Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a Normal human bronchial epithelial cell (BEAS-2B), KRAS Mut cell lines (H358 and H460), KRAS WT and EGFR WT cell lines (HCC1666 and H522), and EGFR Mut cell lines (HCC827 and PC9) were collected with lysis buffer, and immunoblotted with anti-SIRT1 and β-actin antibody. b Immunohistochemical staining for SIRT1 with the lung from LSL-Kras G12D Tg mouse at 16 weeks after administration of adenovirus Cre recombinase induction. Representative images are shown. Scale bar, 100 μm. High-magnification images correspond to the areas marked by the black box. c SIRT1 mRNA expression was measured by RT–qPCR with the same cell lines as in a . RPL32 was used as internal control and for normalization. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. d The mRNA expression of Sirt1 was analyzed by RT–qPCR with the cancerous and adjacent noncancerous lung tissues of LSL- Kras G12D Tg mouse. Rpl32 was used as internal control and for normalization. Student’s t -test, mean ± s.e.m.; n = 6, *, P < 0.05. e H358 and H460 cells were transfected with the 2-μg plasmids of pcDNA , KRAS WT and KRAS G12D . The cells were collected with cell lysis buffer and subjected to western blotting with anti-KRAS, anti-SIRT1 and β-actin antibodies. f H358 and H460 cells were transfected with siCon and siKRAS (80 nM). The cells were collected with cell lysis buffer and subjected to western blotting with same antibodies in e . g H358 and H460 cell extracts were immunoprecipitated with anti-KRAS and anti-SIRT1, and immunoblotted with anti-KRAS, anti-SIRT1 and β-actin antibodies.

Article Snippet: Human recombinant protein SIRT1 (CUSABIO, CSB-EP822202HU) (10 μM) were incubated in 1× kinase buffer (Cell Signaling), supplemented with 2 mM ATP (Cell Signaling), and 25 ng/μl purified active human JNK1 (Abcam, ab201870).

Techniques: Lysis, Immunohistochemical staining, Staining, Expressing, Quantitative RT-PCR, Control, Transfection, Western Blot, Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a KRAS Mut cell lines (H358, A427, NCIH727, NCIH23 and SKLU-1), EGFR Mut cell lines (HCC827, HCC2279, H1650 and H1975), KRAS WT and EGFR WT cell lines (H322M, H522, Calu-3 and HCC1666) and nontumorigenic cells (HEK-293T and BEAS-2B) were collected with lysis buffer, and SIRT1 activity was measured with cell lysates. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. b Normal human bronchial epithelial cell (BEAS-2B) and KRAS Mut cell lines used in a were collected with lysis buffer, and immunoblotted with anti-pMKK4, MKK, pMKK7, MKK, pJNK1, JNK1 and β-actin antibodies. c KRAS Mut cell lines (H358, A427 and NCIH727) were treated with anisomycin 38 μM (10 μg/ml) (JNK1 activator) and SP600125 20 μM (JNK1 inhibitor) for 2 h. The protein levels of pJNK1, JNK, pSIRT1 S27 , pSIRT1 S47 , pSIRT1 T530 , SIRT1 and β-actin were measured by western blot analysis. d H358 cells were treated with anisomycin and SP600125 under the same condition as in b and then whole-cell lysates were subjected to immunoprecipitation with an anti-JNK1 antibody. Immunoblot analysis was performed using antibodies against SIRT1, pSIRT1 S27 , pSIRT1 S47 , pSIRT1 T530 , pJNK1, JNK and β-actin antibodies. e The recombinant proteins, SIRT1 and JNK1 were incubated in the reaction mixture for phosphorylation at 32 °C for 4 h, and then Ser- and Thr-phosphorylated peptides were identified using anti-pSIRT1 S27 , pSIRT1 S47 , pSIRT1 T530 antibody. f H358 cells were transfected with SIRT1 WT , SIRT1 T530A , SIRT1 S27A&S47A , and then H358 cell extracts were immunoprecipitated with anti-KRAS antibody and RAF-1 agarose beads and immunoblotted with anti-acetylation, anti-SIRT1, anti-KRAS, anti-KRAS–GTP-bound and β-actin antibodies. g KRAS Mut cell lines (H358, A427 and H727) were transfected with SIRT1 WT , SIRT1 S27A , SIRT1 S47A , SIRT1 T530A and SIRT1 S27A,S47A (2 μg), then collected with lysis buffer, and SIRT1 activity was assessed with cell lysates. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05.

Article Snippet: Human recombinant protein SIRT1 (CUSABIO, CSB-EP822202HU) (10 μM) were incubated in 1× kinase buffer (Cell Signaling), supplemented with 2 mM ATP (Cell Signaling), and 25 ng/μl purified active human JNK1 (Abcam, ab201870).

Techniques: Lysis, Activity Assay, Western Blot, Immunoprecipitation, Recombinant, Incubation, Phospho-proteomics, Transfection

Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a Normal human bronchial epithelial cell (BEAS-2B) and KRAS Mut cell lines (H358, A427 and H727) were collected with lysis buffer and subjected to western blotting with anti-pERK, ERK and β-actin antibodies. b A luciferase assay was performed to assess the AP-1-mediated transcriptional regulatory activity with cell lysates in Fig. 4a. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. c TGFB1 mRNA expression was measured by RT–qPCR with same cell lines as in a . RPL32 was used as internal control and for normalization. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. d The medium of four cell lines were changed by FBS-free medium before cell collection at 24 h. Conditioned medium was collected and concentrated using an Amicon Ultra-15 tube, and total TGF-β1 levels were measured by ELISA. e KRAS Mut cell lines (H358, A427 and H727) were transfected with pcDNA , KRAS G12C , G12D and G12V plasmids (2 μg). TGF-β1 levels were measured under the same method as in d . f H358, A427 and H727 cells were transplanted with pcDNA , KRAS G12C , G12D and G12V plasmids, siCon and siSmad2/3 (80 nM) for 48 h, and then the activity of Smad2/3, JNK1 and KRAS was measured. g The cell lysates of each different KRAS Mut cell lines (H358, A427 and H727) under KWN-C with indicated dosage for 24 h were transferred by immunoblotting assay with anti-pSmad2/3, anti-Smad2/3, pJNK1, JNK1, anti-pSIRT1 Ser27 , pSIRT1 Ser47 , SIRT1, KRAS–GTP-bound and β-actin antibodies. h , H358, A427 and H460 cells were treated with DMSO or KWN-C (10 μM), and cell extracts were then immunoprecipitated using immunoglobulin G, anti-KRAS, and RAF-1 agarose bead antibodies. Immunoblotting was performed using anti-acetyl, anti-KRAS–GTP-bound, anti-SIRT1, anti-KRAS and β-actin antibodies.

Article Snippet: Human recombinant protein SIRT1 (CUSABIO, CSB-EP822202HU) (10 μM) were incubated in 1× kinase buffer (Cell Signaling), supplemented with 2 mM ATP (Cell Signaling), and 25 ng/μl purified active human JNK1 (Abcam, ab201870).

Techniques: Lysis, Western Blot, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Transfection, Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a H358 cells harboring stably expressed luciferase plasmid were intratracheally injected into nude mice (1 × 10 6 cells per mouse). Top: representative bioluminescence images 2 months after the injection. The mice were euthanized 2 months after the injection, and lungs were excised and stained with Bouin’s fixative. Bottom: the lung tumor images. Therapeutic candidates were treated with CP (5 mg/kg per day, i.p.), MTA (150 mg/kg twice a week, i.p.) and/or KWN-C (30 mg/kg per day, i.p.). b The photon emission values represent the mean ± s.e.m. of the indicated number of mice. Student’s t -test, mean ± s.e.m.; n = 5, * P < 0.05. c The lung tumor weight from combination CP, MTA and/or KWN-C-treated mice was measured and compared with nontreatment, each single treatment and combined treatment. Student’s t -test, mean ± s.e.m.; n = 5, * P < 0.05. d The number of colonies formed in the lungs were measured under microscopy under the same conditions as in c . Student’s t -test, mean ± s.e.m.; n = 5, * P < 0.05. e KRAS–GTP (active form of KRAS) was pulled down by Raf-1–RBD from tumor tissue lysates, followed by western blot using KRAS antibody. Expression levels of anti-pSIRT1 S27 , pSIRT1 S47 , SIRT1, KRAS–GTP-bound, KRAS, pERK, ERK pAkt, Akt, PARP, cleaved PARP, pro-caspase-3, cleaved-caspase-3 and β-actin were analyzed by western blot in tumor tissues.

Article Snippet: Human recombinant protein SIRT1 (CUSABIO, CSB-EP822202HU) (10 μM) were incubated in 1× kinase buffer (Cell Signaling), supplemented with 2 mM ATP (Cell Signaling), and 25 ng/μl purified active human JNK1 (Abcam, ab201870).

Techniques: Stable Transfection, Luciferase, Plasmid Preparation, Injection, Staining, Microscopy, Western Blot, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a After administration of adenovirus Cre recombinase in KRAS G12D mice for 10 weeks, mice were treated using the same method as in Fig. . Representative H&E staining images and pJNK1, cleaved caspase-3, cleaved PARP, TUNEL and Ki-67 were analyzed by immunohistochemical staining in tumor tissues at the end of experiments. b Tumor tissue from each drug-treated group was collected with lysis buffer, and SIRT1 activity was measured with cell lysates. Student’s t -test, mean ± s.e.m.; n = 10, * P < 0.05. c Tumor area was quantified using ImageJ software. Student’s t -test, mean ± s.e.m.; n = 10, * P < 0.05. d Tumor numbers per lung area were counted under the microscope in specimens collected from mice treated with drugs. Student’s t -test, mean ± s.e.m.; n = 10, * P < 0.05 . e Survival rates of mice treated with drugs (log-rank test). f Median survival days and P values were calculated using the log-rank test and the Gehan–Breslow–Wilcoxon test, respectively, based on Student’s t -test. g A schematic overview of the mechanism of enhanced SIRT1 activity in KRAS Mut lung cancer and definition of a rational combination strategy between SIRT1 activity inhibitor and conventional chemotherapy.

Article Snippet: Human recombinant protein SIRT1 (CUSABIO, CSB-EP822202HU) (10 μM) were incubated in 1× kinase buffer (Cell Signaling), supplemented with 2 mM ATP (Cell Signaling), and 25 ng/μl purified active human JNK1 (Abcam, ab201870).

Techniques: Staining, TUNEL Assay, Immunohistochemical staining, Lysis, Activity Assay, Software, Microscopy

Journal: Cardiovascular Research

Article Title: Sirtuin-1 directly binds and deacetylates hepatic PCSK9 thereby promoting the inhibition of LDL receptor degradation

doi: 10.1093/cvr/cvaf087

Figure Lengend Snippet: Exogenous SIRT1 treatment reduces plasma LDL-cholesterol and protects against atherosclerosis in ApoE −/− mice. Eight-week-old ApoE −/− mice were fed on a high cholesterol diet (1.25% w/w) for 4 weeks and randomized to be treated with rmSIRT1 ( n = 6) or vehicle (PBS containing 0.1% BSA) ( n = 6) for another 4 weeks. ( A ) Plasma concentration of SIRT1 measured using ELISA of C57BL/6J wild-type mice ( n = 6), ApoE −/− mice + vehicle ( n = 6) and ApoE −/− mice + rmSIRT1 ( n = 6). ( B ) Weekly measurements of body weight of ApoE −/− mice + vehicle ( n = 6) and ApoE −/− mice + rmSIRT1 ( n = 6). ( C ) Graph showing cholesterol distribution in the different lipoprotein sub-fractions separated by gel filtration chromatography; ( D ) Bar graph of plasma total cholesterol and ( E ) LDL-cholesterol concentrations. ( F ) Representative pictures (left) and quantifications of ( G ) thoracic-abdominal aortae en face , ( H ) aortic root cross sections stained with ORO and ( I ) immunohistochemically for macrophages (CD68). Scale bars in photomicrographs: 1 mm (for E ) and 500 μm (for F , G ). Grey bars represent vehicle treatment and green bars represent rmSIRT1 treatment. ORO, Oil-Red O. Values are represented as means ± SEM. Statistical significance was performed using Student’s unpaired t -test.

Article Snippet: Where indicated, cells were treated with vehicle control (Phosphate buffer saline containing 0.1% BSA) or recombinant human SIRT1 (rhSIRT1, 1 μmol/L) (CSB-EP822202HU, Cusabio Biotech, LubioScience, Switzerland) or recombinant human PCSK9 (rhPCSK9, ab198471, Abcam, concentrations in Figure legends) for 2 h.

Techniques: Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Filtration, Chromatography, Staining

Journal: Cardiovascular Research

Article Title: Sirtuin-1 directly binds and deacetylates hepatic PCSK9 thereby promoting the inhibition of LDL receptor degradation

doi: 10.1093/cvr/cvaf087

Figure Lengend Snippet: Exogenous SIRT1 increases hepatic LDLR by post-translational modification of PCSK9 in ApoE −/− mice. Eight-week-old ApoE −/− mice were fed on a high cholesterol diet (1.25% w/w) for 4 weeks and were randomized to be treated with rmSIRT1 ( n = 6) or with vehicle (PBS with 0.1% BSA) ( n = 6) for another 4 weeks. ( A ) Western blot of LDLR (120 kDa) and PCSK9 (∼62 kDa) in hepatic tissue lysates of mice ( n = 5). β-actin (45 kDa) was used as loading control. Bar graph of ELISA of ( B ) total PCSK9 and ( C ) acetylated PCSK9 in mice plasma. Statistical significance was performed using Student’s unpaired two-sample t -test. ( D , E , F ) Human hepatoma Huh7 cells [three independent triplicate experiments ( n = 3)] were treated with either 1 μmol/L of recombinant human SIRT1 (rhSIRT1) or vehicle (PBS containing 0.1% BSA) for 2 h at 37°C. The cell culture media was collected, and cells were lysed to perform ELISA to detect PCSK9. Bar graph of ELISA of ( D ) total cellular PCSK9, ( E ) total media PCSK9 and ( F ) acetylated PCSK9 in the media of cultured Huh7 cells. Grey bars represent vehicle treatment and green bars represent rmSIRT1 or rhSIRT1 treatment as indicated. Data are represented as means ± SEM. Statistical significance was performed using Student’s unpaired t -test.

Article Snippet: Where indicated, cells were treated with vehicle control (Phosphate buffer saline containing 0.1% BSA) or recombinant human SIRT1 (rhSIRT1, 1 μmol/L) (CSB-EP822202HU, Cusabio Biotech, LubioScience, Switzerland) or recombinant human PCSK9 (rhPCSK9, ab198471, Abcam, concentrations in Figure legends) for 2 h.

Techniques: Modification, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Recombinant, Cell Culture

Journal: Cardiovascular Research

Article Title: Sirtuin-1 directly binds and deacetylates hepatic PCSK9 thereby promoting the inhibition of LDL receptor degradation

doi: 10.1093/cvr/cvaf087

Figure Lengend Snippet: Exogenous SIRT1 inhibits PCSK9-mediated LDLR degradation in hepatocytes. HuH-7 (hereafter Huh7) cells were transfected with a specific siRNA against LDLR or with non-silencing control siRNA (NS control). Assays were performed 72 h post transfection. Cells were then pre-treated with 1 μmol/L of rhSIRT1 for 2 h ( A ) Specific cellular binding of 125 I-LDL was measured at 4°C ( B ) Specific cellular association of 125 I-LDL was measured at 37°C. ( C , D , E ) Huh7 cells were co-incubated with rhPCSK9 (2 μg/mL) with rhSIRT1 (1 μmol/L) for 2 h at 37°C ( C ) Representative Western blots ( n = 3) and quantification of blot density of LDLR (130 kDa) degradation in Huh7 cells. Vinculin (140 kDa) was used as the loading control. ( D ) Specific cellular binding of 125 I-LDL was measured at 4°C ( E ) Specific cellular association of 125 I-LDL was measured at 37°C. Values are represented as means ± SEM of three independent triplicate or more experiments ( n = 3). Statistical significance was performed using one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: Where indicated, cells were treated with vehicle control (Phosphate buffer saline containing 0.1% BSA) or recombinant human SIRT1 (rhSIRT1, 1 μmol/L) (CSB-EP822202HU, Cusabio Biotech, LubioScience, Switzerland) or recombinant human PCSK9 (rhPCSK9, ab198471, Abcam, concentrations in Figure legends) for 2 h.

Techniques: Transfection, Control, Binding Assay, Incubation, Western Blot, Comparison

Journal: Cardiovascular Research

Article Title: Sirtuin-1 directly binds and deacetylates hepatic PCSK9 thereby promoting the inhibition of LDL receptor degradation

doi: 10.1093/cvr/cvaf087

Figure Lengend Snippet: Exogenous SIRT1 inhibits PCSK9 activity through direct deacetylation in hepatocytes—specific mutation of these deacetylation sites restores PCSK9 activity. Huh7 cells were incubated with rhSIRT1 (1 μmol/L) for 2 h at 37°C. ( A ) Western blot analysis of PCSK9 immunoprecipitated Huh7 cells lysates with total PCSK9 antibody (∼62 kDa) and acetyl lysine antibody (Ac-K-PCSK9, ∼62 kDa). ( B ) Direct binding assay interaction between rhSIRT1 and rhPCSK9 using Surface Plasmon Resonance. The equilibrium dissociation constant was 34 nM. ( C ) Schematic diagram of PCSK9 domains and an overview of lysine(K) sites on PCSK9 deacetylated by SIRT1. Representative Western blot and quantification of LDLR (130 kDa) and Vinculin (140 kDa) expression in Huh7 cells expressing WT, 3KR, 3KQ mutants of PCSK9 for 72 h ( n = 3). (E )V. means empty vector ( D ) Specific cellular binding of 125 I-LDL was measured at 4°C ( E ) Specific cellular association of 125 I-LDL was measured at 37°C. ( F ) Binding of acetylated and deacetylated PCSK9 to EGF-A of LDLR using ELISA. SP—signal peptide, Pro—pro-domain, Catalytic—catalytic domain, CHRD—cysteine, histidine-rich domain. Values are represented as means ± SEM of three independent triplicate or more experiments ( n = 3). Statistical significance was performed using one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: Where indicated, cells were treated with vehicle control (Phosphate buffer saline containing 0.1% BSA) or recombinant human SIRT1 (rhSIRT1, 1 μmol/L) (CSB-EP822202HU, Cusabio Biotech, LubioScience, Switzerland) or recombinant human PCSK9 (rhPCSK9, ab198471, Abcam, concentrations in Figure legends) for 2 h.

Techniques: Activity Assay, Mutagenesis, Incubation, Western Blot, Immunoprecipitation, Binding Assay, SPR Assay, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Comparison