Journal: Autophagy

Article Title: Autophagy and post-ischemic conditioning in retinal ischemia

doi: 10.1080/15548627.2020.1767371

Figure Lengend Snippet: MTOR pathway in retina with ischemia and post-C. (A) Changes in immunostaining for the MTOR pathway (from left to right) in normal, ischemia + post-C + scrambled, and ischemia + post-C + Atg5 siRNA. Retinal cryosections were prepared 24 h after post-C (that is, 48 h after ischemia), and examined using confocal microscopy. From top to bottom are staining for MTOR, p-RPS6KB1, and p-EIF4EBP1. These are representative images from N = 3 per group. Orientation is shown on far right; magnification 40x. (B) Localization of MTOR pathway proteins in retinal cells. These cryosections are all taken from the same group, ischemia + post-C + scrambled Atg5 siRNA, at 24 h after post-C (that is, 48 h after ischemia). Nuclei were stained blue using DAPI. From top to bottom are staining (red) for MTOR, p-RPS6KB1, and p-EIF4EBP1. From left to right (staining green) are: GFAP (Muller cells); VIM (for astrocytes); RBPMS (retinal ganglion cells); STX1A (amacrine cells); PRKCA (bipolar cells). For GFAP, white arrows indicate yellow overlap of MTOR (red), p-RPS6KB1: red; and p-EIF4EBP1: red with GFAP: green, in Muller cell endplates and projections. For VIM, white arrows indicate yellow overlap of MTOR (red) and VIM (green) in the superficial inner retina. For RBPMS, white arrows indicate yellow overlap of p-RPS6KB1: red, and p-EIF4EBP1: red with RBPMS: green in retinal ganglion cells. There was no evident overlap with the MTOR pathway for STX1A (amacrine cells), and PRKCA (bipolar cells). Orientation on far left. RGC = retinal ganglion cells, IPL = inner plexiform layer, INL = inner nuclear layer, ONL = outer nuclear layer, RPE = retinal pigment epithelium. Representative images from N = 3 per group; magnification 40x

Article Snippet: Antibody Manufacturer Type Catalog Number Dilution ATG5 Santa Cruz Biotechnology Mouse Monoclonal sc-133158 1:500 ATG7 Santa Cruz Biotechnology Mouse Monoclonal sc-376212 1:1000 BECN1 MBL International Rabbit Polyclonal JM-3663-100 1:1000 ACTB Sigma-Aldrich Mouse Monoclonal A3854-200UL 1:25000 ARRB2 Proteintech Rabbit Polyclonal 10171-1-AP 1:500 TUBB3 Sigma-Aldrich Mouse Monoclonal T8660-.2 ML 1:250 CALB1 Proteintech Group Rabbit Polyclonal 14479-1-AP 1:50 CASP3 (cleaved) Cell Signaling Technology Rabbit Polyclonal 9661 S 1:1000 ITGAM, ITGAX BD Pharmingen Mouse Monoclonal 550299 1:500 GFAP Agilent Technologies Rabbit Polyclonal Z033429-2 1:1000 LC3-I &II MBL International Rabbit Polyclonal PD014 MS 1:1000 LC3B Proteintech Group Rabbit Polyclonal 18725-1-AP 1:500 MTOR Proteintech Group Rabbit Polyclonal 20657-1-AP 1:500 SQSTM1 Sigma-Aldrich Rabbit Polyclonal p0067 1:1000 PRKCA BD Biosciences Mouse Monoclonal 610107 1:250/1:500 p-EIF4EBP1/2/3 (Thr45) Bioss Antibodies Rabbit Polyclonal BS-5672 R-A647 1:500 RBPMS GeneTex Rabbit Polyclonal GTX118619 1:1000 p-RPS6KB1 (Ser421) Bioss Antibodies Rabbit Polyclonal BS-6421RA555 1:500 NFH Covance Mouse Monoclonal SMI32-P 1:250 STX1A Sigma-Aldrich Mouse Monoclonal S0664-.2 ML 1:250/1:2000 VIM Santa Cruz Biotechnology Mouse Monoclonal sc-6260 1:50/1:1000 TH Iowa Developmental Studies Hybridoma Bank Mouse Monoclonal aTH-s 1:500 Open in a separate window Primary antibodies used in the study.

Techniques: Immunostaining, Confocal Microscopy, Staining

Journal: Autophagy

Article Title: Autophagy and post-ischemic conditioning in retinal ischemia

doi: 10.1080/15548627.2020.1767371

Figure Lengend Snippet: Primary antibodies used in the study

Article Snippet: Antibody Manufacturer Type Catalog Number Dilution ATG5 Santa Cruz Biotechnology Mouse Monoclonal sc-133158 1:500 ATG7 Santa Cruz Biotechnology Mouse Monoclonal sc-376212 1:1000 BECN1 MBL International Rabbit Polyclonal JM-3663-100 1:1000 ACTB Sigma-Aldrich Mouse Monoclonal A3854-200UL 1:25000 ARRB2 Proteintech Rabbit Polyclonal 10171-1-AP 1:500 TUBB3 Sigma-Aldrich Mouse Monoclonal T8660-.2 ML 1:250 CALB1 Proteintech Group Rabbit Polyclonal 14479-1-AP 1:50 CASP3 (cleaved) Cell Signaling Technology Rabbit Polyclonal 9661 S 1:1000 ITGAM, ITGAX BD Pharmingen Mouse Monoclonal 550299 1:500 GFAP Agilent Technologies Rabbit Polyclonal Z033429-2 1:1000 LC3-I &II MBL International Rabbit Polyclonal PD014 MS 1:1000 LC3B Proteintech Group Rabbit Polyclonal 18725-1-AP 1:500 MTOR Proteintech Group Rabbit Polyclonal 20657-1-AP 1:500 SQSTM1 Sigma-Aldrich Rabbit Polyclonal p0067 1:1000 PRKCA BD Biosciences Mouse Monoclonal 610107 1:250/1:500 p-EIF4EBP1/2/3 (Thr45) Bioss Antibodies Rabbit Polyclonal BS-5672 R-A647 1:500 RBPMS GeneTex Rabbit Polyclonal GTX118619 1:1000 p-RPS6KB1 (Ser421) Bioss Antibodies Rabbit Polyclonal BS-6421RA555 1:500 NFH Covance Mouse Monoclonal SMI32-P 1:250 STX1A Sigma-Aldrich Mouse Monoclonal S0664-.2 ML 1:250/1:2000 VIM Santa Cruz Biotechnology Mouse Monoclonal sc-6260 1:50/1:1000 TH Iowa Developmental Studies Hybridoma Bank Mouse Monoclonal aTH-s 1:500 Open in a separate window Primary antibodies used in the study.

Techniques:

Journal: Aging (Albany NY)

Article Title: Geniposide-mediated protection against amyloid deposition and behavioral impairment correlates with downregulation of mTOR signaling and enhanced autophagy in a mouse model of Alzheimer's disease

doi: 10.18632/aging.101759

Figure Lengend Snippet: Geniposide treatment decreases mTOR activation markers in brains of APP/PS1 mice. Hippocampal expression of Akt, mTOR, and 4E-BP1, and their respective phosphorylated forms was detected by western blot. The expression of p-Akt ( A ) and p-mTOR ( B ) was enhanced in APP/PS1 mice compared to WT, and geniposide attenuated this increase. The expression of p-4E-BP1 ( C ) in APP/PS1 mice was reduced compared to WT, and geniposide partly restored this decrease. Data are presented as mean ± SEM (n = 6). *** p < 0.001, ** p < 0.01, * p < 0.05 vs. WT; # p < 0.05 vs. APP/PS1 mice (one-way ANOVA, Tukey's Multiple Comparison Test). WT: wild-type mice. GP: geniposide.

Article Snippet: The membranes were blocked in 5% bovine serum albumin in TBST (Tris-buffered saline with 0.05% Tween-20) for 1h, and incubated overnight at 4°C with primary antibodies directed against: Akt (1:1,000), p-Akt (1:2,000), mTOR (1:1,000), p-mTOR (1:1,000), 4E-BP1 (1:1,000), or p-4E-BP1 (1:2,000), followed by incubation at 4°C for 2h with a goat-anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China).

Techniques: Activation Assay, Expressing, Western Blot, Comparison

Journal: eLife

Article Title: Dual leucine zipper kinase-dependent PERK activation contributes to neuronal degeneration following insult

doi: 10.7554/eLife.20725

Figure Lengend Snippet: ( a ) Schematic of sciatic nerve crush (SNC) and optic nerve crush (ONC). Mouse lumbar level 3 and 4 dorsal root ganglia (DRG) and retina were isolated to assess the neuronal stress response after SNC and ONC, respectively. ( b ) Microarray cross-comparison of injury-regulated mRNAs following SNC or ONC (n = 5 per condition) identifies multiple ISR-associated genes (blue), including Eif4ebp1 , Atf4 , Chac1 and Eif2ak3 (PERK), upregulated by both insults. ( c–d ) mRNAs within the ‘ISR-related’ gene set (see Materials and Methods) are observed more frequently amongst upregulated mRNAs than expected by the overall distribution of mRNA expression changes assessed in each microarray study (‘complete gene set’) following SNC (p=2.4 × 10 −5 , ( c )) or ONC (p=9.9 × 10 −7 , ( d )), suggesting the selective activation of the ISR. ( e–f ) Immunoblots reveal upregulation of the ISR (p-PERK, p-eIF2α, and ATF4), in addition to the JNK-pathway (p-c-Jun) in L3/L4 DRG lysates after SNC ( e ), and in retina lysates after ONC ( f ). The time post-injury is indicated in hours. ( g ) Primary e12.5 mouse DRG cultures deprived of NGF (3 h) or treated with the ER stress inducer thapsigargin (Tgn) in the vehicle DMSO. NGF deprivation engages phosphorylation of PERK (p-PERK/PERK), p-eIF2α and ATF4. Protein levels were normalized to GAPDH and non-NGF deprived vehicle control (n = 6–7/condition, four independent experiments). ( h ) siRNA targeting each of the four eIF2α kinases differentially impacts ISR activation at 3 h after NGF withdrawal from embryonic DRG neuronal cultures, with only siRNA targeting Eif2ak3 consistently reducing ATF4 protein levels. ( i ) Representative TUJ-1 immunostainings 42 hr after isolation and siRNA-transfection of adult DRG neurons. ( j ) OnTarget Plus siRNA-mediated knockdown of Atf4 or Eik2ak3 , but not other eIF2α kinases, enhances adult sensory axon regrowth in vitro (n ≥ 8 wells/condition). Molecular weight indicated in kilodaltons (kDa). Data are represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with post-hoc Bonferroni test. DOI: http://dx.doi.org/10.7554/eLife.20725.002

Article Snippet: Predesigned TaqMan primers were obtained from Applied Biosystems, including those for Chac1 (Prod No. Mm00509926_m1), Ddit3 (Prod No. Mm01135937_g1), Ppp1r15a (Prod No. Mm01205601_g1), Eif4ebp1 (Prod No. Mm04207378_g1) and Gapdh (Prod No. Mm03302249_g1).

Techniques: Isolation, Microarray, Comparison, Expressing, Activation Assay, Western Blot, Phospho-proteomics, Control, Transfection, Knockdown, In Vitro, Molecular Weight

Journal: eLife

Article Title: Dual leucine zipper kinase-dependent PERK activation contributes to neuronal degeneration following insult

doi: 10.7554/eLife.20725

Figure Lengend Snippet: ( a ) qRT-PCR of L4 DRG confirms the upregulation of each of four ISR-related mRNAs ( Chac1 , Eif4ebp1, Ppp1r15a, and Ddit3 ) over the first 3 days after SNC (n = 3/condition). ( b ) Real-time qPCR for four putative ATF4 target genes in ATF4-null and wild-type mouse DRGs 24 h following SNC (n = 3/condition). ( c–d ) Dosing with ISRIB reduces induction of ATF4 protein and the mRNA of a subset of its putative target genes in associated DRGs following SNC. ( c ) Immunoblots of L3/L4 DRGs from ISRIB-treated mice 16 h post-SNC (n = 3/condition). ( d ) Real-time qPCR of mouse DRGs 16 h following SNC (n = 3/condition). Mice were dosed with vehicle or ISRIB (10 mg/kg, 1 h pre-SNC and 12 h post-crush). ( e ) qPCR of NGF-deprived embryonic DRG cultures reveals upregulation of mRNA of four putative ATF4 target genes, blocked by 400 nM ISRIB (n = 6–8/condition, two independent experiments). Data are represented as mean ± SEM. One-way ANOVA with post-hoc Bonferroni was used for statistical comparisons. *p<0.05, **p<0.01, ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.20725.008

Article Snippet: Predesigned TaqMan primers were obtained from Applied Biosystems, including those for Chac1 (Prod No. Mm00509926_m1), Ddit3 (Prod No. Mm01135937_g1), Ppp1r15a (Prod No. Mm01205601_g1), Eif4ebp1 (Prod No. Mm04207378_g1) and Gapdh (Prod No. Mm03302249_g1).

Techniques: Quantitative RT-PCR, Western Blot

Journal: eLife

Article Title: Dual leucine zipper kinase-dependent PERK activation contributes to neuronal degeneration following insult

doi: 10.7554/eLife.20725

Figure Lengend Snippet: ( a–b ) Global expression analysis indicates an enrichment in ISR-related genes upregulated 4.5 h after NGF withdrawal from embryonic DRG cultures in the presence of DMSO vehicle (n = 5/condition). ( a ) Density plot showing ‘ISR-related’ genes (blue, see Materials and methods) are more frequently upregulated compared to the distribution of all mRNAs expression changes (‘complete gene set’) (p<1 × 10 −5 , one-tailed Student-t test). ( b ) Scatterplot of gene expression levels (nRPKM) in NGF-containing and NGF-deprived samples. Transcripts of ATF4 target genes Chac1 , Eif4ebp1 , Ddit3 , and Ppp1r15a are among 581 robustly upregulated (red) RNAs following NGF deprivation ( n = 5 per condition, >1.5 fold, adjusted-p<0.001, nRPKM = Reads Per Kilobase per Million mapped reads) ( c ) Venn diagrams reveal the portions of these NGF-responsive genes that exhibit DLKi (GNE-3511, 400 nM) and/or ISRIB (400 nM) sensitivity (see Materials and methods). ( d ) ISRIB-sensitive and ISRIB-insensitive RNAs are among those represented in a heat map of top 50 transcripts strongly upregulated in a DLKi-sensitive manner following NGF withdrawal. DOI: http://dx.doi.org/10.7554/eLife.20725.018 10.7554/eLife.20725.019 Figure 5—source data 1. RNA-seq analysis of primary sensory neurons following NGF deprivation, in the presence of ISRIB or DLK inhibitor GNE-3511. DOI: http://dx.doi.org/10.7554/eLife.20725.019

Article Snippet: Predesigned TaqMan primers were obtained from Applied Biosystems, including those for Chac1 (Prod No. Mm00509926_m1), Ddit3 (Prod No. Mm01135937_g1), Ppp1r15a (Prod No. Mm01205601_g1), Eif4ebp1 (Prod No. Mm04207378_g1) and Gapdh (Prod No. Mm03302249_g1).

Techniques: Expressing, One-tailed Test, Gene Expression, RNA Sequencing

Journal: Journal of Cellular and Molecular Medicine

Article Title: m 6 A methyltransferase METTL3 promotes retinoblastoma progression via PI3K/AKT/mTOR pathway

doi: 10.1111/jcmm.15736

Figure Lengend Snippet: Methyltransferase‐like 3 (METTL3) influences the expression of the PI3K/AKT/mTOR pathway. A, Down‐regulated METTL3 decreases the expression of PI3K‐p85, AKT, mTOR and P70S6K but increases 4EBP1 mRNA levels. B, Western blot data show that the expression of p‐PI3K‐p85, p‐AKT, p‐mTOR and p‐P70S6K is decreased, but p‐4EBP1 is increased; non‐phosphorylated PI3K‐p85, AKT, mTOR, P70S6K and 4EBP1 have no difference. C, The optical density ratio of the Western blot. D, Up‐regulated METTL3 stimulates PI3K‐p85, AKT, mTOR and P70S6K expression at the mRNA level but reduces 4EBP1. E, The protein levels of p‐PI3K‐p85, p‐AKT, p‐mTOR and p‐P70S6K are elevated, but p‐4EBP1 is decreased; the level of non‐phosphorylated total proteins is steady. F, The optical density ratio of the Western blot. Data are shown as the average ± SD (n = 3). P < 0.05(*), P < 0.01(**), and P < 0.001(***)

Article Snippet: The primary antibodies were as follows: METTL3 (ab195352, 1:1000; Abcam, UK, London), p‐PI3K‐p85α (Tyr607) (AP0153, 1:500; Bioworld, China, Beijing), p‐AKT (S473) (CST, USA, New York, #4058, 1:1000), p‐mTOR (Ser2448) (CST, USA, New York, #5536, 1:1000), p‐P70S6K (Ser371) (CST, USA, New York, #9208, 1:1000), p‐4EBP1 (Ser65 + Thr70) (Bioss, China, Beijing, bs‐3720R, 1:500), β‐Tubulin (Bioworld, China, Beijing, AP0064, 1:2000), and GAPDH (Bioworld, China, Beijing 1:5000).

Techniques: Expressing, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: m 6 A methyltransferase METTL3 promotes retinoblastoma progression via PI3K/AKT/mTOR pathway

doi: 10.1111/jcmm.15736

Figure Lengend Snippet: Methyltransferase‐like 3 (METTL3) regulates cell proliferation, apoptosis, migration and invasion via the PI3K/AKT/mTOR pathway. A, Rapamycin inhibits the expression of p‐PI3K‐p85, p‐AKT, p‐mTOR and p‐P70S6K but elevates the expression of p‐4EBP1. The total protein levels of PI3K‐p85, AKT, mTOR, P70S6K and 4EBP1 were not different. B, The statistical histogram of the phosphorylated proteins/total proteins. C, The cell proliferation results show that the stimulatory function of METTL3 is lost after rapamycin treatment. D and E, The apoptosis ratio changes were higher in the METTL3 Rapa group than in the METTL3 group and NC group. F, The cell migration and invasion abilities in the METTL3 Rapa groups were weaker than those in the METTL3 groups and had no statistical difference with NC groups. G and H, Quantification of migratory and invasive areas was analysed by ImageJ software. Data are shown as the average ± SD (n = 3). P < 0.05(*), P < 0.01(**), P < 0.001(***), P < 0.0001(****) and P > 0.05(#). The scale bars represent 50 μm

Article Snippet: The primary antibodies were as follows: METTL3 (ab195352, 1:1000; Abcam, UK, London), p‐PI3K‐p85α (Tyr607) (AP0153, 1:500; Bioworld, China, Beijing), p‐AKT (S473) (CST, USA, New York, #4058, 1:1000), p‐mTOR (Ser2448) (CST, USA, New York, #5536, 1:1000), p‐P70S6K (Ser371) (CST, USA, New York, #9208, 1:1000), p‐4EBP1 (Ser65 + Thr70) (Bioss, China, Beijing, bs‐3720R, 1:500), β‐Tubulin (Bioworld, China, Beijing, AP0064, 1:2000), and GAPDH (Bioworld, China, Beijing 1:5000).

Techniques: Migration, Expressing, Software