Journal: International Journal of Molecular Medicine

Article Title: HELLS inhibits autophagy-dependent ferroptosis in nasopharyngeal carcinoma by modulating the Nrf2/HO-1/GPX4 pathway

doi: 10.3892/ijmm.2026.5788

Figure Lengend Snippet: Effects of HELLS on autophagy-dependent ferroptosis. (A) Relative viability of the sh-HELLS, sh-NC, sh-HELLS + Rapa, sh-NC + Rapa, sh-HELLS + 3-MA, and sh-NC + 3-MA groups at 0, 24, 48 and 72 h. Levels of (B) GSH, (C) MDA and (D) Fe 2+ in the six groups. (E) Representative images illustrating immunofluorescence staining of the ROS probes in the six groups. (F) ROS levels in the six groups. (G and H) Relative mRNA expression levels of (G) ACSL4 and (H) PTGS2 in the six groups. (I) Representative images of immunoblots of 4-HNE, ACSL4, and PTGS2. (J) Relative expression levels of 4-HNE in the six groups. Relative protein expression levels of (K) ACSL4 and (L) PTGS2 in the six groups. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001. HELLS, lymphoid-specific helicase; sh-, short hairpin; NC, negative control; Rapa, rapamycin; 3-MA, 3-Methyladenine; GSH, glutathione; MDA, malondialdehyde; ROS, reactive oxygen species; 4-HNE, 4-hydroxynonenal; ACSL4, acyl-CoA synthetase long-chain family member 4; PTGS2, prostaglandin-endoperoxide synthase 2.

Article Snippet: Furthermore, to investigate apoptosis-dependent ferroptosis, cells were initially stimulated with erastin and subsequently treated with either 500 nM rapamycin (Rapa; cat. no. HY-10219; MedChemExpress) or 5 mM 3-methyladenine (3-MA; cat. no. HY-19312; MedChemExpress) for 24 h ( , ).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Negative Control

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