Journal: International Journal of Molecular Sciences

Article Title: Maackiain Ameliorates 6-Hydroxydopamine and SNCA Pathologies by Modulating the PINK1/Parkin Pathway in Models of Parkinson’s Disease in Caenorhabditis elegans and the SH-SY5Y Cell Line

doi: 10.3390/ijms21124455

Figure Lengend Snippet: Maackiain (MK) significantly diminished the intracellular reactive oxygen species (ROS) level and increased pink1 and pdr-1 expression in 6-hydroxydopamine (6-OHDA)-exposed N2 C. elegans . MK-pretreated or untreated L3 stage worms were exposed to 6-OHDA for 1 h and were then cultured in the NGM plate for 3 days. ( A ) Thirty randomly selected worms from each experimental group were transferred to the well of a 96-well plate. The intracellular ROS level was evaluated. # shows significant differences between 6-OHDA-exposed and control worms (# p < 0.01); * shows significant differences between the MK-untreated 6-OHDA-exposed worms and MK-pretreated 6-OHDA-exposed worms (** p < 0.01). ( B ) The expression level of PD-associated genes in C. elegans was quantified by qPCR. # shows significant differences between 6-OHDA-exposed and control worms (# p < 0.05); * shows significant differences between the MK-untreated 6-OHDA-exposed worms and MK-pretreated 6-OHDA-exposed worms (* p < 0.05, ** p < 0.01).

Article Snippet: Monoclonal antibodies to PINK1, parkin, and β-tubulin and HRP goat anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Maackiain Ameliorates 6-Hydroxydopamine and SNCA Pathologies by Modulating the PINK1/Parkin Pathway in Models of Parkinson’s Disease in Caenorhabditis elegans and the SH-SY5Y Cell Line

doi: 10.3390/ijms21124455

Figure Lengend Snippet: Parkin-siRNA reversed the anti-apoptotic effect of maackiain (MK) in the 6-hydroxydopamine (6-OHDA)-exposed SH-SY5Y cell line. SH-SY5Y cells were transfected with control siRNA or parkin-siRNA for 24 h. Transfected cells were incubated with 1 μM MK for 24 h and then exposed to 100 μM 6-OHDA for 18 h. ( A ) The loss in mitochondrial membrane potential (MMP) was observed by using DiOC6 dye (1 μM). Top, representative phase contrast images and fluorescent images; bottom, the fluorescence intensity of DiOC6 was analyzed by using ImageJ. The relative fold fluorescence intensity is represented as the ratio relative to the control experiment. ( B ) Nuclear condensation was observed by using Hoechst 33258 staining. Top, representative phase contrast images and fluorescent images; bottom, the fluorescence intensity of Hoechst 33258 (5 μg/mL) was analyzed by using ImageJ. The relative fold fluorescence intensity is represented as the ratio relative to the control experiment. ( C ) Western blotting analysis was used to quantify the protein level of PINK1 and parkin. One representative result is shown. The expression of β-tubulin was used as an internal control. The relative fold protein level is represented as the ratio relative to the control experiment. In the above experiments, # shows significant differences between 6-OHDA-exposed and control groups (# p < 0.05, ## p < 0.01, ### p < 0.001); * shows significant differences between the MK-pretreated 6-OHDA-exposed and MK-untreated 6-OHDA-exposed groups (* p < 0.05, ** p < 0.01).

Article Snippet: Monoclonal antibodies to PINK1, parkin, and β-tubulin and HRP goat anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Transfection, Control, Incubation, Membrane, Fluorescence, Staining, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Maackiain Ameliorates 6-Hydroxydopamine and SNCA Pathologies by Modulating the PINK1/Parkin Pathway in Models of Parkinson’s Disease in Caenorhabditis elegans and the SH-SY5Y Cell Line

doi: 10.3390/ijms21124455

Figure Lengend Snippet: Parkin-siRNA reversed the clearance ability of maackiain (MK) in the α-synuclein (α-Syn)-overexpressing SH-SY5Y cell line. α-Syn-overexpressing SH-SY5Y cells were transfected with control siRNA or parkin-siRNA for 24 h. Transfected Cells were incubated with 1 μM MK for 24 h. ( A ) A representative fluorescence image of α-Syn-overexpressing SH-SY5Y Cells. Recombinant α-Syn was detected by Myc antibody (Green). Hoechst 33258 was used as a marker of nuclear morphology (blue). ( B ) Suc-Leu-Leu-Val-Tyr-AMC was used as a substrate to measure proteasome activity. The relative fold proteasome activity is represented as the ratio relative to the control experiment. ( C ) Autophagic vacuoles were observed by using acidic vesicular organelle (AVO) staining. Top, representative phase contrast images and fluorescent images; bottom, the fluorescence intensity of acridine orange (0.5 μg/mL) was analyzed by using ImageJ. The relative fold fluorescence intensity is represented as the ratio relative to the control experiment. ( D ) Western blotting analysis was used to quantify the protein level of PINK1 and parkin. One representative result is shown. The expression of β-tubulin was used as an internal control. The relative fold protein level is represented as the ratio relative to the control experiment. In the above experiments, # shows significant differences between α-Syn-overexpressing and control (## p < 0.01, ### p < 0.001); * shows significant differences between the MK-treated α-Syn-overexpressing and MK-untreated α-Syn-overexpressing experiments (** p < 0.01, *** p < 0.001).

Article Snippet: Monoclonal antibodies to PINK1, parkin, and β-tubulin and HRP goat anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Transfection, Control, Incubation, Fluorescence, Recombinant, Marker, Activity Assay, Staining, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Maackiain Ameliorates 6-Hydroxydopamine and SNCA Pathologies by Modulating the PINK1/Parkin Pathway in Models of Parkinson’s Disease in Caenorhabditis elegans and the SH-SY5Y Cell Line

doi: 10.3390/ijms21124455

Figure Lengend Snippet: Primers for real time PCR.

Article Snippet: Monoclonal antibodies to PINK1, parkin, and β-tubulin and HRP goat anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques:

Journal: The FASEB Journal

Article Title: Tmx2 Maintains Mitochondrial Function to Support Preimplantation Embryogenesis

doi: 10.1096/fj.202500640r

Figure Lengend Snippet: FIGURE 6 | Tmx2 knockdown inhibits mitophagy and autophagy in morula-stage embryos. (A-D) Immunostaining for PINK1 (A), PARKIN (B), MAP1LC3B (C), and LAMP1 (D) in control and Tmx2-knockdown embryos. Scale bars: 75 μm. (E-H) Quantification of immunofluorescence inten- sity for PINK1 (E), PARKIN (F), MAP1LC3B (G), and LAMP1 (H) in control and Tmx2-knockdown morula-stage embryos. Sample sizes: PINK1: Control = 16, siRNA2 = 14, siRNA3 = 19; PARKIN: Control = 21, siRNA2 = 20, siRNA3 = 23; MAP1LC3B: Control = 22, siRNA2 = 20, siRNA3 = 21; LAMP1: Control = 30, siRNA2 = 32, siRNA3 = 31. Statistical significance: **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Article Snippet: The primary antibodies used included: rabbit anti- TMX2 (Origene, TA341391, 1:50), goat anti- OCT4 (Abcam, ab27985, 1:200), mouse antiCDX2 (Biogenex, MU392A- UC, 1:100), goat anti- SOX17 (R&D Systems, AF1924, 1:100), rabbit anti- NANOG (Abcam, ab80892, 1:50), rabbit anti- PINK1 (Affinity, DF7742, 1:50), rabbit antiPARKIN (Abcepta, AP6402B, 1:50), rabbit anti- MAP1LC3B (CUSABIO, CSB- PA013403GA01HU, 1:50), and rabbit antiLAMP1 (Beyotime, AF7353, 1:100).

Techniques: Knockdown, Immunostaining, Control, Immunofluorescence