Journal: International Journal of Molecular Sciences

Article Title: The Mechanisms Underlying the Cytotoxic Effects of Copper Via Differentiated Embryonic Chondrocyte Gene 1

doi: 10.3390/ijms20205225

Figure Lengend Snippet: Effects of copper sulfate on expression of HIF-1α in normoxic and hypoxic HeLa cells. ( A , B ) HeLa cells were incubated for 6 h with the indicated concentrations of copper sulfate under normoxia and hypoxia (1% O 2 ). ( A ) Cell lysates were subjected to western blot analysis using antibodies against HIF-1α, p53, DEC1, p21, total and phosphorylated (p)-ERK, and total and cPARP. ( B ) RT-PCR analysis for HIF-1α , VEGF , and DEC1 . ACTN was the protein loading control; GAPDH mRNA was the mRNA loading control. ( C ) HeLa cells were incubated for 8 h with the indicated compounds (300 μM), after which cell lysates were subjected to western blot analysis using antibodies against HIF-1α, p53, DEC1, p21, total and p-ERK, and total and cleaved PARP. ACTN was the protein loading control. The results are representative of three independent experiments. Protein and PCR bands were quantified through pixel density scanning and evaluated using ImageJ, version 1.44a ( http://imagej.nih.gov/ij/ ). The fold (shown above the bands) was normalized to the internal control protein (ACTN) or gene ( GAPDH ). Phosphorylated ERK after normalization to total ERK protein and cPARP fragment after normalization to full-length PARP protein are shown as fold changes.

Article Snippet: Proteins in the resultant lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against α-actinin (ACTN), ATF3, c-Fos, FASN HuR, Nrf2, p21, p53, p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetyl-CoA carboxylase (ACC), p-ACC (phosphorylation at Ser 79), ERK, p-ERK, HIF-1α, LC3B, cleaved poly-ADP-ribose polymerase (cPARP) (Cell Signaling, Danvers, MA, USA), Cyclin D1 (Abcam, Cambridge, UK), HO-1 (Enzo Life Sciences, Farmingdale, NY, USA), and DEC1 (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Expressing, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control

Journal: International Journal of Molecular Sciences

Article Title: The Mechanisms Underlying the Cytotoxic Effects of Copper Via Differentiated Embryonic Chondrocyte Gene 1

doi: 10.3390/ijms20205225

Figure Lengend Snippet: Mechanism by which copper sulfate increases HIF-1α expression. HeLa cells were incubated for 8 h with the indicated compounds in the absence (−) or presence (+) of 300 μM copper sulfate. Cell lysates were then subjected to western blot analysis using antibodies against HIF-1α, p53, p21, total and p-ERK, Nrf2, HO-1, total and p-ACC, LC3B, and p62. ACTN served as the protein loading control. The results are representative of three independent experiments. Protein bands were quantified through pixel density scanning and evaluated using ImageJ, version 1.44a ( http://imagej.nih.gov/ij/ ). The fold (shown above the bands) was normalized to the internal control (ACTN). Phosphorylated ERK and ACC after normalization of their respective total proteins are shown as fold changes.

Article Snippet: Proteins in the resultant lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against α-actinin (ACTN), ATF3, c-Fos, FASN HuR, Nrf2, p21, p53, p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetyl-CoA carboxylase (ACC), p-ACC (phosphorylation at Ser 79), ERK, p-ERK, HIF-1α, LC3B, cleaved poly-ADP-ribose polymerase (cPARP) (Cell Signaling, Danvers, MA, USA), Cyclin D1 (Abcam, Cambridge, UK), HO-1 (Enzo Life Sciences, Farmingdale, NY, USA), and DEC1 (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Expressing, Incubation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: The Mechanisms Underlying the Cytotoxic Effects of Copper Via Differentiated Embryonic Chondrocyte Gene 1

doi: 10.3390/ijms20205225

Figure Lengend Snippet: Copper sulfate acts via DEC1 to increase expression of related proteins. After DEC1 knockdown in HeLa cells, the cells were incubated for 6 h with the indicated concentrations of copper sulfate. Cell lysates were then subjected to western blotting with antibodies against DEC1, HIF-1α, p53, total and p-ERK, and p62. ACTN served as the loading control. The results are representative of three independent experiments. Protein bands were quantified through pixel density scanning and evaluated using ImageJ, version 1.44a ( http://imagej.nih.gov/ij/ ). The fold (shown above the bands) was normalized to internal control (ACTN). Phosphorylated ERK after normalization to total ERK protein is shown as the fold change.

Article Snippet: Proteins in the resultant lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against α-actinin (ACTN), ATF3, c-Fos, FASN HuR, Nrf2, p21, p53, p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetyl-CoA carboxylase (ACC), p-ACC (phosphorylation at Ser 79), ERK, p-ERK, HIF-1α, LC3B, cleaved poly-ADP-ribose polymerase (cPARP) (Cell Signaling, Danvers, MA, USA), Cyclin D1 (Abcam, Cambridge, UK), HO-1 (Enzo Life Sciences, Farmingdale, NY, USA), and DEC1 (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Expressing, Knockdown, Incubation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: The Mechanisms Underlying the Cytotoxic Effects of Copper Via Differentiated Embryonic Chondrocyte Gene 1

doi: 10.3390/ijms20205225

Figure Lengend Snippet: Effects of copper sulfate are cell-type specific. HeLa, HEC-1A, HEK293, A549, and Beas-2 cells were incubated for 8 h with the indicated concentrations of copper sulfate. Cell lysates were then subjected to western blot analysis using antibodies against HIF-1α, p53, and DEC1. HuR served as the protein loading control. The results are representative of three independent experiments. Protein bands were quantified through pixel density scanning and evaluated using ImageJ, version 1.44a ( http://imagej.nih.gov/ij/ ). The fold (shown above the bands) was normalized to the internal control (HuR).

Article Snippet: Proteins in the resultant lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against α-actinin (ACTN), ATF3, c-Fos, FASN HuR, Nrf2, p21, p53, p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetyl-CoA carboxylase (ACC), p-ACC (phosphorylation at Ser 79), ERK, p-ERK, HIF-1α, LC3B, cleaved poly-ADP-ribose polymerase (cPARP) (Cell Signaling, Danvers, MA, USA), Cyclin D1 (Abcam, Cambridge, UK), HO-1 (Enzo Life Sciences, Farmingdale, NY, USA), and DEC1 (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Incubation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: The Mechanisms Underlying the Cytotoxic Effects of Copper Via Differentiated Embryonic Chondrocyte Gene 1

doi: 10.3390/ijms20205225

Figure Lengend Snippet: The mechanisms of copper-induced cytotoxicity in cancer cells. The cytotoxicity of copper sulfate was manifested by three parts. First, it could decrease respiration and glycolysis in cancer cells, resulting in mitochondrial dysfunction (dark blue arrows). Second, it could increase ROS levels, leading to mitochondrial dysfunction, autophagy, senescence, and upregulation of HIF-1α and p53 protein levels. Finally, it could act via DEC1 to increase expression of related proteins, such as HIF-1α and p53 (green arrows).

Article Snippet: Proteins in the resultant lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against α-actinin (ACTN), ATF3, c-Fos, FASN HuR, Nrf2, p21, p53, p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetyl-CoA carboxylase (ACC), p-ACC (phosphorylation at Ser 79), ERK, p-ERK, HIF-1α, LC3B, cleaved poly-ADP-ribose polymerase (cPARP) (Cell Signaling, Danvers, MA, USA), Cyclin D1 (Abcam, Cambridge, UK), HO-1 (Enzo Life Sciences, Farmingdale, NY, USA), and DEC1 (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Expressing

Journal: Advanced Science

Article Title: EM2, a Natural Product MST1/2 Kinase Activator, Suppresses Non‐Small Cell Lung Cancer via Hippo Pathway Activation

doi: 10.1002/advs.202510508

Figure Lengend Snippet: The role of YAP activity in EM2 anti‐NSCLC. A,B) After EM2 treatment with H520 and A549, representative images A) and quantification B) of YAP localization were detected by IF staining. C,D) Cytoplasmic and nuclear distributions of YAP in H520 and A549 cells treated with or without EM2 for 12 h. Histone 2B and GAPDH were used as endogenous references for nuclear and cytosolic fractions C). The indicated protein expression levels were quantified D). E) Transcriptional activity of YAP/TEAD complex in H520 and A549 cells. Cells were transfected with the reporter plasmids and treated with EM2 at the indicated concentrations for 24 h. F) mRNA levels of C CN1 and CCN2 in H520 cells treated with EM2. RT‐qPCR data were normalized to GAPDH levels and presented as fold‐change compared with control cells. G) Western blotting was used to detect the expressions of YAP and p‐YAP in YAP WT and YAP S127A H520 cells. H) Cell viability of A549 and H520 cells transfected with YAP WT and YAP S127A were treated with 0, 2.5, 5, 10, and 20 µM EM2 for 24 h, and the cell viability was detected by MTT. I,J) Representative images and quantification of EM2 on the migration I) and invasion J) in H520 cells transfected with YAP WT and YAP S127A. Following treatment with indicated concentrations of EM2, the cells were subjected to Transwell migration and invasion assays. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against MST1 (#3682), LATS1 (#3477), YAP (#12395), YAP/TAZ (#8418), phospho‐MST1 (Thr183)/MST2 (Thr180) (#49332), phospho‐LATS1 (Ser909) (#9157), phospho‐YAP (Ser127) (#13008), phospho‐TAZ (Ser89) (#59971), phospho‐Mob1 (Thr35) (#8699), CTGF (#86641), CYR61 (#14479) Phospho‐Rb(Ser780) (#2808), CDK4 (#12790), CDK6 (#3136), and Cyclin D1 (#55506) were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Staining, Expressing, Transfection, Quantitative RT-PCR, Control, Western Blot, Migration

Journal: Advanced Science

Article Title: EM2, a Natural Product MST1/2 Kinase Activator, Suppresses Non‐Small Cell Lung Cancer via Hippo Pathway Activation

doi: 10.1002/advs.202510508

Figure Lengend Snippet: The anti‐tumor activity of EM2 was counteracted by MST1/2 inhibitor in vitro. A) Cell viability of H520 and A549 cells treated by EM2 (8 µM) with or without XMU‐MP‐1 (3 µM). B,C) Representative images B) and quantification C) of colony formation assay of H520 and A549 cells treated by EM2 (2 µM) with or without XMU‐MP‐1 (0.5 µM). D) EdU assay of H520 and A549 cells treated by EM2 (2 µM) with or without XMU‐MP‐1 (0.5 µM). E) Quantification of Edu positive in H520 and A549 cells. F) Immunoblot analysis of p‐MST, p‐LATS1/2, p‐YAP, and p‐TAZ in H520 and A549 cells treated by EM2 (8 µM) with or without XMU‐MP‐1 (3 µM). β‐tubulin served as the loading control. G) Quantification of the protein levels indicated in H520 and A549 cells. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against MST1 (#3682), LATS1 (#3477), YAP (#12395), YAP/TAZ (#8418), phospho‐MST1 (Thr183)/MST2 (Thr180) (#49332), phospho‐LATS1 (Ser909) (#9157), phospho‐YAP (Ser127) (#13008), phospho‐TAZ (Ser89) (#59971), phospho‐Mob1 (Thr35) (#8699), CTGF (#86641), CYR61 (#14479) Phospho‐Rb(Ser780) (#2808), CDK4 (#12790), CDK6 (#3136), and Cyclin D1 (#55506) were purchased from Cell Signaling Technology.

Techniques: Activity Assay, In Vitro, Colony Assay, EdU Assay, Western Blot, Control

Journal: Advanced Science

Article Title: EM2, a Natural Product MST1/2 Kinase Activator, Suppresses Non‐Small Cell Lung Cancer via Hippo Pathway Activation

doi: 10.1002/advs.202510508

Figure Lengend Snippet: The role and mechanism of EM2 in inhibiting the tumor growth of NSCLC in vivo. Representative images of the subcutaneous tumor model in mice of control and EM2 (5 or 10 mg kg −1 ) group. B) Tumor volume in control and EM2 (5 or 10 mg kg −1 ) group. C) Quantification and analysis of the tumor weight in control and EM2 (5 or 10 mg kg −1 ) group. D) The expression levels of Ki67, YAP, CTGF, and CYR61 in tumors assayed by IHC in control and EM2 (5 or 10 mg kg −1 ) group. E) Expression levels of MST1, LATS1, and YAP and their phosphorylation forms along with downstream protein CTGF and CYR61 were examined by Western blotting in control and EM2 (5 or 10 mg kg −1 ) group. F) Representative images of tumors in subcutaneous mouse model, grouped by treatment with EM2 with or without XMU‐MP‐1. G) Tumor volume in each group of EM2 with or without XMU‐MP‐1. H) Quantification and analysis of the tumor weight in each group of EM2 with or without XMU‐MP‐1. I,J) Representative images I) and quantification J) of the expression levels of Ki67, YAP, CTGF, and CYR61 in tumors assayed by IHC in each group of EM2 with or without XMU‐MP‐1. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against MST1 (#3682), LATS1 (#3477), YAP (#12395), YAP/TAZ (#8418), phospho‐MST1 (Thr183)/MST2 (Thr180) (#49332), phospho‐LATS1 (Ser909) (#9157), phospho‐YAP (Ser127) (#13008), phospho‐TAZ (Ser89) (#59971), phospho‐Mob1 (Thr35) (#8699), CTGF (#86641), CYR61 (#14479) Phospho‐Rb(Ser780) (#2808), CDK4 (#12790), CDK6 (#3136), and Cyclin D1 (#55506) were purchased from Cell Signaling Technology.

Techniques: In Vivo, Control, Expressing, Phospho-proteomics, Western Blot

Journal: Advanced Science

Article Title: EM2, a Natural Product MST1/2 Kinase Activator, Suppresses Non‐Small Cell Lung Cancer via Hippo Pathway Activation

doi: 10.1002/advs.202510508

Figure Lengend Snippet: Schematic illustration of EM2‐induced suppression of cancer through targeting MST1/2 and activation of the Hippo signaling pathway. EM2 directly targets MST1/2, enhancing its kinase activity to promote LATS and YAP phosphorylation. This cascade reduces YAP nuclear translocation and diminishes CTGF and CYR61 mRNA translation, thereby exerting a potent inhibitory effect on NSCLC progression.

Article Snippet: Antibodies against MST1 (#3682), LATS1 (#3477), YAP (#12395), YAP/TAZ (#8418), phospho‐MST1 (Thr183)/MST2 (Thr180) (#49332), phospho‐LATS1 (Ser909) (#9157), phospho‐YAP (Ser127) (#13008), phospho‐TAZ (Ser89) (#59971), phospho‐Mob1 (Thr35) (#8699), CTGF (#86641), CYR61 (#14479) Phospho‐Rb(Ser780) (#2808), CDK4 (#12790), CDK6 (#3136), and Cyclin D1 (#55506) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Activity Assay, Phospho-proteomics, Translocation Assay