Journal: Molecules and Cells

Article Title: Endoplasmic Reticulum Stress Induces CAP2 Expression Promoting Epithelial-Mesenchymal Transition in Liver Cancer Cells

doi: 10.14348/molcells.2021.0031

Figure Lengend Snippet: (A) SNU423 and Huh7 cells are treated with/without thapsigargin (Thap; 50 nM and 100 nM, respectively), tunicamycin (Tuni; 50 ng/ml), or DTT (1 mM) for 24 h. Con, control. Expression of CAP2 mRNA is measured by qRT-PCR and normalized to β-actin mRNA. Data represent mean ± SEM of 4 independent experiments. (B) SNU423 and Huh7 cells are treated with thapsigargin (50 nM or 100 nM, respectively) for indicated times. Cell lysates are examined by western blotting with the indicated antibodies. CAP2 is indicated by asterisks (*). (C) Schematic representation of the CAP2 luciferase reporters. ChIP primer sets are shown. (D and E) Huh7 cells are transiently co-transfected with pTurbo-GFP and deleted constructs (D) or ATF2 binding site mutated construct (ATF2-Mut) (E) of the CAP2 promoter. After treatment with 100 nM thapsigargin for 24 h, total RNAs are extracted. Luciferase expression is monitored by qRT-PCR; Luciferase expression levels are normalized by that of GFP mRNA (right). Data represent mean ± SEM of the three independent experiments. (F) Cross-linked chromatin preparation from control and thapsigargin (100 nM, 24 h) treated cells are immunoprecipitated with anti-ATF2, anti-Histone H3 (H3), or normal rabbit IgG (IgG) antibodies. The ATF2 binding sites on the immunoprecipitated DNA are determined by RT-PCR using their corresponding primers. Amplificons of the input chromatin (input) prior to immunoprecipitation were served as controls for each chromatin extraction and PCR amplification. Chromatin immunoprecipitation using a Histone H3 antibody is served as a positive control (P.C) and a non-specific antibody (normal rabbit IgG) is served as a negative control (N.C). (G and H) Huh7 cells are transiently transfected with siNegative control ( siNeg ) or siATF2 . After 24 h, thapsigargin (100 nM) is treated for 24 h and the CAP2 expression levels are measured by qRT-PCR (G) and western blotting (H). CAP2 is indicated by asterisk (*). The data represent mean ± SEM of three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Knockdown experiments for ATF2 or PKC ε were performed using 100 nM of negative control siRNA ( siNeg , catalog No. sc-37007; Santa Cruz Biotechnology), siATF2 (catalog No. sc-29205; Santa Cruz Biotechnology) or siPKC ε (catalog No. sc-36251; Santa Cruz Biotechnology).

Techniques: Control, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Transfection, Construct, Binding Assay, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Extraction, Amplification, Chromatin Immunoprecipitation, Positive Control, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: The Attenuated Secretion of Hyaluronan by UVA-Exposed Human Fibroblasts Is Associated with Up- and Downregulation of HYBID and HAS2 Expression via Activated and Inactivated Signaling of the p38/ATF2 and JAK2/STAT3 Cascades

doi: 10.3390/ijms22042057

Figure Lengend Snippet: Effects of UVA on phosphorylation and protein levels of ATF2/c-Jun ( a ) and MSK1(Thr581/Ser360) ( b ). HDFs were exposed to UVA at the indicated doses in culture and cell lysates prepared at the indicated times post-irradiation were subjected to western blotting as described in the Materials and Methods section. Representative immunoblots from three independent experiments are shown.

Article Snippet: On day 1, HDFs were transfected with a control siRNA (MISSION ® siRNA Universal Negative Control, Sigma Aldrich), a c-Fos siRNA (Mission siRNA, Sigma–Aldrich), an ATF2 siRNA (Santa Cruz), or an PTP-MEG2 siRNA (Santa Cruz) using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol.

Techniques: Phospho-proteomics, Irradiation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Attenuated Secretion of Hyaluronan by UVA-Exposed Human Fibroblasts Is Associated with Up- and Downregulation of HYBID and HAS2 Expression via Activated and Inactivated Signaling of the p38/ATF2 and JAK2/STAT3 Cascades

doi: 10.3390/ijms22042057

Figure Lengend Snippet: Effects of transfection of c-Fos ( a ) or ATF-2 ( b ) siRNA on the UVA-induced increase in the HYBID protein level in HDFs. ( a ) Effect of transfecting a c-Fos siRNA on the expression of c-Fos and HYBID proteins. ( b ) Effect of transfecting an ATF-2 siRNA on the expression of ATF-2 and HYBID proteins. HDFs were exposed to UVA at the indicated dose in culture and cell lysates prepared at the indicated times post-irradiation were subjected to western blotting as described in the Materials and Methods section. Representative immunoblots from three independent experiments are shown. Data represent means ± SD., n = 3, **: p < 0.01, *: p < 0.05.

Article Snippet: On day 1, HDFs were transfected with a control siRNA (MISSION ® siRNA Universal Negative Control, Sigma Aldrich), a c-Fos siRNA (Mission siRNA, Sigma–Aldrich), an ATF2 siRNA (Santa Cruz), or an PTP-MEG2 siRNA (Santa Cruz) using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol.

Techniques: Transfection, Expressing, Irradiation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Attenuated Secretion of Hyaluronan by UVA-Exposed Human Fibroblasts Is Associated with Up- and Downregulation of HYBID and HAS2 Expression via Activated and Inactivated Signaling of the p38/ATF2 and JAK2/STAT3 Cascades

doi: 10.3390/ijms22042057

Figure Lengend Snippet: Effects of the transfection of a protein tyrosine phosphatase (PTPMEG2) siRNA on the UVA-induced decrease in the phosphorylation of STAT3 at Tyr 705 and JAK2 at Tyr 221 in HDFs. ( a ) Effect of transfecting a PTPMEG2 siRNA on the expression level of PTPMEG2 mRNA in UVA- or non-exposed HDFs at 3 h post-irradiation. Cell lysates were prepared at 3 h post-irradiation and were subjected to RT-qPCR analysis as described in the Materials and Methods section. Data represent means ± SD., n = 3, ** p < 0.01. ( b ) Effect of transfecting a PTPMEG2 siRNA on the expression level of PTPMEG2 protein and the phosphorylation level of STAT3 (Tyr705) and ERK in UVA- or non-exposed HDFs at 15 min post-irradiation. ( c ) Effect of transfecting a PTPMEG2 siRNA on the expression level of PTPMEG2 protein and the phosphorylation level of JAK2 (Tyr221) in UVA- or non-exposed HDFs at 5 min post-irradiation. Representative immunoblots from three independent experiments are shown.

Article Snippet: On day 1, HDFs were transfected with a control siRNA (MISSION ® siRNA Universal Negative Control, Sigma Aldrich), a c-Fos siRNA (Mission siRNA, Sigma–Aldrich), an ATF2 siRNA (Santa Cruz), or an PTP-MEG2 siRNA (Santa Cruz) using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol.

Techniques: Transfection, Phospho-proteomics, Expressing, Irradiation, Quantitative RT-PCR, Western Blot

Journal: Science Advances

Article Title: Extracellular vesicle tetraspanin-8 level predicts distant metastasis in non–small cell lung cancer after concurrent chemoradiation

doi: 10.1126/sciadv.aaz6162

Figure Lengend Snippet: ( A ) Representative Western blots and ( B ) quantification of 393P and 344SQ WCL expression of ITSN2, CD49d, Tspan8, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). ( C ) Representative Western blot and ( D ) quantification of Western blots of Tspan8, ITSN2, TSG101, HSP70, and GAPDH expression in EVs and WCLs of 393P and 393P cells overexpressing ITSN2 (393P Itsn2+ ). Data are means ± SEM from three independent experiments. ** P < 0.01.

Article Snippet: Antibodies used for Western blotting included 1:500 Tspan8 (sc-292058, Santa Cruz Biotechnology Inc.), 1:1000 TSG101 (ab83, Abcam), 1:1000 CD9 (ab92726, Abcam), 1:1000 GM130 (sc-55591, Santa Cruz Biotechnology Inc.), 1:1000 HSP70 (sc-32239, Santa Cruz Biotechnology Inc.), 1:1000 galectin-3–binding protein (sc-74970, Santa Cruz Biotechnology Inc.), 1:1000 clusterin (sc-8354, Santa Cruz Biotechnology Inc.), 1:1000 filamin-A (sc-28284, Santa Cruz Biotechnology Inc.), 1:1000 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118L, Cell Signaling Technology), 1:1000 Itsn2 (ab133854, Abcam), and 1:1000 CD49d (PA520595, Invitrogen).

Techniques: Western Blot, Expressing

Journal: Science Advances

Article Title: Extracellular vesicle tetraspanin-8 level predicts distant metastasis in non–small cell lung cancer after concurrent chemoradiation

doi: 10.1126/sciadv.aaz6162

Figure Lengend Snippet: Kaplan-Meier plots of freedom from distant metastasis (DM) in 106 patients with stage III NSCLC segregated by Tspan8 Low and Tspan8 High serum levels (Tspan8 cutoff = 0.08), with P values calculated with log-rank tests. NR, not reached; m, months.

Article Snippet: Antibodies used for Western blotting included 1:500 Tspan8 (sc-292058, Santa Cruz Biotechnology Inc.), 1:1000 TSG101 (ab83, Abcam), 1:1000 CD9 (ab92726, Abcam), 1:1000 GM130 (sc-55591, Santa Cruz Biotechnology Inc.), 1:1000 HSP70 (sc-32239, Santa Cruz Biotechnology Inc.), 1:1000 galectin-3–binding protein (sc-74970, Santa Cruz Biotechnology Inc.), 1:1000 clusterin (sc-8354, Santa Cruz Biotechnology Inc.), 1:1000 filamin-A (sc-28284, Santa Cruz Biotechnology Inc.), 1:1000 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118L, Cell Signaling Technology), 1:1000 Itsn2 (ab133854, Abcam), and 1:1000 CD49d (PA520595, Invitrogen).

Techniques: