Journal: Oxidative Medicine and Cellular Longevity

Article Title: LRRK2 Kinase Inhibitor Rejuvenates Oxidative Stress-Induced Cellular Senescence in Neuronal Cells

doi: 10.1155/2021/9969842

Figure Lengend Snippet: Rotenone-induced LRRK2 kinase activation promotes cellular senescence in the differentiated human neuroblastoma cell line. Western blotting analysis of dSH cells treated with 1 μ M rotenone or 1 μ M GSK2578215A (GSK-KI), an LRRK2 kinase inhibitor, for 48 h using antibodies against the target proteins (a). The densities of the proteins were normalized to those of β -actin (b–g). n = 3. (h) dSH cells treated with 1 μ M rotenone or 1 μ M GSK-KI for 48 h were subjected to proximity ligation assay (PLA) of phospho-S1292 LRRK2 and total LRRK2 (PLA-pS1292) and immunofluorescence (IF) analysis of β -galactosidase. Nuclei were stained with Hoechst 33342. The controls used in PLA and IF staining revealed the validity of the results (right two panels). The intensities of PLA-pS1292 (i) and β -galactosidase (j) were normalized to those of 4′,6-diamidino-2-phenylindole. n = 4; number of cells = 12–17.

Article Snippet: The following antibodies were used for the western blotting: rabbit anti-LRRK2 phospho-S935 monoclonal (ab133450, Abcam, Cambridge, UK), rabbit anti-LRRK2 phospho-S1292 monoclonal (ab203181, Abcam), mouse anti-LRRK2 monoclonal (N241A/34, NeuroMab, UC Davis, CA, USA), mouse anti-p53 monoclonal (for human p53: sc-126; for mouse p53: sc-393031; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-p21 Waf1/Cip1 (p21) monoclonal (CM5131, ECM Biosciences, Versailles, KY, USA), mouse anti-Rb monoclonal (#9309S, Cell Signaling Technology (CST), Danvers, MA, USA), anti-phosphoRb (Ser807/811) (#9308S, CST), mouse anti- β -galactosidase monoclonal (sc-377257, Santa Cruz Biotechnology), mouse anti-p62 monoclonal (ab56416, Abcam), mouse anti- β -actin monoclonal (sc-47778; Santa Cruz Biotechnology), rabbit anti-LC3B polyclonal (#2775S; CST), mouse anti- α -syn (clone 42) monoclonal (610786; BD Biosciences, San Jose, CA, USA), anti-phospho-threonine-arginine (#2351S, CST), anti-p53 (SC-99, Santa Cruz Biotechnology), anti-LaminB (SC-6216, Santa Cruz Biotechnology), goat peroxidase-conjugated AffiniPure anti-mouse IgG (H+L) (#115-035-003; Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA), and goat peroxidase-conjugated AffiniPure anti-rabbit IgG (H+L) (#111-035-144; Jackson Immunoresearch Laboratories Inc.) antibodies.

Techniques: Activation Assay, Western Blot, Proximity Ligation Assay, Immunofluorescence, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: LRRK2 Kinase Inhibitor Rejuvenates Oxidative Stress-Induced Cellular Senescence in Neuronal Cells

doi: 10.1155/2021/9969842

Figure Lengend Snippet: LRRK2 kinase inhibition mitigates rotenone-induced cellular senescence and promotes the autophagic clearance of α -synuclein in the rat primary cortical neurons. The experiments performed using dSH treated with α -synuclein fibril (70 nM), rotenone (1 μ M), and GSK-KI (1 μ M) for 48 h were repeated with rat primary cortical neurons. Immunoreactive signals in the western blot (a) were visualized using antibodies against the target proteins. The densities of all proteins, except LC3B II/I, were normalized to those of β -actin (b–i). (j) The lysates of (a) were subjected to the senescence-associated β -galactosidase activity assay. n = 3.

Article Snippet: The following antibodies were used for the western blotting: rabbit anti-LRRK2 phospho-S935 monoclonal (ab133450, Abcam, Cambridge, UK), rabbit anti-LRRK2 phospho-S1292 monoclonal (ab203181, Abcam), mouse anti-LRRK2 monoclonal (N241A/34, NeuroMab, UC Davis, CA, USA), mouse anti-p53 monoclonal (for human p53: sc-126; for mouse p53: sc-393031; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-p21 Waf1/Cip1 (p21) monoclonal (CM5131, ECM Biosciences, Versailles, KY, USA), mouse anti-Rb monoclonal (#9309S, Cell Signaling Technology (CST), Danvers, MA, USA), anti-phosphoRb (Ser807/811) (#9308S, CST), mouse anti- β -galactosidase monoclonal (sc-377257, Santa Cruz Biotechnology), mouse anti-p62 monoclonal (ab56416, Abcam), mouse anti- β -actin monoclonal (sc-47778; Santa Cruz Biotechnology), rabbit anti-LC3B polyclonal (#2775S; CST), mouse anti- α -syn (clone 42) monoclonal (610786; BD Biosciences, San Jose, CA, USA), anti-phospho-threonine-arginine (#2351S, CST), anti-p53 (SC-99, Santa Cruz Biotechnology), anti-LaminB (SC-6216, Santa Cruz Biotechnology), goat peroxidase-conjugated AffiniPure anti-mouse IgG (H+L) (#115-035-003; Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA), and goat peroxidase-conjugated AffiniPure anti-rabbit IgG (H+L) (#111-035-144; Jackson Immunoresearch Laboratories Inc.) antibodies.

Techniques: Inhibition, Western Blot, Activity Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: LRRK2 Kinase Inhibitor Rejuvenates Oxidative Stress-Induced Cellular Senescence in Neuronal Cells

doi: 10.1155/2021/9969842

Figure Lengend Snippet: Rotenone promotes cellular senescence in the mouse midbrain by upregulating the expression of LRRK2 kinase. (a) A scheme of the mouse model experiment. (b) The measurements of falling time from the rotarod are represented. The falling times of each mouse in the experimental group ( n = 4) were recorded thrice. The midbrain lysates were subjected to senescence-associated β -galactosidase activity assay (c), western blotting (d–e), and enzyme-linked immunosorbent assay for detecting filamentous oligomer α -synuclein (f). n = 4. ∗ indicates the results of two-way analysis of variance (ANOVA), followed by Tukey's post hoc test. # indicates the results of one-way ANOVA, followed by Tukey's post hoc test. ## p < 0.01 and ### p < 0.001.

Article Snippet: The following antibodies were used for the western blotting: rabbit anti-LRRK2 phospho-S935 monoclonal (ab133450, Abcam, Cambridge, UK), rabbit anti-LRRK2 phospho-S1292 monoclonal (ab203181, Abcam), mouse anti-LRRK2 monoclonal (N241A/34, NeuroMab, UC Davis, CA, USA), mouse anti-p53 monoclonal (for human p53: sc-126; for mouse p53: sc-393031; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-p21 Waf1/Cip1 (p21) monoclonal (CM5131, ECM Biosciences, Versailles, KY, USA), mouse anti-Rb monoclonal (#9309S, Cell Signaling Technology (CST), Danvers, MA, USA), anti-phosphoRb (Ser807/811) (#9308S, CST), mouse anti- β -galactosidase monoclonal (sc-377257, Santa Cruz Biotechnology), mouse anti-p62 monoclonal (ab56416, Abcam), mouse anti- β -actin monoclonal (sc-47778; Santa Cruz Biotechnology), rabbit anti-LC3B polyclonal (#2775S; CST), mouse anti- α -syn (clone 42) monoclonal (610786; BD Biosciences, San Jose, CA, USA), anti-phospho-threonine-arginine (#2351S, CST), anti-p53 (SC-99, Santa Cruz Biotechnology), anti-LaminB (SC-6216, Santa Cruz Biotechnology), goat peroxidase-conjugated AffiniPure anti-mouse IgG (H+L) (#115-035-003; Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA), and goat peroxidase-conjugated AffiniPure anti-rabbit IgG (H+L) (#111-035-144; Jackson Immunoresearch Laboratories Inc.) antibodies.

Techniques: Expressing, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Neurochemical Research

Article Title: The VGVAPG Peptide Regulates the Production of Nitric Oxide Synthases and Reactive Oxygen Species in Mouse Astrocyte Cells In Vitro

doi: 10.1007/s11064-019-02746-z

Figure Lengend Snippet: Effect of 10 nM and 1 µM of the VGVAPG peptide on DCF fluorescence (ROS measurement) in astrocytes; a after 6 h and b after 24. White bars represent cells transfected by scrambled siRNA, black bars represent cells transfected by Glb1 siRNA. Each point represents the mean ± SD of three independent experiments, each of which consisted of six replicates per treatment group. *P < 0.05, **P < 0.01, versus the control cultures

Article Snippet: Glb1 gene siRNA (sc-61342) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Fluorescence, Transfection, Control

Journal: Neurochemical Research

Article Title: The VGVAPG Peptide Regulates the Production of Nitric Oxide Synthases and Reactive Oxygen Species in Mouse Astrocyte Cells In Vitro

doi: 10.1007/s11064-019-02746-z

Figure Lengend Snippet: Effect of 10 nM and 1 µM of the VGVAPG peptide on DAF-FM fluorescence (NO measurement) in astrocytes; a after 6 h and b after 24. White bars represent cells transfected by scrambled siRNA, black bars represent cells transfected by Glb1 siRNA. Each point represents the mean ± SD of three independent experiments, each of which consisted of six replicates per treatment group. ***P < 0.001, versus the control cultures

Article Snippet: Glb1 gene siRNA (sc-61342) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Fluorescence, Transfection, Control

Journal: Neurochemical Research

Article Title: The VGVAPG Peptide Regulates the Production of Nitric Oxide Synthases and Reactive Oxygen Species in Mouse Astrocyte Cells In Vitro

doi: 10.1007/s11064-019-02746-z

Figure Lengend Snippet: Effect of 10 nM and 1 µM of the VGVAPG peptide on mRNA expression of nNos ( a ), iNos ( b ) and eNos ( c ) after 6 h of exposure. White bars represent cells transfected by scrambled siRNA, black bars represent cells transfected by Glb1 siRNA. mRNA expression was normalised to the Actb gene. Data are expressed as means ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle control

Article Snippet: Glb1 gene siRNA (sc-61342) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Control

Journal: Neurochemical Research

Article Title: The VGVAPG Peptide Regulates the Production of Nitric Oxide Synthases and Reactive Oxygen Species in Mouse Astrocyte Cells In Vitro

doi: 10.1007/s11064-019-02746-z

Figure Lengend Snippet: Effect of 10 nM and 1 µM of the VGVAPG peptide on protein expression of nNos ( a ), iNos ( b ) and eNos ( c ) after 24 h of exposure. White bars represent cells transfected by scrambled siRNA, black bars represent cells transfected by Glb1 siRNA. Protein expression was normalised to the total protein level. Data are expressed as means ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle control

Article Snippet: Glb1 gene siRNA (sc-61342) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Control

Journal: IUBMB life

Article Title: B4GALT1 gene knockdown inhibits the hedgehog pathway and reverses multidrug resistance in the human leukemia K562/adriamycin-resistant cell line.

doi: 10.1002/iub.1080

Figure Lengend Snippet: Figure 2. b1, 4-Gal-T1 activity is determined with a phosphatase-coupled method. The product formation was calculated by using the conversion factor determined from the phosphate standard curve (A, B). Enzymatic activity was lower in K562 cells, whereas enhanced in K562/ADR cells. A reduction of enzyme activity was shown in K562/ADR cells treated with shRNA, compared with that in K562/ADR cells (C). Asterisk and hash signs indicate significant difference from the groups without signs (P \ 0.05).

Article Snippet: Then, cells were transfected with 0.4 lM B4GALT1-specific shRNA lentiviral particles (Santa Cruz Biotech, Santa Cruz, CA sc-40616-V) according to the manufacturer’s instruction.

Techniques: Activity Assay, shRNA

Journal: IUBMB life

Article Title: B4GALT1 gene knockdown inhibits the hedgehog pathway and reverses multidrug resistance in the human leukemia K562/adriamycin-resistant cell line.

doi: 10.1002/iub.1080

Figure Lengend Snippet: Figure 3. FITC–RCA120-binding profiles of four cell groups show differences. Histograms of fluorescence intensities of cells with the exposed galactosyl residues were determined by flow cytometry by using FITC-RCA120-lectin, the binding to four types of cells followed the same tendency with the change of enzyme activity, less intensely in K562 cells, increased in K562/ADR cells, and downregulated in K562/ADR cells treated with shRNA. The data represented an average of three independent experiments (A, B). Asterisk and hash signs indicate significant difference from the groups without signs (P \ 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Then, cells were transfected with 0.4 lM B4GALT1-specific shRNA lentiviral particles (Santa Cruz Biotech, Santa Cruz, CA sc-40616-V) according to the manufacturer’s instruction.

Techniques: Binding Assay, Cytometry, Activity Assay, shRNA

Journal: IUBMB life

Article Title: B4GALT1 gene knockdown inhibits the hedgehog pathway and reverses multidrug resistance in the human leukemia K562/adriamycin-resistant cell line.

doi: 10.1002/iub.1080

Figure Lengend Snippet: Figure 5. B4GALT silencing affects antitumor activity of MDR- related drugs in vivo. Nude mice bearing four types of tumors were administrated adriamycin (7 mg/kg) or normal saline for 3 weeks. (A, B) Mean tumor weight was lower in the K562 mice group with adriamycin, compared with that in the K562 mice group without adriamycin (P \ 0.05). Mean tumor weight was increased in K562/ADR mice group, compared with that in K562 group, when exposed to adriamycin (P \ 0.01). A decrease of mean tumor weight in mice group with B4GALT1 shRNA tumors was observed, as compared with that in K562/ADR group and K562/ADR–control shRNA group (P \ 0.05). Within K562/ ADR-B4GALT1 shRNA group, an increase of tumor growth was found in group without adriamycin, compared to that with adria- mycin (P \ 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Then, cells were transfected with 0.4 lM B4GALT1-specific shRNA lentiviral particles (Santa Cruz Biotech, Santa Cruz, CA sc-40616-V) according to the manufacturer’s instruction.

Techniques: Activity Assay, In Vivo, Saline, shRNA, Control

Journal: IUBMB life

Article Title: B4GALT1 gene knockdown inhibits the hedgehog pathway and reverses multidrug resistance in the human leukemia K562/adriamycin-resistant cell line.

doi: 10.1002/iub.1080

Figure Lengend Snippet: Figure 6. B4GALT1 knockdown inhibits the activity of Hh signaling pathway. With real-time PCR and Western blot analysis, the main signal molecules of Hh pathway, including Shh, Smo, ptch, and GLI-1, were found to be downregulated at both gene (A) and protein level (B, C) in K562/ADR cells with B4GALT1 shRNA, as compared with those in K562/ADR cells (*P \ 0.05 vs. K562/ ADR cells and K562/ADR cells with control shRNA).

Article Snippet: Then, cells were transfected with 0.4 lM B4GALT1-specific shRNA lentiviral particles (Santa Cruz Biotech, Santa Cruz, CA sc-40616-V) according to the manufacturer’s instruction.

Techniques: Knockdown, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, shRNA, Control

Journal: IUBMB life

Article Title: B4GALT1 gene knockdown inhibits the hedgehog pathway and reverses multidrug resistance in the human leukemia K562/adriamycin-resistant cell line.

doi: 10.1002/iub.1080

Figure Lengend Snippet: Figure 7. The protein levels of B4GALT1 and Shh signaling molecules were determined by immunohistochemistry staining in xen- ograft tumors derived from K562, K562/ADR, K562/ADR–control shRNA, or K562/ADR–B4GALT1 shRNA cells (4003). Differ- ential protein levels of B4GALT1, Shh, and GLI-1 were shown from three independent experiments (#, P \ 0.05 vs. K562/ADR cells; *, P \ 0.05 vs. K562/ADR cells and K562/ADR cells with control shRNA). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Then, cells were transfected with 0.4 lM B4GALT1-specific shRNA lentiviral particles (Santa Cruz Biotech, Santa Cruz, CA sc-40616-V) according to the manufacturer’s instruction.

Techniques: Immunohistochemistry, Staining, Derivative Assay, Control, shRNA

Journal: IUBMB life

Article Title: B4GALT1 gene knockdown inhibits the hedgehog pathway and reverses multidrug resistance in the human leukemia K562/adriamycin-resistant cell line.

doi: 10.1002/iub.1080

Figure Lengend Snippet: Figure 8. Suppression of the activity of the Hh pathway influences drug resistance of K562/ADR cells. (A) The levels of GLI-1 and Shh were deregulated following transfection of GLI-1 siRNA in K562/ADR cells by real-time PCR. (B) MTT assays were used to evaluate the effect of paclitaxel, vincristine, and adriamycin on the survival of four cell lines. A significant enhancement in the chemosensitivity of K562/ADR–GLI-1-siRNA cells group was observed, compared with that in K562/ADR cells group and K562/ADR–control siRNA cells group (#, P \ 0.001 vs. K562/ADR cells; *, P \ 0.05 vs. K562/ADR cells and K562/ADR cells with control siRNA).

Article Snippet: Then, cells were transfected with 0.4 lM B4GALT1-specific shRNA lentiviral particles (Santa Cruz Biotech, Santa Cruz, CA sc-40616-V) according to the manufacturer’s instruction.

Techniques: Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Control