Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: An enriched polyphenolic extract obtained from the by-product of Rosa damascena hydrodistillation activates antioxidant and proteostatic modules.

doi: 10.1016/j.phymed.2021.153757

Figure Lengend Snippet: Fig. 2. Oral administration of the enriched polyphenolic extract of R. damascena in Drosophila flies does not affect flies’ feeding rate and induces the upregulation of main proteostatic components. (A) Representative stereoscopic images (A1) and quantification of relative abdomen redness (A2) following a gustatory assay in starved (for 20 h) (or not) flies exposed for 2 h to culture medium containing 100 μg/ml of the R. damascena extract mixed with 0.2% sulforhodamine B sodium salt (Acid-Red). (B) Q-RT-PCR mRNA expression analyses of the autophagy lysosome-related genes Atg8a and cathD, as well as of Trxr-1 (antioxidant response) and Hsp70 molecular chaperone genes. (C1) Q-RT-PCR mRNA expression analyses of the 20S Prosα7, Prosβ5 and the 19S Rpn11 proteasome genes. (C2) Representative immunoblot analyses of protein samples probed with antibodies against 26S-α, p54/Rpn10 and Prosβ5 proteasome subunits. (D) Relative (%) cathepsins B, L activity in Drosophila dissected somatic tissues. (E) Enzymatic activities of the three (LLVY/β5, LLE/β1 and LRR/β2) proteasome peptidases activities in middle-aged flies. Cathepsins (D) and proteasome (E) activities were expressed in fluorescence units per μg of input protein vs. controls set to 100%. Data in (B-E) refer to somatic tissues of wild type Oregon R Drosophila flies treated with the indicated doses of RDet for 7–8 days; in (B-D), data refer to young flies. RpL32/rp49 gene expression (B, C1) and Gapdh probing (C2) were used as reference for total RNA and protein input, respectively. Bars, ± SD (n ≥2); *, p < 0.05; **, p < 0.01 vs. controls.

Article Snippet: Primary antibodies against α7 (sc-100456), β5 (sc-55009), 26S-α (sc65755), 26S p42A (Rpn7) (sc-65750) and 26S p54 (Rpn10) (sc-65748) proteasomal subunits; BECN1 (sc-11427), ubiquitin (Ub) (sc-8017) and the HRP-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology, Inc.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Activity Assay, Fluorescence, Gene Expression

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: An enriched polyphenolic extract obtained from the by-product of Rosa damascena hydrodistillation activates antioxidant and proteostatic modules.

doi: 10.1016/j.phymed.2021.153757

Figure Lengend Snippet: Fig. 3. Ubiquitous cncC/NRF2 knock down (cncCRNAi) abolishes the induction of proteostatic modules observed in Drosophila’s somatic tissues after treatment with the enriched polyphenolic extract of R. damascena. (A) CSLM visualization and quantification of intracellular cytosolic H2O2 levels (fluorescence intensities ratio, 405 nm/488 nm) in the inner cavity of the intestinal wall isolated from young female flies ubiquitously expressing the fluorescent cyto-roGFP2-Orp1 probe after treatment (or not) with 100 μg/ml RDet and ubiquitous (or not) cncCRNAi for 7 days. (B) Representative immunoblot analyses of protein lysates probed with antibody against ubiquitinated (Ub) proteins. (C) Q-RT-PCR mRNA expression analyses of the cncC transcription factor, the 20S Prosα7, Prosβ5, and 19S Rpn11 proteasome genes and of the antioxidant Sod1 gene. (D) Immunoblotting analyses of protein lysates probed with antibodies against 26S-α, PSMD11/Rpn6, p42a/Rpn7 and p54/ Rpn10 proteasome subunits. (E) Relative (%) enzymatic activities of proteasome peptidases (LLVY/β5 and LLE/β1). Proteasome activities were expressed in fluo rescence units per μg of input protein vs. controls set to 100%. In (B-E) data refer to somatic tissues of young Drosophila flies after treatment (or not) with the indicated doses of RDet for 7–8 days and ubiquitous cncCRNAi. cncC downregulation was induced by culturing young flies with 320 μМ RU486 for 7–8 days. Flies of the same genotype cultured without RU486 were used as controls. Gapdh protein (B, D) and RpL32/rp49 gene (C) expression were used as reference for protein input and total RNA, respectively. Bars, ± SD (n ≥2); *, p < 0.05; **, p < 0.01 vs. controls.

Article Snippet: Primary antibodies against α7 (sc-100456), β5 (sc-55009), 26S-α (sc65755), 26S p42A (Rpn7) (sc-65750) and 26S p54 (Rpn10) (sc-65748) proteasomal subunits; BECN1 (sc-11427), ubiquitin (Ub) (sc-8017) and the HRP-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology, Inc.

Techniques: Knockdown, Fluorescence, Isolation, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: Genome Biology

Article Title: Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein

doi: 10.1186/gb-2014-15-1-r4

Figure Lengend Snippet: Validation of new Smaug targets. Extracts were prepared from 0- to 1-, 1- to 2- and 2- to 3-hour-old wild-type and smaug -mutant embryos and assayed for the levels of (A) Rpn7, (B) Su(z)12 and (C) Bicaudal C proteins via western blots.

Article Snippet: Antibodies against Rpn7 (Santa Cruz Biotechnology, Dallas, Texas, USA; catalogue #SC-65750), Su(z)12 [ ] and Bicaudal C [ ] were used in standard western blot assays.

Techniques: Biomarker Discovery, Mutagenesis, Western Blot

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: An enriched polyphenolic extract obtained from the by-product of Rosa damascena hydrodistillation activates antioxidant and proteostatic modules.

doi: 10.1016/j.phymed.2021.153757

Figure Lengend Snippet: Fig. 2. Oral administration of the enriched polyphenolic extract of R. damascena in Drosophila flies does not affect flies’ feeding rate and induces the upregulation of main proteostatic components. (A) Representative stereoscopic images (A1) and quantification of relative abdomen redness (A2) following a gustatory assay in starved (for 20 h) (or not) flies exposed for 2 h to culture medium containing 100 μg/ml of the R. damascena extract mixed with 0.2% sulforhodamine B sodium salt (Acid-Red). (B) Q-RT-PCR mRNA expression analyses of the autophagy lysosome-related genes Atg8a and cathD, as well as of Trxr-1 (antioxidant response) and Hsp70 molecular chaperone genes. (C1) Q-RT-PCR mRNA expression analyses of the 20S Prosα7, Prosβ5 and the 19S Rpn11 proteasome genes. (C2) Representative immunoblot analyses of protein samples probed with antibodies against 26S-α, p54/Rpn10 and Prosβ5 proteasome subunits. (D) Relative (%) cathepsins B, L activity in Drosophila dissected somatic tissues. (E) Enzymatic activities of the three (LLVY/β5, LLE/β1 and LRR/β2) proteasome peptidases activities in middle-aged flies. Cathepsins (D) and proteasome (E) activities were expressed in fluorescence units per μg of input protein vs. controls set to 100%. Data in (B-E) refer to somatic tissues of wild type Oregon R Drosophila flies treated with the indicated doses of RDet for 7–8 days; in (B-D), data refer to young flies. RpL32/rp49 gene expression (B, C1) and Gapdh probing (C2) were used as reference for total RNA and protein input, respectively. Bars, ± SD (n ≥2); *, p < 0.05; **, p < 0.01 vs. controls.

Article Snippet: Primary antibodies against α7 (sc-100456), β5 (sc-55009), 26S-α (sc65755), 26S p42A (Rpn7) (sc-65750) and 26S p54 (Rpn10) (sc-65748) proteasomal subunits; BECN1 (sc-11427), ubiquitin (Ub) (sc-8017) and the HRP-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology, Inc.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Activity Assay, Fluorescence, Gene Expression

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: An enriched polyphenolic extract obtained from the by-product of Rosa damascena hydrodistillation activates antioxidant and proteostatic modules.

doi: 10.1016/j.phymed.2021.153757

Figure Lengend Snippet: Fig. 3. Ubiquitous cncC/NRF2 knock down (cncCRNAi) abolishes the induction of proteostatic modules observed in Drosophila’s somatic tissues after treatment with the enriched polyphenolic extract of R. damascena. (A) CSLM visualization and quantification of intracellular cytosolic H2O2 levels (fluorescence intensities ratio, 405 nm/488 nm) in the inner cavity of the intestinal wall isolated from young female flies ubiquitously expressing the fluorescent cyto-roGFP2-Orp1 probe after treatment (or not) with 100 μg/ml RDet and ubiquitous (or not) cncCRNAi for 7 days. (B) Representative immunoblot analyses of protein lysates probed with antibody against ubiquitinated (Ub) proteins. (C) Q-RT-PCR mRNA expression analyses of the cncC transcription factor, the 20S Prosα7, Prosβ5, and 19S Rpn11 proteasome genes and of the antioxidant Sod1 gene. (D) Immunoblotting analyses of protein lysates probed with antibodies against 26S-α, PSMD11/Rpn6, p42a/Rpn7 and p54/ Rpn10 proteasome subunits. (E) Relative (%) enzymatic activities of proteasome peptidases (LLVY/β5 and LLE/β1). Proteasome activities were expressed in fluo rescence units per μg of input protein vs. controls set to 100%. In (B-E) data refer to somatic tissues of young Drosophila flies after treatment (or not) with the indicated doses of RDet for 7–8 days and ubiquitous cncCRNAi. cncC downregulation was induced by culturing young flies with 320 μМ RU486 for 7–8 days. Flies of the same genotype cultured without RU486 were used as controls. Gapdh protein (B, D) and RpL32/rp49 gene (C) expression were used as reference for protein input and total RNA, respectively. Bars, ± SD (n ≥2); *, p < 0.05; **, p < 0.01 vs. controls.

Article Snippet: Primary antibodies against α7 (sc-100456), β5 (sc-55009), 26S-α (sc65755), 26S p42A (Rpn7) (sc-65750) and 26S p54 (Rpn10) (sc-65748) proteasomal subunits; BECN1 (sc-11427), ubiquitin (Ub) (sc-8017) and the HRP-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology, Inc.

Techniques: Knockdown, Fluorescence, Isolation, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: Cell reports

Article Title: cindr, the Drosophila Homolog of the CD2AP Alzheimer’s Disease Risk Gene, Is Required for Synaptic Transmission and Proteostasis

doi: 10.1016/j.celrep.2019.07.041

Figure Lengend Snippet:

Article Snippet: mouse anti-26S Proteasome p50 (112)-RPT5 , Santa Cruz , RRID: SC_65745.

Techniques: Activity Assay, Recombinant

Journal: Frontiers in Pharmacology

Article Title: Inhibition of HDAC3- and HDAC6-Promoted Survivin Expression Plays an Important Role in SAHA-Induced Autophagy and Viability Reduction in Breast Cancer Cells

doi: 10.3389/fphar.2016.00081

Figure Lengend Snippet: SAHA increases the expression of 26S proteasome and decreases the expression of Hsp90 in breast cancer cells. (A) Breast cancer cells were treated with the pharmacological inhibitor of proteasome, MG132, for 48 h. Expression of survivin was determined by Western blotting. (B) Breast cancer cells were treated with indicated concentrations of SAHA for 72 h and the expression of 26S proteasome was analyzed by Western blotting. (C) Breast cancer cells were treated with SAHA for 72 h and the intracellular proteasome activity in the treated cells were assessed using proteasome activity fluorometric assay kit. Experiment was repeated three times. A statistically significant difference in the proteasome activity in cells treated with SAHA vs. without SAHA (control) is denoted by “ * ” ( p < 0.05). (D) MCF7 cells were treated with SAHA for indicated durations and expression of Hsp90 was determined by Western blotting. Signals in the Hsp90 blots (of all repeats) were quantitated and a graph was generated to show the effect of SAHA on the expression of Hsp90. A statistically significant difference in the expression of Hsp90 in cells treated with SAHA vs. without SAHA (0 h) is denoted by “ ** ” ( p < 0.01). (E) Breast cancer cells were transfected with scramble, HDAC3, HDAC1, or HDAC2 siRNA for 72 h. Expression of various proteins was determined by Western blotting. (F) Breast cancer cells were treated with BML281 for 24–72 h. Expression of different proteins was determined by Western blotting. Signals in the Hsp90 blots (of all repeats) were quantitated and a graph was generated to show the effect of BML281 on the expression of Hsp90. A statistically significant difference in the expression of Hsp90 in cells treated with BML281 vs. without BML281 (0 h) is denoted by either “ * ” ( p < 0.01) or “ *** ” ( p < 0.001).

Article Snippet: Primary antibodies used in this study are listed as follows: mouse antibodies against β-actin and acetylated tubulin were obtained from Millipore (cat# MAB1501 and cat# 05829); antibodies against lamin A/C were from Santa Cruz (cat# sc-7292); antibodies against proteasome 26S and Hsp90 were from Abcam (cat# AB58115-100); and Enzo (cat# SPA-830), respectively.

Techniques: Expressing, Western Blot, Activity Assay, Control, Generated, Transfection