Journal: Frontiers in Pharmacology

Article Title: Detrimental Effects of Chronic L-Arginine Rich Food on Aging Kidney

doi: 10.3389/fphar.2020.582155

Figure Lengend Snippet: Prolonged L-arginine diet does not affect p16 INK4a , Arg-II or S6K1 pathways. Aging marker p16 INK4a mRNA expression was evaluated in male (A) and female (C) kidney of control and L-arginine supplemented mice. rps12 was used as reference gene. Immunoblotting analysis of Arg-II and S6 protein (S6-S240/244) and total S6 in kidney lysates of control and supplemented male (B) and female (D) mice. n indicates the number of animals used in the experimental groups. Dot plots show quantifications of the markers. Tubulin was used as loading control. The values shown are mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: Reagents were purchased from the following sources: Mouse antibody against S6 (#2317s) and rabbit antibodies against phospho S6-S240/244 (#5364) and Arg-II (#55003S) were purchased from Cell Signaling Technology; Mouse antibody against Tubulin (T5168) and Goat Serum Donor Herd (G6767) were from MilliporeSigma (Burlington, MA, United States); Mouse antibody against p16 (sc-81156) was purchased from Santa Cruz Technology Inc. (Dallas, United States); Secondary antibodies IRDye 800-conjugated affinity purified goat anti-rabbit IgG was purchased from BioConcept (Alschwil, Switzerland) and Alexa fluor 680-conjugated goat anti-mouse IgG was from Invitrogen (Lucerne, Switzerland); Mouse IgG blocking reagent (MKB-2213) was from Vector Laboratories (Peterborough, United Kingdom); Alexa Fluor 488 conjugated-Goat anti-mouse IgG(H + L) secondary antibody (A-11001) was from Thermo Fisher Scientific (Waltham, MA United States); Dihydroethidium (DHE) was from Molecular Probes/Invitrogen (Lucerne, Switzerland), and the membrane-permeable 4,5-diaminofluoresceine diacetate (DAF-2DA) was from VWR international SA (Dietikon, Switzerland).

Techniques: Marker, Expressing, Control, Western Blot

Journal: Oncology reports

Article Title: The combination of temsirolimus and chloroquine increases radiosensitivity in colorectal cancer cells.

doi: 10.3892/or.2019.7134

Figure Lengend Snippet: Figure 1. Ionizing radiation activates the mTOR pathway and induces autophagy in human colorectal cancer cells. HT‑29 and SW480 cells were treated with ionizing radiation at a dose of 4 Gy and mTOR pathway activation and autophagy induction were investigated. (A) Western blot analysis of the mTOR pathway‑related proteins, p‑S6, total S6, p‑4E‑BP1, and total 4E‑BP1; and autophagy‑related proteins LC3B‑II/I and SQSTM1/p62 in cell lines 24 h after radiotherapy. β‑actin was used as the control. (B) Bar plots represent the relative expression levels of each protein, as determined via densitometric analysis. The mTOR pathway‑related proteins were presented as the phosphorylated isoform ratios normalized by the respective total proteins. The autophagy‑related proteins were presented as LC3‑II/LC3‑I ratios and the p62 expression levels were normalized to β‑actin levels. The data are expressed as the mean ± SD from the results of three independent experiments (n=3). *P<0.05. Unpaired Student's t‑tests were used. (C) p‑S6, total S6, and LC3B‑II/I levels at different intervals after radiotherapy. (D) Line graphs represent the changes in the expression levels of p‑S6/total S6 and LC3‑II/LC3‑I ratio, as determined via densi- tometric analysis. The data are expressed as the mean ± SD (n=3). (E) Acridine orange staining and fluorescence microscopy results for the accumulation of acidic vesicular organelles (bright red fluorescence) in cell lines 24 h after radiotherapy. Scale bar, 20 µm. (F) Bar plots represent flow cytometric results for formation of acidic vesicular organelles in cell lines 24 h after radiotherapy. The values of each therapy group were standardized by the value of the control, without any treatment. The data are expressed as the mean ± SD (n=3). *P<0.05, **P<0.01. One‑way ANOVA was used. p‑S6, phospho‑S6 ribosomal protein; p‑4E‑BP1, phospho‑eukaryotic translation initiation factor 4E‑binding protein 1; SQSTM1, sequestosome‑1.

Article Snippet: After blocking with the iBind solution (Life Technologies; Thermo Fisher Scientific, Inc.) at room temperature for 5 min, western blotting was performed using the iBind Western System (Life Technologies; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions; the membranes were incubated with rabbit antibodies against phospho-S6 ribosomal protein (Ser235/236; dilution 1:1,000; cat. no. 4857), S6 ribosomal protein (5G10; dilution 1:1,000; cat. no. 2217), phospho-4E-BP1 (Thr37/46; dilution 1:1,000; cat. no. 2855), 4E-BP1 (dilution 1:1,000; cat. no. 9452) all from CST; LC3B (dilution 1:1,000; cat. no. PM036), p62, also known as sequestosome-1 (SQSTM1) (dilution 1:1,000; cat. no. PM045), or β-actin (dilution 1:1,000; cat. no. PM053) all from MBL at room temperature for 3 h, followed by alkaline phosphatase-labeled goat anti-rabbit antibody (dilution 1:2,000; cat. no. ab97048; Abcam) at room temperature for 2 h. The membranes were incubated in CDP-Star solution (Life Technologies; Thermo Fisher Scientific, Inc.) for 5 min, and chemiluminescence was detected using ChemiDoc XRS system (Bio-Rad Laboratories, Inc., Tokyo, Japan).

Techniques: Activation Assay, Western Blot, Control, Expressing, Staining, Fluorescence, Microscopy

Journal: Oncology reports

Article Title: The combination of temsirolimus and chloroquine increases radiosensitivity in colorectal cancer cells.

doi: 10.3892/or.2019.7134

Figure Lengend Snippet: Figure 3. Combination of TEM, CQ and IR downregulates mTOR pathway‑related proteins and autophagy‑related proteins in SW480 and HT‑29 cells. The cells were treated with 80 nM TEM and/or 20 µM CQ followed by 4 Gy radiotherapy and examined 24 h after radiotherapy. (A) Western blot analysis of mTOR pathway‑ and autophagy‑related proteins. (B) Bar plots represent the relative expression levels of mTOR‑related proteins, p‑S6/total S6, and p‑4E‑BP1/total 4E‑BP1, as determined via densitometric analysis. The data are expressed as the mean ± SD (n=3). *P<0.05, **P<0.01. One‑way ANOVA was used. (C) Bar plots represent the relative expression levels of autophagy‑related proteins, LC3‑II/LC3‑I ratios, and p62 levels. The data are expressed as the mean ± SD (n=3). *P<0.05, **P<0.01. One‑way ANOVA was used. (D) Bar plots represent flow cytometric results revealing the formation of acridine orange‑stained acidic vesicular organelles in cells 24 h after radiotherapy. The values of each group were standardized by the value of the control without any treatment. The data are expressed as the mean ± SD (n=3). *P<0.05, **P<0.01. One‑way ANOVA was used. (E) Fluorescence microscopy images showing acridine orange‑stained autophagic vesicles in cells. Scale bar, 20 µm. NS, not significant; TEM, temsirolimus; CQ, chloroquine; IR, ionizing radiation; p‑S6, phospho‑S6 ribosomal protein; p‑4E‑BP1, phospho‑eukaryotic translation initiation factor 4E‑binding protein 1.

Article Snippet: After blocking with the iBind solution (Life Technologies; Thermo Fisher Scientific, Inc.) at room temperature for 5 min, western blotting was performed using the iBind Western System (Life Technologies; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions; the membranes were incubated with rabbit antibodies against phospho-S6 ribosomal protein (Ser235/236; dilution 1:1,000; cat. no. 4857), S6 ribosomal protein (5G10; dilution 1:1,000; cat. no. 2217), phospho-4E-BP1 (Thr37/46; dilution 1:1,000; cat. no. 2855), 4E-BP1 (dilution 1:1,000; cat. no. 9452) all from CST; LC3B (dilution 1:1,000; cat. no. PM036), p62, also known as sequestosome-1 (SQSTM1) (dilution 1:1,000; cat. no. PM045), or β-actin (dilution 1:1,000; cat. no. PM053) all from MBL at room temperature for 3 h, followed by alkaline phosphatase-labeled goat anti-rabbit antibody (dilution 1:2,000; cat. no. ab97048; Abcam) at room temperature for 2 h. The membranes were incubated in CDP-Star solution (Life Technologies; Thermo Fisher Scientific, Inc.) for 5 min, and chemiluminescence was detected using ChemiDoc XRS system (Bio-Rad Laboratories, Inc., Tokyo, Japan).

Techniques: Western Blot, Expressing, Control, Fluorescence, Microscopy

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: PKM2 Knockdown Induces Autophagic Cell Death via AKT/mTOR Pathway in Human Prostate Cancer Cells.

doi: 10.33594/000000107

Figure Lengend Snippet: Fig. 6. Expression patterns of different signaling pathway proteins after PKM2 knockdown by siRNAs (100 nM for 72 h). (A) The effect of PKM2 knockdown on anticancer signaling pathways in DU145 cells. The cells were transfected with the indicated siRNAs, and western blotting was performed to determine the expression levels of the different signaling pathway proteins. Representative blots are shown. (B) The intensities of the bands were measured and depicted in the bar graph by the ratio of GAPDH. (C) The ratio of p-Akt/Akt, p-mTOR/mTOR, p-p70S6K/ p70S6K and p-GSK3β/ GSK3β were measured and depicted in the bar graph. One-way ANOVA was used to compare the means of different groups. Differences between the means were considered to be significant at P<0.05 by using Tukey multiple comparison tests; *P<0.05, **P<0.01, and ***P<0.001 compared with the normal control cells.

Article Snippet: Mouse monoclonal antibodies against phosphorylated P70S6K (p-P70S6K; T389) (cat. no. 9206) were purchased from Cell Signaling Technology, Inc.,.

Techniques: Expressing, Knockdown, Protein-Protein interactions, Transfection, Western Blot, Comparison, Control

Journal: Cell

Article Title: Ribosome ADP-ribosylation inhibits translation and maintains proteostasis in cancers.

doi: 10.1016/j.cell.2021.07.005

Figure Lengend Snippet: Figure 2. NMNAT-2-dependent MARylation of ribosomal proteins inhibits protein synthesis (A) MARylation, but not PARylation, is detected in ribosomal fractions. Western blot analysis for MAR and PAR of ribosomal fractions or whole cell extracts prepared from OVCAR3 cells. RPS6 and SNRP70 were used as the markers for ribosomal and nuclear fractions, respectively. (B) FK866 treatment reduces both MARylation and PARylation in OVCAR3 cells. Western blot analysis of ribosomal fractions or whole cell extracts isolated from OVCAR3 cells treated with 20 nM FK866 for 48 h. (C) NMNAT-2 depletion inhibits ribosomal protein MARylation. Western blot analysis of ribosomal fractions or whole cell extracts isolated from OVCAR3 cells subjected to NMNAT2 knockdown. (D) NMNAT-2 catalytic activity is required for ribosomal protein MARylation. Western blot analysis of ribosomal fractions or whole cell extracts prepared from OVCAR3 cells subjected to Dox-induced expression of wild-type (WT) or catalytically dead (H24D) mouse NMNAT-2 (Nmnat2; siRNA-resistant) followed by siRNA-mediated knockdown of NMNAT2. (E) NMNAT-2 depletion enhances protein synthesis in OVCAR3 cells. Western blot analysis of puromycin incorporation assays from OVCAR3 cells subjected to NMNAT1 or NMNAT2 knockdown. (F) Ectopic expression of NMNAT-2 overexpression inhibits protein synthesis. Western blot analysis of puromycin incorporation assays prepared from OVCAR3 cells subjected to Dox-induced expression of wild-type (WT) or catalytically dead (H24D) mouse NMNAT-2 (Nmnat2; siRNA-resistant) followed by siRNA- mediated knockdown of NMNAT2. See also Figure S1.

Article Snippet: The other antibodies used were as follows: rabbit polyclonal against PARP-16 (Abcam, ab84641 and GeneTex, GTX123450), mousemonoclonal against NMNAT-2 (Abcam, ab56980), b-tubulin (Abcam, ab6046), SNRP70 (Abcam, ab83306), RPS6 (Cell Signaling Technologies, 2317S), RPL10 (Biorad, VPA00362), RPS5 (Santa Cruz, sc390935), RPS19 (Santa Cruz, sc-100836), Puromycin (Millipore, MABE343), Flag (Sigma-Aldrich, F3165), COX20 (Proteintech, 25752-1-AP and Sigma, HPA045490), phospho-eIF2a (Cell signaling, 9721), eIF2a (Cell signaling, 9722), RPL24 (Proteintech, 17082-1-AP), eIF6 (Cell signaling, 3833T), rabbit monoclonal against RPS6 (Cell signaling, 2217S), cleaved caspase-3 (Cell signaling, 9661S), mouse monoclonal against HA (Sigma-Aldrich, H3663), rabbit polyclonal against HA (Abcam, ab9110), rabbit polyclonal against Flag (Invitrogen, PA1-984B), rabbit IgG (Invitrogen, 10500C), goat anti-rabbit HRP-conjugated IgG (Pierce, 31460), and goat anti-mouse HRP-conjugated IgG (Pierce, 31430). siRNAs-mediated knockdown The siRNA oligos used to knockdown the PARP enzymes were as follows: d PARP6 (siRNA1: SASI_Hs02_0035 3150, siRNA2: SASI_Hs01_00036196) d PARP7 (siRNA1: SASI_Hs01_00202925, siRNA2: SASI_Hs01_00202926) d PARP8 (siRNA1: SASI_Hs01_00139643, siRNA2: SASI_Hs01_00139644) e4 Cell 184, 4531–4546.e1–e14, August 19, 2021 ll

Techniques: Western Blot, Isolation, Knockdown, Activity Assay, Expressing, Over Expression

Journal: Cell

Article Title: Ribosome ADP-ribosylation inhibits translation and maintains proteostasis in cancers.

doi: 10.1016/j.cell.2021.07.005

Figure Lengend Snippet: Figure 5. Ribosomal protein MARylation regulates polysome function through 30 UTR stem-loop structures in mRNAs (A) Ribosomal protein MARylation is enriched in the monosome and polysome fractions of OVCAR3 cells. Western blot analysis for MAR and PARP-16 of the sucrose density gradient fractions prepared from OVCAR3 cells. RPS6 and RPL10 were used as markers for the small and large ribosomal subunits, respectively. (B and C) Depletion of NMNAT-2 or PARP-16 alters mRNA loading on polysomes. RNA-sequencing assay of mRNAs associated with polysomes isolated from OVCAR3 cells subjected to NMNAT2 or PARP16 knockdown. (B) Heatmap representation of mRNAs that exhibited altered loading on the polysomes when NMNAT-2 or PARP-16 were depleted. (C) Gene ontology (GO) analysis of these mRNAs. (D) Identification of a transferable stem-loop motif in the 30UTRs of mRNAs enriched on polysomes after NMNAT2 or PARP16 knockdown. Top panel: sequence of the motif with the highest score. Bottom panels: the stem-loop motif in the 30UTR of Flag-luciferase mRNA is required for translational regulation by PARP-16 and

Article Snippet: The other antibodies used were as follows: rabbit polyclonal against PARP-16 (Abcam, ab84641 and GeneTex, GTX123450), mousemonoclonal against NMNAT-2 (Abcam, ab56980), b-tubulin (Abcam, ab6046), SNRP70 (Abcam, ab83306), RPS6 (Cell Signaling Technologies, 2317S), RPL10 (Biorad, VPA00362), RPS5 (Santa Cruz, sc390935), RPS19 (Santa Cruz, sc-100836), Puromycin (Millipore, MABE343), Flag (Sigma-Aldrich, F3165), COX20 (Proteintech, 25752-1-AP and Sigma, HPA045490), phospho-eIF2a (Cell signaling, 9721), eIF2a (Cell signaling, 9722), RPL24 (Proteintech, 17082-1-AP), eIF6 (Cell signaling, 3833T), rabbit monoclonal against RPS6 (Cell signaling, 2217S), cleaved caspase-3 (Cell signaling, 9661S), mouse monoclonal against HA (Sigma-Aldrich, H3663), rabbit polyclonal against HA (Abcam, ab9110), rabbit polyclonal against Flag (Invitrogen, PA1-984B), rabbit IgG (Invitrogen, 10500C), goat anti-rabbit HRP-conjugated IgG (Pierce, 31460), and goat anti-mouse HRP-conjugated IgG (Pierce, 31430). siRNAs-mediated knockdown The siRNA oligos used to knockdown the PARP enzymes were as follows: d PARP6 (siRNA1: SASI_Hs02_0035 3150, siRNA2: SASI_Hs01_00036196) d PARP7 (siRNA1: SASI_Hs01_00202925, siRNA2: SASI_Hs01_00202926) d PARP8 (siRNA1: SASI_Hs01_00139643, siRNA2: SASI_Hs01_00139644) e4 Cell 184, 4531–4546.e1–e14, August 19, 2021 ll

Techniques: Western Blot, RNA Sequencing, Isolation, Knockdown, Sequencing, Luciferase

Journal: International Journal of Nephrology and Renovascular Disease

Article Title: Induction monotherapy with sirolimus has selected beneficial effects on glomerular and tubulointersititial injury in nephrotoxic serum nephritis

doi: 10.2147/IJNRD.S64202

Figure Lengend Snippet: Quantitative assessment of glomerular injury and phosphorylated S6 immunostaining in the experimental groups

Article Snippet: Blots were blocked overnight at 4°C in 5% powdered skim milk in tris-buffered saline and incubated for 60 minutes at room temperature with a rabbit monoclonal antibody against phosphorylated S6 ribosomal protein antibody (Cell Signaling), followed by alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma-Aldrich).

Techniques: Immunostaining, Control, Staining

Journal: International Journal of Nephrology and Renovascular Disease

Article Title: Induction monotherapy with sirolimus has selected beneficial effects on glomerular and tubulointersititial injury in nephrotoxic serum nephritis

doi: 10.2147/IJNRD.S64202

Figure Lengend Snippet: Quantitative assessment of tubulointerstitial injury and phosphorylated S6 immunostaining in the experimental groups

Article Snippet: Blots were blocked overnight at 4°C in 5% powdered skim milk in tris-buffered saline and incubated for 60 minutes at room temperature with a rabbit monoclonal antibody against phosphorylated S6 ribosomal protein antibody (Cell Signaling), followed by alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma-Aldrich).

Techniques: Immunostaining, Control, Staining

Journal: International Journal of Nephrology and Renovascular Disease

Article Title: Induction monotherapy with sirolimus has selected beneficial effects on glomerular and tubulointersititial injury in nephrotoxic serum nephritis

doi: 10.2147/IJNRD.S64202

Figure Lengend Snippet: Effect of sirolimus (S) on the renal expression of phosphorylated S6 (phospho-S6) ribosomal protein (Ser 235/236) in rats with nephrotoxic serum nephritis. Notes: ( A ) Western blot showing the expression of phosphorylated S6 ribosomal protein in renal cortical homogenates; ( B ) Representative photomicrographs showing immunohistochemistry for phosphorylated S6 ribosomal protein in the cortex (×100), glomerulus (×400), and tubulointerstitum (×200). Cells positive for S6 are revealed by DAB-positive (dark brown) cytoplasmic staining (arrows); sections were counterstained with methyl green. Abbreviations: DAB, 3.3′-diaminobenzidine tetrahydrochloride; NSN, nephrotoxic serum nephritis; V, vehicle.

Article Snippet: Blots were blocked overnight at 4°C in 5% powdered skim milk in tris-buffered saline and incubated for 60 minutes at room temperature with a rabbit monoclonal antibody against phosphorylated S6 ribosomal protein antibody (Cell Signaling), followed by alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma-Aldrich).

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining

Journal: International Journal of Nephrology and Renovascular Disease

Article Title: Induction monotherapy with sirolimus has selected beneficial effects on glomerular and tubulointersititial injury in nephrotoxic serum nephritis

doi: 10.2147/IJNRD.S64202

Figure Lengend Snippet: Molecular mechanisms by which sirolimus inhibits cell growth and proliferation. Notes: The target of rapamycin complex 1 (TORC1) is a key regulator of mitogenesis in mammalian cells. In the cytosol, TORC1 exists as a complex consisting of TOR, raptor and GβL. Phosphorylation (+P) of TORC1 by upstream signal transduction pathways (not shown), in response to mitogenic and other stimuli, leads to cell proliferation and growth, primarily through the activation of two key downstream substrates that control protein synthesis/ribosome biogenesis and cap-dependent translation: 1) the ribosomal S6 kinase (S6K) family and 2) the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Activation of p70 S6 kinase and subsequent phosphorylation of S6 ribosomal protein leads to the translation of ribosome proteins and components of the translational machinery. In parallel, the phosphorylation 4E-BP1 releases it from the translation factor, eIF-4E, and allows the latter to initiate the translation of cyclin D1 and D3 messenger RNA (mRNA). Both of these events mediate G 1 -S phase cell cycle progression. TORC1 is also a negative regulator of autophagy via autophagy-related proteins (ATG), and this regulates cell growth. In the cytosol, sirolimus binds to FK506-binding protein-12 (not shown), and this complex is a highly specific and irreversible allosteric inhibitor of the kinase activities of TORC1, and thus causes G 1 -phase cell cycle arrest and suppression of proliferation. In some cell types, such as podocytes, prolonged exposure to sirolimus also inhibits TORC2 and promotes apoptosis via Akt2 (not shown).

Article Snippet: Blots were blocked overnight at 4°C in 5% powdered skim milk in tris-buffered saline and incubated for 60 minutes at room temperature with a rabbit monoclonal antibody against phosphorylated S6 ribosomal protein antibody (Cell Signaling), followed by alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma-Aldrich).

Techniques: Phospho-proteomics, Transduction, Activation Assay, Control, Binding Assay