Journal: Cell reports

Article Title: Methylglyoxal Scavengers Resensitize KRAS-Mutated Colorectal Tumors to Cetuximab.

doi: 10.1016/j.celrep.2020.01.012

Figure Lengend Snippet: Figure 6. Carnosine Specifically Inhibits KRASG12V Cell Survival through AKT Inhibition (A) Determination of half-maximal inhibitory concentration (IC50) for LY294002, BYL719, and MK2206 in SW48 CRC cells as described in STAR Methods. Data were analyzed using an unpaired Student’s t test and shown as mean values ± SEM of three independent experiments. (B) Apoptosis analysis in CRC cells treated with LY294002 (50 mM, 8 h), BYL719 (40 mM, 24 h), and MK2206 (40 mM, 24 h). Annexin-V-positive cells were quantified and shown as the percentage of apoptotic cells. All data are presented as mean values ± SEM of three independent experiments and were analyzed using two- way ANOVA followed by Bonferroni’s test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Argpyrimidine (Oya et al., 1999) Cat# mAb6B b-actin Sigma-Aldrich Cat# A5441; RRID: AB_476744 Glyoxalase 1 BioMAC Cat# 02-14 MG-Hs (3D11) Cell Biolabs Cat# STA-011 P-AKT Cell Signaling Cat# 4060; RRID: AB_2315049 AKT Cell Signaling Cat# 9272; RRID: AB_329827 P-PI3K Cell Signaling Cat# 4228; RRID: AB_659940 PI3K Cell Signaling Cat# 4257 GLUT1 Cell Signaling Cat# 12939; RRID: AB_2687899 P-PDH Abcam Cat# 92696; RRID: AB_10711672 PDH Abcam Cat# 168379 P-PKM2 Cell Signaling Cat# 3827; RRID: AB_1950369 PKM2 Cell signaling Cat# 4053; RRID: AB_1904096 P-P70S6K Cell signaling Cat# 2708; RRID: AB_390722 P-GSK3beta Cell signaling Cat# 9331; RRID: AB_329830 GSK3beta BD Biosciences Cat# 610202; RRID: AB_397601 Hsp27 Enzo Life Sciences Cat# ADI-SPA-803-D; RRID: AB_10615084 Cleaved caspase 3 Cell Signaling Cat# 9664; RRID: AB_2070042 Cleaved PARP Cell Signaling Cat# 5625; RRID: AB_10699459 HKII Cell Signaling Cat# 2867; RRID: AB_2232946 PFKFB3 Cell Signaling Cat# 13123; RRID: AB_2617178 Rictor Cell Signaling Cat# 2114; RRID: AB_2179963 mTOR Cell Signaling Cat# 2983; RRID: AB_2105622 Hsc70 Santa Cruz Cat# Sc-7298; RRID: AB_627761 Bacterial and Virus Strains pSPAX2 Addgene #12260 VSV-G encoding vector Emi et al., 1991 N/A GLO1#1 shRNA plasmid Sigma-Aldrich TRCN0000118627 GLO1#4 shRNA plasmid Sigma-Aldrich TRCN0000118631 Non-target shRNA plasmid Sigma-Aldrich SHC005 Chemicals, Peptides, and Recombinant Proteins L-carnosine Sigma-Aldrich Cat# C-9625 Aminoguanidine Sigma-Aldrich Cat# 396494 Deoxyglucose Sigma-Aldrich Cat# D-8375 Methylglyoxal Sigma-Aldrich Cat# M-0252 Cycloheximide Sigma-Aldrich Cat# C-7698 Human recombinant Hsp27 Enzo Life Sciences Cat# ADI-SPP-715-D Torin 1 Selleckchem Cat# S2827 SC79 Selleckchem Cat# S7863 LY294002 Selleckchem Cat# S1105 BYL719 Selleckchem Cat# S2814 MK2206 Selleckchem Cat# S1078 Rapamycin Calbiochem Cat# 553210 Cetuximab Merck C225 (Continued on next page) Cell Reports 30, 1400–1416.e1–e6, February 4, 2020 e1

Techniques: Inhibition, Concentration Assay

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 1. Protective effect of D4476 and possible association with autophagy in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 (commercially available stable RPE cell line) and primary mouse RPE cells. Cytotoxicity of ARPE-19 cells treated with (A) varying concentrations of CQ (10–100 μM) alone for 24 h; (B) 100 μM CQ with vehicle or different D4476 concentrations (1–20 μM) for 24 h; (C) vehicle, 100 μM CQ, CQ+D4476 and 10 μM D4476 for different time-points. (D and E) Relative survival rate of ARPE-19 cells treated with casein kinase 1 (CK1) inhibitor, IC261, and selective activin receptor-like kinase (ALK)5 inhibitor, SB525334, instead of D4476 with the same combination of drugs for 24 h and (F) 100 μM CQ with vehicle and with 0.5 μM rapamycin (Rap) for 24 h. Cytotoxicity assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay unless otherwise specified. (G) Phase-contrast and fluorescence photo micrographs of primary mouse RPE cells. After 3 weeks of culture, cells were stained with RPE65, marker protein of RPE, and Na+/K+-ATPase, marker of plasma membrane. Scale bar, 20 μm. (H and I) Relative survival rate of primary mouse RPE cells. Cells were treated with vehicle, 100 μM CQ, CQ+D4476, 10 μM D4476, CQ+IC261, 1 μM IC261, CQ+SB525334, 1 μM SB525334, CQ+Rap, and 0.5 μM Rap for 24 h. Mean ± standard deviation (SD), n = 3; ##P < 0.01 and ###P < 0.001 compared to vehicle and *P < 0.05, **P < 0.01, ***P < 0.001 compared to CQ alone. ns, not significant, compared to vehicle or CQ-treated cells.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: MTT Assay, Fluorescence, Staining, Marker, Clinical Proteomics, Membrane, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 2. Attenuation of chloroquine (CQ)-induced inhibition of autophagy by D4476 in adult retinal pigment epithelial (ARPE)-19 cells. (A) Phase-contrast photo micrograph and (B) confocal images of green fluorescent protein-light chaing 3 (GFP-LC3, green)-transfected cells after immunostaining with anti-lysosomal asso ciated membrane protein (LAMP)-1 (red) antibody. Cells were treated with vehicle, 100 μM CQ alone, 10 μM D4476 alone, or 10 μM D4476 + 100 μM CQ for 6 h. 4ʹ,6-Diamidino-2-phenylindole (DAPI, blue) was used to counterstain nuclei. Scale bar, 10 μm. Quantification of LC3-positive vacuole (C) size, (D) number (green), and (E) number of LC3-positive (green) vacuoles colocalized (yellow) to LAMP-1 (red). One-hundred cells per experimental condition were analyzed using fluo rescence microscopy. (F) Western blot and quantitative analyses of Beclin 1, p62, and LC3 A/B expression in ARPE-19 cell lysates 6 h after treatment with vehicle, 100 μM CQ alone, 10 μM D4476 + 100 μM CQ, 10 μM D4476 alone, 0.5 μM rapamycin (Rap) plus 100 μM CQ, and 0.5 μM Rap alone. α-Tubulin was used as a loading control. Mean ± standard deviation (SD), n = 3; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to vehicle; *P < 0.05 and **P < 0.01 compared to CQ alone. ns, not significant, compared to vehicle or Bafilomycin A1 (BA1) alone-treated cells.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Transfection, Immunostaining, Membrane, Microscopy, Western Blot, Expressing, Control, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 5. Effect of D4476 on chloroquine (CQ)-induced interaction of Beclin 1 B-cell lymphoma 2 (Bcl-2) homology 3 (BH3) domain with Bcl-2 and cell proliferation- associated signaling in adult retinal pigment epithelial (ARPE)-19 cells. (A) immunoblotted or (B) immunoprecipitated ARPE-19 cells. Cells were immunoprecipitated with anti-mouse IgG, as an isotype control, or anti-Bcl-2 antibody, then immunoblotted with anti-Beclin 1 or anti-Bcl-2 antibody. (C and D) Densitometric analysis of Immunoblots with Beclin 1 and Bcl-2. Quantitative analysis performed using ImageJ software. (E) Western blot and (F) quantitative densitometric analyses of phosphorylated phosphoinositide 3-kinase (p-PI3K), PI3K, p-AKT, AKT, p-mechanistic target of rapamycin (Rap, mTOR), mTOR, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, p-c-Jun N terminal kinase (JNK), and JNK in APRE-19 cells 6 h after treatment with vehicle, 100 μM CQ alone, 10 μM D4476 + 100 μM CQ, 10 μM D4476 alone, 0.5 μM Rap+100 μM CQ, and 0.5 μM Rap alone. Data are means ± standard deviation (SD), n = 3; #P < 0.05 and ##P < 0.01 compared to vehicle and *P < 0.05 and **P < 0.01 compared to CQ alone. ns, not significant, compared to vehicle or CQ alone.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Control, Western Blot, Software, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 7. c-Jun N terminal kinase (JNK) inhibitor-induced reproduction of D4476 effects in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells. (A) Relative survival percentage of APRE-19 cells treated with 100 μM CQ alone, 100 μM CQ+10 μM SP600125, and 10 μM SP600125 alone for 24 h. (B) immu noblotted or (C) immunoprecipitated ARPE-19 cells. Cell lysates immunoprecipitated with anti-mouse Ig G or anti-B-cell lymphoma 2 (Bcl-2) antibody were immunoblotted with anti-Beclin 1 or anti-Bcl-2 antibody. Bars denote densitometric analysis of Immunoblots. (D) Western blots and (E and F) quantitative densi tometric analyses for phosphorylated mechanistic target of rapamycin (p-mTOR), mTOR, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, p-JNK, JNK, light chain 3 (LC3) A/B, p62, and Bcl-xL in APRE-19 cells 6 h after treatment as in (A). Data are means ± standard deviation (SD), n = 3; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to vehicle and *P < 0.05, **P < 0.01, and ***P < 0.001 compared to CQ alone. ns, not significant, compared to vehicle or CQ alone.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 6. p38 Mitogen-activated protein kinase (MAPK) inhibitor-induced reproduction of D4476 effects in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells. (A) Relative survival rate of APRE-19 cells 24 h after treatment with vehicle, 100 μM CQ alone, 100 μM CQ+20 μM SB203580, 20 μM SB203580 alone. (B) immunoblotted or (C) immunoprecipitated ARPE-19 cells. Cell lysates immunoprecipitated using anti-mouse Ig G or anti-B-cell lymphoma 2 (Bcl-2) antibody were immunoblotted with anti-Beclin 1 and anti-Bcl-2 antibodies. Bars denote densitometric analyses of Immunoblots. (D) Western blot and (E and F) quantitative densitometric analyses of phosphorylated mechanistic target of rapamycin (p-mTOR), mTOR, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, p-c-Jun N terminal kinase (JNK), JNK, light chain 3 (LC3) A/B, p62, and Bcl-2 extra-large (Bcl-xL) in APRE-19 cells 6 h after treatment as in (A). Data are means ± standard deviation (SD), n = 3; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to vehicle and *P < 0.05, **P < 0.01, and ***P < 0.001 compared to CQ alone. ns, not significant, compared to vehicle or CQ alone.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 8. Schematic representation of D4476 effects on crosstalk between autophagy and apoptosis in chlo roquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells. D4476 inhibits CQ-induced increase of mechanistic target of rapamycin (mTOR), c-Jun N terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) activities. This inhibitory ef fect alters CQ-mediated interaction between Beclin 1 and B-cell lymphoma 2 (Bcl-2) via the Bcl-2 homol ogy 3 (BH3) domain, resulting in release of Beclin 1 from Bcl-2 and activating autophagy. Beclin 1, light chain 3 (LC3) A/B, and p62 are all associated with autophagosome formation and autophagy flux. D4476 may play an important role in the gateway of intersection between autophagy and apoptosis during CQ-induced toxicity, mitigating damage through reinstating cellular homeostasis.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques:

Journal: Acta Pharmaceutica Sinica. B

Article Title: MicroRNA-34c-5p provokes isoprenaline-induced cardiac hypertrophy by modulating autophagy via targeting ATG4B

doi: 10.1016/j.apsb.2021.09.020

Figure Lengend Snippet: MiR-34c-5p promotes cardiac hypertrophy via modulating autophagy. (A) and (B) Cultured NRCMs were transfected respectively with miR-34c-5p mimic and inhibitor for 24 h. The protein level of P62 and LC3-II were measured by Western blot. Data are shown as mean ± SD, n = 5; ∗ P < 0.05, ∗∗ P < 0.01 vs. control group; # P < 0.05 vs. NC mimic or NC inhibitor group. (C) and (D) NRCMs were treated with 3-MA or rapamycin accompanying with ISO treatment for 24 h. The cell surface area was measured ( n = 6). The levels of autophagic and hypertrophic markers were detected by Western blot ( n = 3). Data are shown as mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01 vs. control group; # P < 0.05 vs. ISO group. (E) and (F) NRCMs with miR-34c-5p mimic transfection were submitted to rapamycin treatment for 24 h. The cell surface area ( n = 6) and expression of autophagic and hypertrophic markers ( n = 5) was determined. Data are shown as mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01 vs. control group; # P < 0.05, ## P < 0.01 vs. NC mimic group; $ P < 0.05, $$ P < 0.01 vs. miR-34c-5p mimic group. (G) and (H) NRCMs were transfected with miR-34c-5p inhibitor, and then incubated with ISO and 3-MA for 24 h. The cell surface area ( n = 6) and expression of autophagic and hypertrophic markers ( n = 5) was determined. Data are shown as mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01 vs. control group; # P < 0.05, ## P < 0.01 vs. ISO + NC inhibitor group; $ P < 0.05, $$ P < 0.01 vs. ISO + miR-34c-5p inhibitor group.

Article Snippet: Chloroquine (CQ) was purchased from Sangon Biotech; 3-methyladenine (3-MA), rapamycin and bafilomycin A1 (Baf A1) were obtained from TargetMol (Boston, MA, USA).

Techniques: Cell Culture, Transfection, Western Blot, Expressing, Incubation

Journal: American Journal of Cancer Research

Article Title: NRSN2 promotes osteosarcoma cell proliferation and growth through PI3K/Akt/MTOR and Wnt/β-catenin signaling

doi:

Figure Lengend Snippet: NRSN2 regulates PI3K/Akt/GSK3β axis and Wnt/β-catenin signaling in osteosarcoma cells. A. The level of phosphorylated Akt, mTOR, p-GSK3β and nuclear β-catenin (nu-β-catenin) are positively correlated with the level of NRSN2 in U2OS and MG63 cells. B. Luciferase reporter assays revealed that NRSN2 could regulate Wnt/β-catenin signaling in U2OS and MG63 cells. C. Knockdown NRSN2 inhibits the expression of CCND1 and c-myc in U2OS cells. D. Overexpression of NRSN2 elevates the mRNA levels of CCND1 and c-myc in MG63 cells. E. The pro-proliferation effect was reversed when treated with IWR-1-endo, a inhibitor of β-catenin. *p<0.05, **p<0.01.

Article Snippet: The following antibodies were used in this study: NRSN2 (1:1000, Proteintech), GAPDH (1:5000, Proteintech), p-Akt, Akt, p-mTOR, mTOR, p-GSK3β, GSK3β, β-catenin and the second antibodies were purchased from Cell Signaling Technology and with 1:1000 dilutions.

Techniques: Luciferase, Expressing, Over Expression