDKO mice. Scale bars, 200 μm. (C) Bar graph for LW/BW (± SEM) at 24 h post PH shows comparable liver restoration in FL6.13-treated controls, EC-Wnt2-9b-DKO, and EC-Wls-KO mice, but not LRP5-6-LDKO mice. n.s., not significant, ∗∗p < 0.01. n = 5, 3, 3, 3 mice).

Article Snippet: Rabbit monoclonal anti-Cyclin D1 , Abcam , Cat#ab134175; RRID: AB_2750906.

Techniques:

Journal: Cell Reports Medicine

Article Title: Single-cell spatial transcriptomics reveals a dynamic control of metabolic zonation and liver regeneration by endothelial cell Wnt2 and Wnt9b

doi: 10.1016/j.xcrm.2022.100754

Figure Lengend Snippet: Single-cell spatial transcriptomics profiling of FL6.13-treated control mice reveals dynamic changes in zonation and promotes regeneration after partial hepatectomy (A) UMAP plots of single-cell transcriptomic analysis of hepatocytes by Molecular Cartography showing gain of specific clusters in FL6.13-treated livers. (B) Stacked bar chart depicting distribution of various hepatocyte clusters showing specific gain of C3 and C4 after FL6.13 treatment (right bar) compared with controls (left bar). (C) Feature plots from Molecular Cartography analysis of specific representative genes showing ectopic expression of pericentral genes ( Lect2 , Cyp2e1 , Rgn ) in midzonal and periportal cluster after FL6.13 treatment (red) compared with controls (blue), while periportal genes ( G6pc , Arg1 , Atp5a1 ) continued to be expressed in these clusters and hence remained unchanged. (D) Molecular Cartography visualization of tissue section of the same genes as indicated in (C) showing ectopic expression of pericentral genes in midzonal and periportal regions, while periportal genes were unchanged after FL6.13 treatment. PC, pericentral; PP, periportal. (E) qPCR using bulk mRNA from livers of controls or FL6.13-treated mice shows significant increase in the expression of several pericentral Wnt target genes by FL6.13. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. n = 6, 5 mice. (F) qPCR using bulk mRNA from livers of controls or FL6.13-treated mice shows no statistically significant effect on expression of several known periportal genes by FL6.13. Corresponding p values are indicated. n = 6, 5 mice. (G) Representative IHC verify ectopic localization of CYP2E1 and RGN proteins in midzonal and periportal regions after FL6.13 treatment, while CYP2F2 and PIGR protein locations remained unchanged. PV, portal vein. Scale bars, 200 μm. (H) Representative qPCR using whole-liver RNA and WB using whole-liver lysate shows dramatic increase in Ccnd1 expression and cyclin D1 protein after FL6.13 treatment compared with controls. qPCR: n = 6, 5 mice. (I) Violin plot of Seurat clustering of the Molecular Cartography from (A) shows appearance of periportal Ccnd1 expression (C4 and C5 clusters; green) while its expression in C2 and C3 clusters, where it is normally expressed in controls (red), remains unchanged after FL6.13 treatment. (J) Representative IHC for cyclin D1 and BrdU showing FL6.13 induces periportal hepatocyte G1 to S-phase transition and hepatocyte proliferation, respectively, compared with controls. Scale bars, 100 μm. (K) Quantification of BrdU-positive hepatocytes (± SEM) in (J) shows that FL6.13 induces significant increase in hepatocyte proliferation at baseline compared with control IgG. ∗∗∗∗p < 0.0001. n = 6, 6 mice. (L) Representative IHC showing that FL6.13 accelerates liver regeneration at 24 h post PH by promoting the number of cyclin D1-positive periportal and midzonal hepatocytes and concurrently increasing BrdU-positive proliferating periportal and midzonal hepatocytes compared with control IgG treatment. Scale bars, 100 μm. (M) Quantification of BrdU-positive hepatocytes (± SEM) in (L) shows that FL6.13 induces significant increase in hepatocyte proliferation at 24 h post hepatectomy compared with control IgG treatment. ∗∗∗∗p < 0.0001. n = 6, 9 mice. (N) LW/BW (± SEM) at 24 h post PH depicts an advantage of liver mass restoration in the FL6.13 versus control IgG-treated group. ∗p < 0.05. n = 6, 5 mice.

Article Snippet: Rabbit monoclonal anti-Cyclin D1 , Abcam , Cat#ab134175; RRID: AB_2750906.

Techniques: Expressing, Sublimation

Journal: Cell Reports Medicine

Article Title: Single-cell spatial transcriptomics reveals a dynamic control of metabolic zonation and liver regeneration by endothelial cell Wnt2 and Wnt9b

doi: 10.1016/j.xcrm.2022.100754

Figure Lengend Snippet: Late treatment with FL6.13 promotes liver repair after APAP injury through induction of hepatocyte proliferation (A) Study design showing administration of a single dose of 5 mg/kg pan-FZD agonist FL6.13 or isotype control IgG at 32 h post 600 mg/kg i.p. APAP injection. Mice were sacrificed at 60 h for analysis. (B) Gross images of livers from FL6.13- and control IgG-treated groups after APAP showing decreased necrosis and congestion at 60 h in the FL6.13 treatment group. (C) Serum ALT, AST, direct and total bilirubin, and ALP levels (± SEM) at 60 h after APAP showing significantly reduced serum ALT and AST (trending favorably) in FL6.13 compared with control IgG. n.s., not significant, ∗p < 0.05. n = 5, 4 mice. (D) Representative IHC showing decreased necrotic areas by hematoxylin and eosin (H&E) staining, pan-lobular cyclin D1 staining, increased periportal cell proliferation by Ki67 staining, and a more localized immune cell response by CD45 staining after FL6.13 treatment compared with control IgG. CV, central vein; PV, portal vein. Scale bars, 200 μm. (E) Quantification of H&E and Ki67 showing significant decrease in necrotic areas and increase in hepatocyte proliferation in the FL6.13 group. ∗∗p < 0.01. Necrotic area: n = 5, 4 mice. Ki67: n = 4, 4 mice.

Article Snippet: Rabbit monoclonal anti-Cyclin D1 , Abcam , Cat#ab134175; RRID: AB_2750906.

Techniques: Injection, Staining

Journal: Cell Reports Medicine

Article Title: Single-cell spatial transcriptomics reveals a dynamic control of metabolic zonation and liver regeneration by endothelial cell Wnt2 and Wnt9b

doi: 10.1016/j.xcrm.2022.100754

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-Cyclin D1 , Abcam , Cat#ab134175; RRID: AB_2750906.

Techniques: Recombinant, SYBR Green Assay, Plasmid Preparation, Software

Journal: Biomolecules

Article Title: Eicosapentaenoic Acid Inhibits KRAS Mutant Pancreatic Cancer Cell Growth by Suppressing Hepassocin Expression and STAT3 Phosphorylation

doi: 10.3390/biom11030370

Figure Lengend Snippet: Expression level of hepassocin (HPS) in Kirsten rat sarcoma viral oncogene homolog (KRAS) -mutant pancreatic cancer cells. ( A ) Protein expressions of HPS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by hTERT-HPNE, BxPC-3, PANC-1, MIA Paca-2, and SUIT-2 cells were measured by a Western blot analysis. GAPDH was used as the internal control. ( B ) Protein expressions of phosphorylated (p)-STAT3, STAT3, and HPS by HEK293T cells transiently transfected with the RAS wild-type (WT), constitutively active RAS (V12), and dominant negative RAS (N17) plasmids and a control vector were measured by a Western blot analysis. Actin was used as the internal control. The p-STAT3/STAT3 ratio ( C ) and HPS ( D ) expression level of indicated cells were analyzed with ImageJ software, normalized to actin, and evaluated as multiples of change compared to vector-transfected cells. Results are shown as the mean ± SD of three independent experiments. ** p < 0.01 by a one-way ANOVA followed by Tukey’s post-hoc test.

Article Snippet: Membranes were blocked with 5% skim milk in TBST (Tris-buffered saline containing 0.1% Tween-20) for 30 min and then incubated overnight at 4 °C with the indicated primary antibodies: β-actin (Millipore, Billerica, MA, USA; 1:7500), α-tubulin (ABclonal, Cambridge, MA, USA; 1:7500), GAPDH (GeneTex, Irvine, CA, USA; 1:7500), HPS (FGL1; Proteintech Group, Chicago, IL, USA; 1:1000), STAT3 (Cell Signaling, Beverly, MA, USA; 1:5000), phosphorylated (p)-STAT3 (Tyr705, D3A7, Cell Signaling; 1:5000), cyclin B1 (Cell Signaling; 1:1000), cyclin D1 (Cell Signaling; 1:1000), cyclin E (Cell Signaling; 1:1000), acetyl coenzyme A carboxylase (ACC)-1 (Cell Signaling; 1:1000), and FA synthase (FAS; Cell Signaling; 1:1000).

Techniques: Expressing, Mutagenesis, Western Blot, Control, Transfection, Dominant Negative Mutation, Plasmid Preparation, Software

Journal: Biomolecules

Article Title: Eicosapentaenoic Acid Inhibits KRAS Mutant Pancreatic Cancer Cell Growth by Suppressing Hepassocin Expression and STAT3 Phosphorylation

doi: 10.3390/biom11030370

Figure Lengend Snippet: Knockdown of hepassocin (HPS) decreases pancreatic cancer cell growth in SUIT-2 cells. ( A ) SUIT-2 cells were infected with a lentivirus carrying the short hairpin (sh) control vectors of Scramble, Void, and pLKO, and shHPS vectors #20, #21, and #90, and protein expressions of STAT3, phosphorylated (p)-STAT3, HPS, and GAPDH of stably transfected SUIT-2 cells were measured by a Western blot analysis. GAPDH was used as the internal control. ( B ) HPS expression levels of the indicated SUIT-2 cells were analyzed with ImageJ software, normalized to GAPDH, and evaluated as multiples of change compared to SUIT-2 cells. Results are shown as the mean ± SD of three independent experiments. * p < 0.05 vs. Scramble, # p < 0.05 vs. Void, and † p < 0.05 vs. pLKO control (by a one-way ANOVA followed by Tukey’s post-hoc test). ( C ) The indicated SUIT-2 cells were seeded and cultured for a period of time (24, 48, 72, and 96 h), and cell viability was subsequently evaluated by an MTT assay. Multiple changes of cell viability were compared to each 24-h group. Results are shown as the mean ± SD of three independent experiment, each performed in triplicate. * p < 0.05 vs. Scramble, # p < 0.05 vs. Void, and † p < 0.05 vs. pLKO control (by a one-way ANOVA followed by Tukey’s post-hoc test). ( D ) The cell clonogenicity effect of HPS knockdown on SUIT-2 cells was measured by a clonogenic assay. ( E ) Total cell numbers were measured as the number of colonies Size (pixel*2) = 100-infinity calculated by ImageJ. * p < 0.05 vs. the Scramble control.

Article Snippet: Membranes were blocked with 5% skim milk in TBST (Tris-buffered saline containing 0.1% Tween-20) for 30 min and then incubated overnight at 4 °C with the indicated primary antibodies: β-actin (Millipore, Billerica, MA, USA; 1:7500), α-tubulin (ABclonal, Cambridge, MA, USA; 1:7500), GAPDH (GeneTex, Irvine, CA, USA; 1:7500), HPS (FGL1; Proteintech Group, Chicago, IL, USA; 1:1000), STAT3 (Cell Signaling, Beverly, MA, USA; 1:5000), phosphorylated (p)-STAT3 (Tyr705, D3A7, Cell Signaling; 1:5000), cyclin B1 (Cell Signaling; 1:1000), cyclin D1 (Cell Signaling; 1:1000), cyclin E (Cell Signaling; 1:1000), acetyl coenzyme A carboxylase (ACC)-1 (Cell Signaling; 1:1000), and FA synthase (FAS; Cell Signaling; 1:1000).

Techniques: Knockdown, Infection, Control, Stable Transfection, Transfection, Western Blot, Expressing, Software, Cell Culture, MTT Assay, Clonogenic Assay

Journal: Biomolecules

Article Title: Eicosapentaenoic Acid Inhibits KRAS Mutant Pancreatic Cancer Cell Growth by Suppressing Hepassocin Expression and STAT3 Phosphorylation

doi: 10.3390/biom11030370

Figure Lengend Snippet: Treatment with n-3 and n-6 polyunsaturated fatty acids (PUFAs) decreases the viability of SUIT-2 cells. SUIT-2 and hTERT-HPNE cells were exposed to different concentrations (0, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μM) of n-3 or n-6 PUFAs for 48 ( A ) and 72 h ( B ), and cell viability was subsequently evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Ethanol (75%) was used to prepare the solution and as serial dilution controls. The percentage of cell viability is shown relative to the untreated controls. Results are shown as the mean ± SD of three independent experiments, each performed in triplicate. * p < 0.05 vs. untreated SUIT-2 cells, # p < 0.05 vs. untreated hTERT-HPNE cells (by a two-way ANOVA followed by Tukey’s post-hoc test). ( C ) Protein expressions of signal transduction and activator of transcription 3 (STAT3), phosphorylated (p)-STAT3, and HPS of the indicated SUIT-2 cells were measured by a Western blot analysis. Tubulin was used as the internal control. ( D ) Expression levels of p-STAT3/STAT3 and HPS were analyzed with ImageJ software, normalized to actin, and evaluated as multiples of change compared to untreated cells. Results are shown as the mean ± SD of three independent experiments. * p < 0.05 (by a one-way ANOVA followed by Tukey’s post-hoc test). ( E ) Protein expressions of ACC-1 and FAS of the indicated SUIT-2 cells were measured by a Western blot analysis. Actin was used as the internal control. ( F ) Expression levels of ACC-1 and FAS were analyzed with ImageJ software, normalized to actin, and evaluated as multiples of change compared to untreated cells. Results are shown as the mean ± SD of three independent experiments. * p < 0.05 (by a one-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: Membranes were blocked with 5% skim milk in TBST (Tris-buffered saline containing 0.1% Tween-20) for 30 min and then incubated overnight at 4 °C with the indicated primary antibodies: β-actin (Millipore, Billerica, MA, USA; 1:7500), α-tubulin (ABclonal, Cambridge, MA, USA; 1:7500), GAPDH (GeneTex, Irvine, CA, USA; 1:7500), HPS (FGL1; Proteintech Group, Chicago, IL, USA; 1:1000), STAT3 (Cell Signaling, Beverly, MA, USA; 1:5000), phosphorylated (p)-STAT3 (Tyr705, D3A7, Cell Signaling; 1:5000), cyclin B1 (Cell Signaling; 1:1000), cyclin D1 (Cell Signaling; 1:1000), cyclin E (Cell Signaling; 1:1000), acetyl coenzyme A carboxylase (ACC)-1 (Cell Signaling; 1:1000), and FA synthase (FAS; Cell Signaling; 1:1000).

Techniques: MTT Assay, Serial Dilution, Transduction, Western Blot, Control, Expressing, Software