Journal: Molecular therapy. Nucleic acids

Article Title: Nuclear-Encoded lncRNA MALAT1 Epigenetically Controls Metabolic Reprogramming in HCC Cells through the Mitophagy Pathway.

doi: 10.1016/j.omtn.2020.09.040

Figure Lengend Snippet: Figure 5. MALAT1 Knockdown Impairs Mitophagy (A) Mitophagy biomarkers were detected by western blot. b-Actin was used as the control. (B) Quantitation of mitophagy biomarker western blot results. ****p < 0.0001, **p < 0.01 compared with the shCT control group. (C) The LC3B II/I ratio. Autophagy was assessed by LC3B-II/I ratio. After knockdown of MALAT1, the LC3B-II/I ratio was significantly decreased. ****p < 0.0001 compared with the shCT control group. (D) Acidic lysosome by Lysotracker staining. The intensity of the fluorescence stained by lysotracker was measured. ****p < 0.0001 compared with the shCT control group.

Article Snippet: Antibodies used for western blot were: Anti-LAMP1 antibody-Lysosome Marker (ab24170), Anti-Cyclophilin F antibody (ab110324), Mitophagy Antibody Sampler Kit (Cell Signaling Technology [CST], #43110), Bcl-2 (124) Mouse monoclonal antibody (mAb) (CST, #15071), Cytochrome c (D18C7) Rabbit mAb (CST, #11940), Caspase-3 antibody (CST, #9662), Bax (D2E11) Rabbit mAb (CST, #5023), b-Actin (8H10D10) Mouse mAb (CST, #3700), and BID antibody (CST, #2002).

Techniques: Knockdown, Western Blot, Control, Quantitation Assay, Biomarker Discovery, Staining

Journal: Molecular therapy. Nucleic acids

Article Title: Nuclear-Encoded lncRNA MALAT1 Epigenetically Controls Metabolic Reprogramming in HCC Cells through the Mitophagy Pathway.

doi: 10.1016/j.omtn.2020.09.040

Figure Lengend Snippet: Figure 6. MALAT1 Affects Cancer Biology Behavior (A) MALAT1 knockdown cells showed decreased cell in- vasion ability. Ordinary one-way ANOVA, followed by Stu- dent’s t test. ****p < 0.0001 compared with the shCT control group. (B) Cell proliferation assay. Total cells were counted after 24 h, 48 h, 72 h, and 96 h. Ordinary one-way ANOVA, followed by Student’s t test. ****p < 0.0001 compared with the shCT control group. (C) Wound healing assay. Scale bar = 50 mm. MALAT1 knockdown cells showed slower wound healing capability indicating slower migration ability. (D) The putative model of MALAT1 as an epigenetic messenger in controlling cancer metabolism. MALAT1 is aberrantly expressed in HCC mitochondria, where it binds to mtDNA and alters mtDNA methylation and mitochondrial function, including mitochondria synthesis, metabolism, mitophagy, ROS regulation, and apoptosis.

Article Snippet: Antibodies used for western blot were: Anti-LAMP1 antibody-Lysosome Marker (ab24170), Anti-Cyclophilin F antibody (ab110324), Mitophagy Antibody Sampler Kit (Cell Signaling Technology [CST], #43110), Bcl-2 (124) Mouse monoclonal antibody (mAb) (CST, #15071), Cytochrome c (D18C7) Rabbit mAb (CST, #11940), Caspase-3 antibody (CST, #9662), Bax (D2E11) Rabbit mAb (CST, #5023), b-Actin (8H10D10) Mouse mAb (CST, #3700), and BID antibody (CST, #2002).

Techniques: Knockdown, Control, Proliferation Assay, Wound Healing Assay, Migration, Methylation

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 1. (A) Hematoxylin and eosin staining and immunohistochemical staining for PKCα and claudin-1 in normal pancreatic ducts, well- and poorly differentiated pancreatic carcinomas. Bar: 50 μm. (B and C) Western blotting for pPKCα, panPKCα (PKCα), Snail, claudin-1, -4, -7 and occludin in normal pancreatic duct epithelial cells (hTERT-HPDEs) and pancreatic cancer cell lines HPAC, HPAF-II, BXPC-3 and PANC-1. Snail (1): exposed to X-ray film for 1 min, Snail (2): exposed to X-ray film for 20 min. The corresponding expression levels of B and C are shown as bar graphs. n = 3, *P < 0.05 and **P < 0.01 versus hTERT-HPDEs.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Staining, Immunohistochemical staining, Western Blot, Expressing

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 2. (A) Western blotting for Snail, claudin-1, -4, -7 and occludin in PANC-1 cells after treatment with 0–2 μg/ml Gö6976 for 24 h. (B) Real-time PCR for Snail, claudin-1 and occludin in PANC-1 cells after treatment with 0–2 μg/ml Gö6976 for 24 h. (C) Western blotting for claudin-1, -4, -7 and occludin in hTERT- HPDEs after treatment with 1 and 2 μg/ml Gö6976 for 24 h. (D) Real-time PCR for claudin-1, -4, -7 and occludin in hTERT-HPDEs after treatment with 1 μg/ ml Gö6976 for 24 h. (E) TER values in hTERT-HPDEs after treatment with 1 μg/ml Gö6976 for 24 h. iPKCα: PKCα inhibitor Gö6976. n = 3, *P < 0.05 and **P < 0.01 versus control (0 μg/ml).

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 3. Western blotting for pMAPK and panMAPK (MAPK) in PANC-1 cells (A) and hTERT-HPDEs (B) after treatment with 0–2 μg/ml Gö6976 for 24 h. Western blotting for claudin-1, occludin, pMAPK, MAPK and Snail in PANC-1 cells (C) and hTERT-HPDEs (D) pretreated with 20 μM MAPK/ERK inhibitor U0126 before treatment with 1 μg/ml Gö6976 for 24 h. Western blotting for claudin-1 in PANC-1 cells (E) and hTERT-HPDEs (F) pretreated with 20 μM MAPK/ERK inhibitor U0126, 10 μM p38 MAPK inhibitor SB203580 (SB), 10 μM PI3K inhibitor LY294002 (LY), 10 μM panPKC inhibitor GF109203X (GF), 10 μM JNK inhibitor SP600125 (SP) and 0.1 μM NF-κB inhibitor IMD-0354 (IMD) before treatment with 1 μg/ml PKCα inhibitor Gö6976 for 24 h. iPKCα: PKCα inhibitor Gö6976.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 4. (A) Western blotting for Snail, pPKCα, panPKCα (PKCα), pMAPK, panMAPK (MAPK), claudin-1, -4, -7 and occludin in PANC-1 cells after treatment with 100 ng/ml TGF-β1 for 24 and 48 h. (B) Western blotting for claudin-1, -4, -7, occludin, Snail, PKCα, pMAPK and MAPK in hTERT-HPDEs after treatment with 20 ng/ml TGF-β1 for 24 and 48 h. (C) Western blotting for Snail and claudin-1 in PANC-1 cells treated with siRNAs of Snail. (D) Western blotting for Snail and claudin-1 in PANC-1 cells treated with siRNAs of Snail and 100 ng/ml TGF-β1 for 48 h. Control cells in Figure 4C and D are transfected with a scrambled siRNA. KD: knockdown.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Control, Transfection, Knockdown

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 5. (A) Western blotting for Snail, claudin-1, -4, -7, occludin, pMAPK and panMAPK (MAPK) in PANC-1 cells pretreated with 1 μg/ml Gö6976 before treatment with 100 ng/ml TGF-β1 for 24 h. (B) Western blotting for claudin-1, -4, -7, occludin, Snail, panPKCα (PKCα), pMAPK and MAPK in hTERT-HPDEs pretreated with 1 μg/ml Gö6976 before treatment with 20 ng/ml TGF-β1 for 24 h. (C) Western blotting for Snail, claudin-1, occludin, pPKCα, PKCα, pMAPK and MAPK in PANC-1 cells with or without 1 μg/ml Gö6976 under hypoxia for 24 h. (D) Western blotting for claudin-1, -4, -7, occludin, pMAPK, MAPK and Snail in hTERT-HPDEs with or without 1 μg/ml Gö6976 under hypoxia for 24 h. iPKCα: PKCα inhibitor Gö6976.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 6. (A) Western blotting for Snail, pPKCα, panPKCα (PKCα), claudin-1, -4, -7 and occludin in HPAC cells after treatment with 100 μg/ml TGF-β1 for 24 and 48 h. (B) Western blotting for Snail, pPKCα, PKCα, claudin-1, -4, -7 and occludin in HPAC cells pretreated with 1 μg/ml Gö6976 before treatment with 100 ng/ml TGF-β1 for 24 h. (C) TER values in HPAC cells pretreated with 100 ng/ml TGF-β1 for 24 h with or without 1 μg/ml Gö6976. (D) Fence function examined by diffusion of labeled BODIPY-sphingomyelin into HPAC cells pretreated with 100 ng/ml TGF-β1 for 24 h with or without 1 μg/ml Gö6976. In HPAC cells treated with TGF-β1, the probe strongly labels the basolateral surface and appears to penetrate the cells (arrowheads), whereas the probe is effectively retained in the apical domain of the control cells and the cells were treated with TGF-β1 and Gö6976. Bar: 20 μm. iPKCα: PKCα inhibitor Gö6976.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Diffusion-based Assay, Labeling, Control