Journal: Free radical biology & medicine

Article Title: Drp1-regulated PARK2-dependent mitophagy protects against renal fibrosis in unilateral ureteral obstruction.

doi: 10.1016/j.freeradbiomed.2019.12.005

Figure Lengend Snippet: Fig. 7. Mdivi-1 inhibited hypoxia-induced PARK2-dependent mitophagy, increased mtROS production, and promoted TGFβ1 signaling in HK-2 cells. (A)Immunoblot analysis of Drp1 in mitochondria of HK-2 cells and (B) quantification of immunoblot analysis (n = 4 per group). (C)HK-2 cells were pretreated with vehicle or Mdivi- 1(5 μM) 1h before hypoxia exposure, and were cultured in hypoxic condition(1%O2) for 24 h. Immunoblot analysis of proteins in mitochondrial fraction of HK- 2 cells, and (D) densitometric analysis (n = 3 per group). (E)Representative images of MitoSOX/Hoechst staining of HK-2 cells and (F)quantification data of fluorescence density (n = 6 per group). Scale bar, 25 μm. (G) MnSOD activity of HK-2 cells analyzed by colorimetric activity assay(n = 6 per group).(H) MnSOD protein level detected by immunoblot analysis and(I) quantification data(n = 4 per group). (J) Immunoblot analysis and densitometry data of the protein level of (K)TGFβ1, (J)phosphorylation of smad2 and (L)smad3 in HK-2 cells (n = 4 per group). Error bars: SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Article Snippet: Living HK-2 cells were incubated in MitoSOX (5 μM) in HBSS and Hoechst (5 μg/ml,HY15631,MedChemExpress) for 30 min at 37 °C, and then observed under fluorescence microscope.

Techniques: Western Blot, Cell Culture, Staining, Activity Assay, Phospho-proteomics

Journal: Free radical biology & medicine

Article Title: Drp1-regulated PARK2-dependent mitophagy protects against renal fibrosis in unilateral ureteral obstruction.

doi: 10.1016/j.freeradbiomed.2019.12.005

Figure Lengend Snippet: Fig. 9. MitoTEMPO treatment attenuated hypoxia-induced mtROS production and TGFβ1 signaling in HK-2 cells. After transfected with siRNAs for 24 h, HK-2 cells were pretreated with vehicle or mitoTEMPO(100 μM) 4h before hypoxia exposure, and were then cultured in hypoxic conditions(1%O2) for 24 h. (A)Representative images of MitoSOX/Hoechst staining of HK-2 cells and (B)quantification data of fluorescence density (n = 3 per group). Scale bar, 25 μm. (C) Immunoblot analysis and densitometry data of phosphor the protein level of (D)TGFβ1, (E)phosphorylation of smad2 and (F)smad3 in HK-2 cells (n = 3 per group). Error bars: SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. NC, negative control; Norm, normoxia; Hypo, Hypoxia for 24 h; mT, mitoTEMPO.

Article Snippet: Living HK-2 cells were incubated in MitoSOX (5 μM) in HBSS and Hoechst (5 μg/ml,HY15631,MedChemExpress) for 30 min at 37 °C, and then observed under fluorescence microscope.

Techniques: Transfection, Cell Culture, Staining, Western Blot, Phospho-proteomics, Negative Control

Journal: Neuron

Article Title: Wireless Optogenetic Stimulation of Oxytocin Neurons in a Semi-natural Setup Dynamically Elevates Both Pro-social and Agonistic Behaviors

doi: 10.1016/j.neuron.2020.05.028

Figure Lengend Snippet:

Article Snippet: Ornithine Vasotocin Analog (OVTA) , Perkin Elmer , NEX254050UC.

Techniques: Virus, Recombinant, Software, Amplification, Adhesive

Journal: Investigative Ophthalmology & Visual Science

Article Title: TFEB-Mediated Lysosomal Restoration Alleviates High Glucose-Induced Cataracts Via Attenuating Oxidative Stress

doi: 10.1167/iovs.63.6.26

Figure Lengend Snippet: Curcumin analog C1 exerted protective effects in HG-induced cataracts. Rat lenses were subjected to control (5.5 mM), HG (50 mM), and HG (50 mM) with C1 (5 µM) medium for 7 days. ( A ) Representative images of cultured rat lenses and the percentage distribution of cataract scores in the control, HG, and HG + C1 groups. ( B ) TEM showed that C1 treatment alleviated abnormally large autophagic vesicles ( arrows ) with massive undegraded substrates in anterior capsular LECs under HG conditions. ( C ) Protein expression levels and quantitative analysis of α-SMA, FN, LC3-I/II, and P62 determined by Western blotting in LECs. ( D ) Protein expression levels and quantitative analysis of LC3-I/II and P62 determined by Western blotting in LFCs. All the data were shown as mean ± standard deviation. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: To study the effects of TFEB activation, we treated lenses with curcumin analog C1 (C1; 5 μM; Selleck) or tetrahydrocurcumin (THC; 5 μM; Selleck) upon HG (50 mM) treatment.

Techniques: Control, Cell Culture, Expressing, Western Blot, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: TFEB-Mediated Lysosomal Restoration Alleviates High Glucose-Induced Cataracts Via Attenuating Oxidative Stress

doi: 10.1167/iovs.63.6.26

Figure Lengend Snippet: C1 enhanced lysosomal degradation via promoting the nuclear translocation of TFEB in HG-cultured LECs. Primary rabbit LECs were exposed to control (5.5 mM), HG (25 mM), and HG (25 mM) with C1 (5 µM) medium for 3 days. ( A ) ROS levels were detected by DCFH-DA assay. ( B , C ) Protein expression levels and quantitative analysis of α-SMA, FN, LC3-I/II, and P62 determined by Western blotting in LECs. ( D ) Representative fluorescence images of mCherry-EGFP-LC3 puncta within LECs and quantification. ( E ) Immunofluorescence staining and Western blot analysis ( F ) showed that C1 increased the expression levels and distributions of TFEB in the cytoplasm and nuclei in LECs. ( G ) Protein expression levels and quantitative analysis of Pro-CTSB, LAMP1, and ubiquitin determined by Western blotting in LECs. ( H ) Lysotracker staining and immunofluorescence staining of LAMP1 puncta in LECs. Relative protein levels of total proteins were standardized to the expression of β-Actin, whereas nuclear proteins were normalized to Lamin B1. All the data were shown as mean ± standard deviation. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: To study the effects of TFEB activation, we treated lenses with curcumin analog C1 (C1; 5 μM; Selleck) or tetrahydrocurcumin (THC; 5 μM; Selleck) upon HG (50 mM) treatment.

Techniques: Translocation Assay, Cell Culture, Control, DCFH-DA Assay, Expressing, Western Blot, Fluorescence, Immunofluorescence, Staining, Ubiquitin Proteomics, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: TFEB-Mediated Lysosomal Restoration Alleviates High Glucose-Induced Cataracts Via Attenuating Oxidative Stress

doi: 10.1167/iovs.63.6.26

Figure Lengend Snippet: Comparison of THC and C1 in alleviating HG-induced cataracts. Primary rabbit LECs were exposed to control (5.5 mM), HG (25 mM), HG (25 mM) with THC (5 µM), and HG (25 mM) with C1 (5 µM) medium for 3 days. ( A ) Protein expression levels and quantitative analysis of TFEB determined by Western blotting in LECs. ( B ) DCFH-DA assay compared ROS levels in the control, HG, HG + THC, and HG + C1 groups. Rat lenses were subjected to control (5.5 mM), HG (50 mM), HG (50 mM) with THC (5 µM), and HG (50 mM) with C1 (5 µM) medium for 7 days. ( C ) Representative images of cultured rat lenses and the percentage distribution of cataract scores in the control, HG, HG + THC, and HG + C1 groups. ( D ) Protein expression levels and quantitative analysis of α-SMA, FN, LC3-I/II, and P62 determined by Western blotting in LECs. All the data were shown as mean ± standard deviation. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: To study the effects of TFEB activation, we treated lenses with curcumin analog C1 (C1; 5 μM; Selleck) or tetrahydrocurcumin (THC; 5 μM; Selleck) upon HG (50 mM) treatment.

Techniques: Comparison, Control, Expressing, Western Blot, DCFH-DA Assay, Cell Culture, Standard Deviation

Journal: Cell reports

Article Title: Physical interactions between MCM and Rad51 facilitate replication fork lesion bypass and ssDNA gap filling by non-recombinogenic functions.

doi: 10.1016/j.celrep.2021.109440

Figure Lengend Snippet: Figure 1. MCM interacts with Rad51 and Rad52 through mechanisms regulated by cell cycle, DNA damage, and Cdc7 (A) Rad51 interacts with Mcm4, independent of Rad52. CoIP was performed in asynchronous cultures. (B) Mcm4 interacts with Rad52. Interactions were detected regardless of whether extracts had been treated or not with MNase I (A) or benzonase (B). CoIP was performed in asynchronous cultures. (C) Mcm4 interacts with Rad51, both with and without 0.015% MMS for 2 h. CoIP was performed in asynchronous cultures. (D) Mcm4 interacts with Rad51 in cdc7D mcm5-bob1 and wild-type cells treated with 0.2 M HU for 2 h. Wild-type cells treated with 0.025% MMS for 2 h were included as controls. CoIP was performed in asynchronous cultures. (E) The Mcm4/Rad51 interaction in cells released into MMS during the S phase depends on Cdc7 kinase activity, and mcm5-bob1 bypasses that requirement. cdc7-as3 cells were synchronized in the G1, released into the S phase in the presence of 0.015% MMS for 90 min, and treated with 15 mM 1NMPP1 30 min after G1 release. (F) Mcm4/Rad51 interaction is eliminated during an unperturbed S phase. G1-synchronized cells were released in the absence or presence of 0.015% MMS for 45 and 90 min, respectively, and were treated or not with 15 mM 1NMPP1 30 min after G1 release (MMS). An over-exposure (o.e.) of the Rad51 gel is shown. (G) The Mcm4/Rad51 interaction occurs in G1, independent of Cdc7 activity. The analysis was performed in G1-arrested cells, either coming from G1 and released into fresh medium with a-factor and 15 mM 1NMPP1 60 min later (G2-to-G1) or maintained in G1 for 30 min with and without an inhibitor (G1). Cells synchronized in G1 and released into fresh medium for 60 min were included as controls (S/G2). Physical interactions of MCM with Rad51 and Rad52 were determined by immunoprecipitation of Mcm4-GFP and western blot analyses. All experiments were repeated at least twice with similar results. The asterisks indicate a degradation product. Dashed lines indicate spliced images.

Article Snippet: N/A a-factor Proteogenix SAS Cat# GM-PT301350-95 Pronase Merck Cat# 10165921001 Nocodazole Merck Cat# M1404-50MG PP1 Analog II (1NMPP1) Merck Cat# 529581-1 PP1 Analog II (1NMPP1) Santa Cruz Cat# sc-203214 MNase I Merck Cat# N3755-200UN benzonase Merck Cat# 70746-4 5-Bromo-20-deoxyuridine (BrdU) Sigma Cat# B5002 Agarose, NuSieve GTG Agarose (low-melting temp) Lonza Cat# 50081 l phosphatase New England Biolabs Cat# P0753S Critical commercial assays GFP trap magnetic beads Chromotek Cat# gtma-100 dynabeads protein G Invitrogen Cat# 10004D QuickChange XL Site-directed Mutagenesis Kit Agilent Cat# 200517 Experimental models: Organisms/strains S. cerevisiae: Strain background: W303 ATCC ATTC: 208353 Other yeast strains: Table S1 This study N/A Oligonucleotides See Table S2 This study N/A Recombinant DNA pRS551-L120A,V181A A gift from Nancy M. Hollingsworth, Stony Brook University N/A pRS51s A gift from Andrés Aguilera, Seville University N/A (Continued on next page) Cell Reports 36, 109440, July 27, 2021 e1

Techniques: Activity Assay, Immunoprecipitation, Western Blot

Journal: Cell reports

Article Title: Physical interactions between MCM and Rad51 facilitate replication fork lesion bypass and ssDNA gap filling by non-recombinogenic functions.

doi: 10.1016/j.celrep.2021.109440

Figure Lengend Snippet: Figure 3. Cell cycle, DNA damage, and Cdc7 regulate the accumulation of MCM, Rad51, and Rad52 in the insoluble fraction (A) Mcm4 and Rad51 accumulate in the insoluble fraction in G1 and are released during the S phase. (B) Mcm4 and Rad51 are maintained in the insoluble fraction during the S phase in the presence of 0.025% MMS. (C) Rad52 is not required for Rad51 binding to the insoluble fraction in G1 and MMS-released cells. (D) Inhibition of Cdc7 kinase activity prevents the maintenance of Rad51 in the insoluble fraction in response to DNA damage. G1-synchronized cells were released into the S phase in the presence of 0.025% MMS; they were treated with 15 mM 1NMPP1 35 min after G1 release. (E) The mcm5-bob1 mutation bypasses the requirement of Cdc7 for Rad51 maintenance in the insoluble fraction under replicative stress. G1-synchronized cells were released into the S phase in the presence of 0.025% MMS; they were treated with 15 mM 1NMPP1 35 min after G1 release. MCM and Rad51 binding to the insoluble fraction was determined by cell fractionation and western blot. See Figure S2 for fractionation controls (A–E). Dashed lines indicate spliced images. Histone H4 was used to normalize the amount of each protein. The mean of 4 (A), 11 for Mcm4-GFP and 14 for Rad51 (B), 2 (C and E) and 11 for 90-min, and 6 for 120-min (D) independent experiments are plotted. Either the SEM (A, B, and D) or range (C and E) are shown. Values are normalized to G1 (A–C) or to the absence of the inhibitor (D and E), taking as 100. Statistically significant differences relative to G1 (A and B) or to the absence of the inhibitor (D) are shown; *p < 0.05, **p < 0.01, ***p < 0.001; one-sample t test.

Article Snippet: N/A a-factor Proteogenix SAS Cat# GM-PT301350-95 Pronase Merck Cat# 10165921001 Nocodazole Merck Cat# M1404-50MG PP1 Analog II (1NMPP1) Merck Cat# 529581-1 PP1 Analog II (1NMPP1) Santa Cruz Cat# sc-203214 MNase I Merck Cat# N3755-200UN benzonase Merck Cat# 70746-4 5-Bromo-20-deoxyuridine (BrdU) Sigma Cat# B5002 Agarose, NuSieve GTG Agarose (low-melting temp) Lonza Cat# 50081 l phosphatase New England Biolabs Cat# P0753S Critical commercial assays GFP trap magnetic beads Chromotek Cat# gtma-100 dynabeads protein G Invitrogen Cat# 10004D QuickChange XL Site-directed Mutagenesis Kit Agilent Cat# 200517 Experimental models: Organisms/strains S. cerevisiae: Strain background: W303 ATCC ATTC: 208353 Other yeast strains: Table S1 This study N/A Oligonucleotides See Table S2 This study N/A Recombinant DNA pRS551-L120A,V181A A gift from Nancy M. Hollingsworth, Stony Brook University N/A pRS51s A gift from Andrés Aguilera, Seville University N/A (Continued on next page) Cell Reports 36, 109440, July 27, 2021 e1

Techniques: Binding Assay, Inhibition, Activity Assay, Mutagenesis, Cell Fractionation, Western Blot, Fractionation

Journal: Frontiers in Endocrinology

Article Title: Legumain is a paracrine regulator of osteoblast differentiation and mediates the inhibitory effect of TGF-β1 on osteoblast maturation

doi: 10.3389/fendo.2024.1445049

Figure Lengend Snippet: Pharmacological inhibition of legumain rescue matrix mineralization in osteogenic cells treated with TGF-β1 and prolegumain. BMSC were differentiated to osteogenic cells for 14 days with or without treatment with 50% prolegumain-rich conditioned medium (165 ng/mL, control: 1.1 ng/mL), TGF-β1 (25 ng/mL) and the legumain inhibitor RR-11a (50 μM). (A) Legumain activity (dF/sec) in cell lysates adjusted for total protein concentration (µg/mL) (n=3-4). (B) Normalized matrix mineralization measured by BoneTag™ fluorescence. Data represent mean ± SEM (n=3-4). Three-way ANOVA (Tukey’s post-hoc ). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: In addition, the BMSC cells were incubated with or without TGF-β1 (25 ng/mL) (R&D systems, MN, USA) and/or 50 μM of the irreversible legumain inhibitor RR-11a analog (referred as RR-11a; MedChemExpress, NJ, USA).

Techniques: Inhibition, Control, Activity Assay, Protein Concentration, Fluorescence

Journal: Frontiers in Chemistry

Article Title: Bulky PP1 analogs exert cellular effects independently from analog-sensitive kinase inhibition

doi: 10.3389/fchem.2026.1812827

Figure Lengend Snippet: PP1 analogs modify HaCaT cell cycle profile. HaCaT cells were treated with either 0.1% DMSO, 5 µM of 1NA- or 1NM-PP1 or 10 µM of 3MB, 3-IB, or 3MSB-PP1 for 48 h. Cell cycle profiles were determined by flow cytometry after propidium iodide labeling of fixed cells, in four independent experiments. p-values were determined by a two-way ANOVA test followed by Dunnet’s multiple-comparison test comparing each inhibitor to DMSO. Significant differences are labelled with asterisks.

Article Snippet: We prepared 10 mM stock solutions of 1NA-PP1 (MedChemExpress, HY-13941), 1NM-PP1 (MedChemExpress, HY-13942), 3MB-PP1 (Aobious, AOB3854), 3IB-PP1(Sigma, 529598) and 3MSB-PP1 in 100% DMSO and we stored aliquots at −20 °C.

Techniques: Flow Cytometry, Labeling, Comparison