Journal: Journal of Cellular and Molecular Medicine

Article Title: Reticulocalbin 1 is required for proliferation and migration of non‐small cell lung cancer cells regulated by osteoblast‐conditioned medium

doi: 10.1111/jcmm.17040

Figure Lengend Snippet: Information of antibodies

Article Snippet: p‐PERK(phosphoT980) , Bioss Bs−3330R , 1:1000 , 4℃ overnight.

Techniques:

Journal: Neuro-Signals

Article Title: Endoplasmic reticulum stress and neurodegeneration in experimental cerebral malaria.

doi: 10.1159/000336970

Figure Lengend Snippet: Fig. 2. a Immunoblot analysis of p-PERK, p-IRE1, ATF6 and - actin in the control and infected mouse brains. An equal amount of protein was electrophoresed by sodium dodecylsulphate poly- acrylamide gel electrophoresis and transferred to nitrocellulose membranes and probed with primary antibody to p-PERK, p- IRE1, ATF6 and -actin. -Actin was used as loading control. The result is representative of 4 independent experiments with similar results. The lanes C and I indicate uninfected control and PbA- infected mouse brains sacrificed on day 7 after infection, respec- tively. b–d Densitometric analysis (arbitrary units, AU) showing significant increases in the levels of p-PERK, p-IRE1 and ATF6 in infected samples as compared to their respective controls. * p ! 0.05, * * p ! 0.001: significant difference relative to the corre- sponding control.

Article Snippet: The primary antibodies used in these experiments included rabbit polyclonal antibodies raised against eIF2- , growth arrest and DNA damage-inducible protein 34 (GADD34), BiP, calregulin, calnexin (Santa Cruz Biotechnology); phospho-IRE1 (p-IRE1), ATF4 (Abcam); p-eIF2 (Epitomics), phospho-PERK (p-PERK), caspase-3, B cell lymphoma protein 2 (BCL-2), BCL-2-associated X protein (BAX), cleaved caspase-7, caspase-12 (Cell Signaling Technology); CHOP/GADD153 (Pierce) and mouse monoclonal antibodies raised against ATF6 (Abcam).

Techniques: Western Blot, Control, Infection, Polyacrylamide Gel Electrophoresis

Journal: Autophagy

Article Title: Autophagy-dependent EIF2AK3 activation compromises ursolic acid-induced apoptosis through upregulation of MCL1 in MCF-7 human breast cancer cells

doi: 10.4161/auto.22805

Figure Lengend Snippet: Figure 1. Ursolic acid (UA) induced cytoprotective EIF2AK3 activation in MCF-7 human breast cancer cells. UA induced cell number reduction (A) and apoptosis (B) in a dose-dependent manner. MCF-7 cells were treated with various concentrations of UA for 24 h, and then overall inhibitory effects and apoptosis were measured by crystal violet staining and annexin V/FITC staining, respectively. (C) Unfolded protein response induced by UA. MCF-7 cells were treated with various concentrations of UA for 24 h and then ER stress associated markers were analyzed by western blotting. (D) Effects of EIF2AK3 inactivation by RNAi on UA-induced apoptosis. The cells were transfected with 50 nmol/L of EIF2AK3 siRNA using siPORT™ NeoFX™ Transfection Agent. At 24 h post-transfection, the cells were treated with 20 μM for 24 h and apoptosis induction was assessed by sub-G1 analysis (n = 3, **p < 0.01). (The blots shown are representative of three independent experiments).

Article Snippet: RNA interference siRNAs targeting EIF2AK3 (sc-36213), ERN1 (sc-40705), HSPA5 (sc-29338), BECN1 (sc-29797) and MCL1 (sc-35877) were purchased from Santa Cruz Biotechnologies. siRNAs targeting ATG5 and nontargeting siRNA were synthesized by Integrated DNA Technologies.

Techniques: Activation Assay, Staining, Western Blot, Transfection

Journal: Autophagy

Article Title: Autophagy-dependent EIF2AK3 activation compromises ursolic acid-induced apoptosis through upregulation of MCL1 in MCF-7 human breast cancer cells

doi: 10.4161/auto.22805

Figure Lengend Snippet: Figure 4. ER stress was an effect rather than a cause of UA-induced autophagy. (A and B) Effects of EIF2AK3 (A) or ERN1 (B) knockdown on UA-induced LC3-I to LC3-II conversion. The cells were transfected with 50 nmol/L of EIF2AK3 siRNA or 50 nmol/L of ERN1 siRNA using siPORT™ NeoFX™ Transfection Agent. At 24 h post-transfection, the cells were treated with 20 μM for 24 h and then EIF2AK3, p-EIF2S1, ERN1 and LC3 were assessed by western blotting. (C and D) Effects of autophagy inhibition by 3-MA (C) or WORT (D) on UA-induced HSPA5 expression and EIF2S1 phosphorylation. The cells were treated with 20 μM UA in the presence or absence of 3-MA or WORT for 24 h and were analyzed by western blotting. (E) Effects of autophagy inhibition by BECN1/ATG5 knockdown on UA-induced HSPA5 expression and EIF2S1 phosphorylation. The cells were simultaneously transfected BECN1 and ATG5 siRNAs using siPORT™ NeoFX™ Transfection Agent. After 24 h transfection, the cells were treated with 20 μM UA for 24 h and were assessed by western blotting. (F) Time-course analysis of key parameters of ER stress, autophagy and apoptosis induced by UA in MCF-7cells. The cells were treated with UA for the indicated time and then LC3, EIF2S1 phosphorylation and PARP1 cleavage were assessed by western blotting. (G) UA induces autophagy-mediated ER stress in MDA-MB-231 cells. The cells were treated with UA for the indicated time and then LC3 and EIF2S1 phosphorylation were assessed by western blotting (left); The cells were pretreated with 3-MA for 1 h and further treated with UA for 6 h and western blotting was used to analyze LC3 and EIF2AK3-EIF2S1 phosphorylation (middle) and sub-G1 analysis was used to assess apoptosis induction by UA in the presence or absence of 3-MA for 24 h (right, n = 3, **p < 0.01). (The blots shown are representative of three independent experiments).

Article Snippet: RNA interference siRNAs targeting EIF2AK3 (sc-36213), ERN1 (sc-40705), HSPA5 (sc-29338), BECN1 (sc-29797) and MCL1 (sc-35877) were purchased from Santa Cruz Biotechnologies. siRNAs targeting ATG5 and nontargeting siRNA were synthesized by Integrated DNA Technologies.

Techniques: Knockdown, Transfection, Western Blot, Inhibition, Expressing, Phospho-proteomics

Journal: Autophagy

Article Title: Autophagy-dependent EIF2AK3 activation compromises ursolic acid-induced apoptosis through upregulation of MCL1 in MCF-7 human breast cancer cells

doi: 10.4161/auto.22805

Figure Lengend Snippet: Figure 5. Upregulation of MCL1 contributed to the prosurvival property of UA-induced, autophagy-dependent EIF2AK3 activation. (A) Effects of UA treatments on MCL1 expression. The cells were treated with various concentrations of UA for 24 h and MCL1 expression was analyzed by western blotting. (B) Effects of EIF2AK3 inactivation by RNAi on MCL1 induction by UA. EIF2AK3 was silenced by siRNA and MCL1 expression was analyzed by western blotting. (C) Effects of BECN1 and ATG5 knockdown on MCL1 induction by UA. BECN1 and ATG5 were simultaneously silenced by a siRNA approach and MCL1 was measured by western blotting. (D) Effects of ATG5 knockdown on phosphorylation of EIF2AK3-EIF2S1 and MCL1 induction. ATG5 was silenced by siRNA approach and phosphorylation of EIF2AK3-EIF2S1 and MCL1 expression were measured by western blotting. (E) Effects of 3-MA on MCL1 induction by UA. The cells were treated with 20 μM UA in the presence or absence of 3-MA for 24 h, MCL1 was analyzed by western blotting. (E) Effects of MCL1 inhibition by siRNA on UA-induced apoptosis measured by sub-G1 analysis (n = 3, **p < 0.01). (The blots shown are representative of three independent experiments).

Article Snippet: RNA interference siRNAs targeting EIF2AK3 (sc-36213), ERN1 (sc-40705), HSPA5 (sc-29338), BECN1 (sc-29797) and MCL1 (sc-35877) were purchased from Santa Cruz Biotechnologies. siRNAs targeting ATG5 and nontargeting siRNA were synthesized by Integrated DNA Technologies.

Techniques: Activation Assay, Expressing, Western Blot, Knockdown, Phospho-proteomics, Inhibition

Journal: Autophagy

Article Title: Autophagy-dependent EIF2AK3 activation compromises ursolic acid-induced apoptosis through upregulation of MCL1 in MCF-7 human breast cancer cells

doi: 10.4161/auto.22805

Figure Lengend Snippet: Figure 8. Signaling pathways underlying UA-induced cytoprotective autophagy in human MCF-7 breast cancer cells. UA at relatively low concentrations induced autophagy which in turn led to activation of UPR including EIF2AK3 pathway. Activation of EIF2AK3 suppressed UA-mediated apoptosis through upregulation of MCL1. UA-induced cytoprotective autophagy was attributed to MAPK1/3 activation but not associated with MTOR inhibition.

Article Snippet: RNA interference siRNAs targeting EIF2AK3 (sc-36213), ERN1 (sc-40705), HSPA5 (sc-29338), BECN1 (sc-29797) and MCL1 (sc-35877) were purchased from Santa Cruz Biotechnologies. siRNAs targeting ATG5 and nontargeting siRNA were synthesized by Integrated DNA Technologies.

Techniques: Protein-Protein interactions, Activation Assay, Inhibition