Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of matrix metalloproteinase expression by selective clearing of senescent dermal fibroblasts attenuates ultraviolet-induced photoaging.

doi: 10.1016/j.biopha.2022.113034

Figure Lengend Snippet: Fig. 2. Selective elimination of senescent cells in UV-irradiated mice skin after treatment with ABT-263 or ABT-737. Hairless mice were exposed to chronic UV irradiation for eight weeks with 100 mJ/cm2 for the first 2 weeks, then 50 mJ/cm2 weekly, then 300 mJ/cm2. Each drug (ABT-263, 5 μM; ABT-737, 10 μM) or vehicle (DMSO) was diluted in PBS (30 μl) and injected intradermally into the dorsal skin for a total of ten doses. (A) Representative images of SA-β-gal staining in mice skin. SA-β-gal (blue color) was detected in 6 µm frozen skin sections from unirradiated control mice injected with vehicle (n = 7, white bars), UVB-irradiated mice injected with vehicle (n = 4, black bars), UVB-irradiated mice injected with ABT-263 (n = 6, blue bars) or ABT-737 (n = 7, red bars). Arrowheads indicate stained cells. (B) Quantitative analysis of the percentage of SA-β-gal positive cells in the indicated mice skin (n = 3–4 mice per group) (C) Western blots and quantification of p21 and p16 protein levels normalized to GAPDH using protein extracts from the skin. (D) p16 and p21 mRNA levels in indicated mice skin, standardized to GAPDH. (E) Representative images of immunofluorescence staining for p16 (red) and DAPI (blue) in the skin of the indicated mice. Right: Quan tification of the percentage of p16-positive cells among DAPI-positive cells in over n = 4 mice per group. Arrowheads indicate stained cells. Data represent means ± SEM, *p < 0.05, * *p < 0.005, * **p < 0.001. p-values were calculated by Student’s t-test. A, Bar = 100 µm; E, Bar = 50 µm.

Article Snippet: The antibodies used included p16 (MA5–17142, Thermo Fisher Scientific; ab189034, Abcam), p21 (556431, BD Pharmingen; ab109199, Abcam), GAPDH (CSB-PA00029A0Rb, Cusabio, Houston, TX, USA), β-actin (CSBPA000350, Cusabio), MMP-1 (Lab Frontier, Seoul, Korea), and type I procollagen (SP1.

Techniques: Irradiation, Injection, Staining, Control, Western Blot, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Abnormal Paraplegin Expression in Swollen Neurites, τ- and α-Synuclein Pathology in a Case of Hereditary Spastic Paraplegia SPG7 with an Ala510Val Mutation

doi: 10.3390/ijms161025050

Figure Lengend Snippet: Pedigree ( a ) and electropherograms ( b ) of the p.Ala510Val SPG7 family. The p.Ala510Val SPG7 variant was observed in a homozygous state in both affected subjects for whom DNA was available, namely in the index patient (arrow) and his sister. She was similarly affected with progressive spastic-ataxic gait disorder and cPEO, starting at age 50 years. The unaffected children of the index patient carried this mutation in a heterozygous state. Pedigree symbols for a : squares: male; circle: female; diamond: gender not specified; black filled symbols: affected by disease; white symbols: healthy; symbols cross by a line: deceased. Color codes for b : G (guanin): black, T (thymine): red, C (cytosine): blue, A (adenine): green.

Article Snippet: In addition immunohistochemistry with antibodies directed against abnormal τ-protein (AT-8, Thermo-Scientific—Pierce Biotechnology, Rockford, IL, USA, 1/1000) three-repeat τ (3rp-τ; RD3, 8E6/C11, Millipore, Temecula, CA, USA, 1/500, formic acid and microwave pretreatment), four-repeat τ (4rp-τ; RD4, 1E1/A6, Millipore, Temecula, CA, USA, 1/1000, formic acid and microwave pretreatment), α-synuclein (KM51, Leica Biosystems—Novocastra, Newcastle, UK, 1/40, formic acid pretreatment), amyloid β-protein (Aβ) (4G8, Covance, Dedham, MA, USA, 1/5000, formic acid pretreatment), p62 (clone number 3/p62 LCK ligand, BD Transduction Laboratories, Mountain View, CA, USA, 1:500), TDP43 (clone 2E2-D3, Novus Biologicals, Littleton, CO, USA, 1:2000, formic acid and microwave pretreatment), paraplegin (polyclonal rabbit, Acris Antibodies, San Diego, CA, USA, 1/100), and the 68 kDa subunit of neurofilaments (NF-L, SPM 204, Zytomed, 1/100, microwave pretreatment; 24 h at 22 °C) was performed.

Techniques: Variant Assay, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Abnormal Paraplegin Expression in Swollen Neurites, τ- and α-Synuclein Pathology in a Case of Hereditary Spastic Paraplegia SPG7 with an Ala510Val Mutation

doi: 10.3390/ijms161025050

Figure Lengend Snippet: Moderate neuron loss and gliosis in the inferior olivary nucleus (ION) (arrows in a , b ) and in the dentate nucleus (dentate nucl.) of the SPG7 case (arrows in c , d ); a , c show overviews of the ION ( a ) and the dentate nucleus ( c ), whereas at increased magnification ( b , d ) a moderately reduced neuron frequency could be observed when comparing with control cases as depicted for control case number three ( e , f ). Arrows in e , f indicate normal neuron densities in ION ( e ) and the dentate nucleus ( f ). Calibration bar in f valid for a : 320 µm; c : 710 µm; b , d – f : 130 µm.

Article Snippet: In addition immunohistochemistry with antibodies directed against abnormal τ-protein (AT-8, Thermo-Scientific—Pierce Biotechnology, Rockford, IL, USA, 1/1000) three-repeat τ (3rp-τ; RD3, 8E6/C11, Millipore, Temecula, CA, USA, 1/500, formic acid and microwave pretreatment), four-repeat τ (4rp-τ; RD4, 1E1/A6, Millipore, Temecula, CA, USA, 1/1000, formic acid and microwave pretreatment), α-synuclein (KM51, Leica Biosystems—Novocastra, Newcastle, UK, 1/40, formic acid pretreatment), amyloid β-protein (Aβ) (4G8, Covance, Dedham, MA, USA, 1/5000, formic acid pretreatment), p62 (clone number 3/p62 LCK ligand, BD Transduction Laboratories, Mountain View, CA, USA, 1:500), TDP43 (clone 2E2-D3, Novus Biologicals, Littleton, CO, USA, 1:2000, formic acid and microwave pretreatment), paraplegin (polyclonal rabbit, Acris Antibodies, San Diego, CA, USA, 1/100), and the 68 kDa subunit of neurofilaments (NF-L, SPM 204, Zytomed, 1/100, microwave pretreatment; 24 h at 22 °C) was performed.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Abnormal Paraplegin Expression in Swollen Neurites, τ- and α-Synuclein Pathology in a Case of Hereditary Spastic Paraplegia SPG7 with an Ala510Val Mutation

doi: 10.3390/ijms161025050

Figure Lengend Snippet: Distribution of neuron loss and astrogliosis, neuritic pathology as detected by anti-paraplegin or anti-NF-L immunohistochemistry, NFT, τ-, and α-synuclein-pathology.

Article Snippet: In addition immunohistochemistry with antibodies directed against abnormal τ-protein (AT-8, Thermo-Scientific—Pierce Biotechnology, Rockford, IL, USA, 1/1000) three-repeat τ (3rp-τ; RD3, 8E6/C11, Millipore, Temecula, CA, USA, 1/500, formic acid and microwave pretreatment), four-repeat τ (4rp-τ; RD4, 1E1/A6, Millipore, Temecula, CA, USA, 1/1000, formic acid and microwave pretreatment), α-synuclein (KM51, Leica Biosystems—Novocastra, Newcastle, UK, 1/40, formic acid pretreatment), amyloid β-protein (Aβ) (4G8, Covance, Dedham, MA, USA, 1/5000, formic acid pretreatment), p62 (clone number 3/p62 LCK ligand, BD Transduction Laboratories, Mountain View, CA, USA, 1:500), TDP43 (clone 2E2-D3, Novus Biologicals, Littleton, CO, USA, 1:2000, formic acid and microwave pretreatment), paraplegin (polyclonal rabbit, Acris Antibodies, San Diego, CA, USA, 1/100), and the 68 kDa subunit of neurofilaments (NF-L, SPM 204, Zytomed, 1/100, microwave pretreatment; 24 h at 22 °C) was performed.

Techniques: Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Abnormal Paraplegin Expression in Swollen Neurites, τ- and α-Synuclein Pathology in a Case of Hereditary Spastic Paraplegia SPG7 with an Ala510Val Mutation

doi: 10.3390/ijms161025050

Figure Lengend Snippet: Paraplegin expression in the frontal neocortex of SPG7 case ( a ) and of a non-diseased control (case No. 2) ( b ); ( c ) depicts the negative control for the SPG7 case by omitting the primary antibody to document the specificity of the immunostainings. Despite an overall increased staining intensity in the SPG7 case that can be explained by varying staining intensities, neurons were slightly stained in both the SPG7 case and in the control (arrowheads in a , b ), whereas neurites exhibited paraplegin only in the SPG7 case (arrows in a ) and not in the healthy control brain. Similarly, neurites exhibit paraplegin in the Purkinje cell layer of the cerebellum in the SPG7 case (arrows in d ) but not in the control (case number four) ( e ); Here only the perikarya of the Purkinje cells were labeled mildly ( e ); The presence of paraplegin positive neurites in the cerebellum was associated with high numbers of neurites accumulating 68 kDa neurofilaments. Some of these neurites appear swollen (arrows in f ); In the control (case number four) there was no neuritic accumulation of the 68 kDa neurofilament protein ( g ); Calibration bar in c valid for a – c : 35 µm; d , e : 40 µm; f , g : 50 µm.

Article Snippet: In addition immunohistochemistry with antibodies directed against abnormal τ-protein (AT-8, Thermo-Scientific—Pierce Biotechnology, Rockford, IL, USA, 1/1000) three-repeat τ (3rp-τ; RD3, 8E6/C11, Millipore, Temecula, CA, USA, 1/500, formic acid and microwave pretreatment), four-repeat τ (4rp-τ; RD4, 1E1/A6, Millipore, Temecula, CA, USA, 1/1000, formic acid and microwave pretreatment), α-synuclein (KM51, Leica Biosystems—Novocastra, Newcastle, UK, 1/40, formic acid pretreatment), amyloid β-protein (Aβ) (4G8, Covance, Dedham, MA, USA, 1/5000, formic acid pretreatment), p62 (clone number 3/p62 LCK ligand, BD Transduction Laboratories, Mountain View, CA, USA, 1:500), TDP43 (clone 2E2-D3, Novus Biologicals, Littleton, CO, USA, 1:2000, formic acid and microwave pretreatment), paraplegin (polyclonal rabbit, Acris Antibodies, San Diego, CA, USA, 1/100), and the 68 kDa subunit of neurofilaments (NF-L, SPM 204, Zytomed, 1/100, microwave pretreatment; 24 h at 22 °C) was performed.

Techniques: Expressing, Negative Control, Staining, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Abnormal Paraplegin Expression in Swollen Neurites, τ- and α-Synuclein Pathology in a Case of Hereditary Spastic Paraplegia SPG7 with an Ala510Val Mutation

doi: 10.3390/ijms161025050

Figure Lengend Snippet: ( a ) Lewy-bodies (arrows) and Lewy neurites (arrowhead) are detected in the locus coeruleus with the anti-α-synunclein (α-syn) antibody; ( b , c ) NFTs are detected in the neurons of the substantia nigra with an antibody directed against abnormal phosphorylated τ-protein (arrow in b ) as well as with the Gallyas silver method (arrow in c ). Neuropil threads were also detected (arrowhead in c ); ( d , e ) the abnormal τ-protein aggregates in the substantia nigra nerve cells (arrowhead in d ) and in neuropil threads contained 4rp τ (arrows in d ) but no 3rp τ ( e ); Lewy bodies (red arrows in d ) occurring inside τ-positive NFTs (arrowhead in d ) did not exhibit immunoreactivity with anti-τ antibodies such as anti-rp4 τ; ( f ) coiled bodies were detected with an antibody raised against abnormal phosphorylated τ-protein in the globus pallidum (arrows); ( g ) neuronal silver-stained inclusions (arrow) as well as neuropil threads (arrowheads) also occurred in the cerebellar dentate nucleus; ( h ) single glial inclusions with a tufted astrocyte-like pattern were detected in the putamen by Gallyas silver staining; ( i ) NFTs (arrowhead) and LBs (large and small arrows) were also detectable with an antibodies directed against p62 as documented in the substantia nigra; ( j ) Although paraplegin was detected in neurites (arrowheads) and neurons of the substantia nigra (arrow), Lewy bodies were not specifically labeled (red arrow); ( k ) neuritic accumulation of paraplegin was also observed in the dentate nucleus; and ( l ) antibodies against the 68 kDa subunit of neurofilaments also showed some neurites with neurofilament accumulation. Calibration bar in f valid for a – f , i – l : 30 µm; g , h : 15 µm.

Article Snippet: In addition immunohistochemistry with antibodies directed against abnormal τ-protein (AT-8, Thermo-Scientific—Pierce Biotechnology, Rockford, IL, USA, 1/1000) three-repeat τ (3rp-τ; RD3, 8E6/C11, Millipore, Temecula, CA, USA, 1/500, formic acid and microwave pretreatment), four-repeat τ (4rp-τ; RD4, 1E1/A6, Millipore, Temecula, CA, USA, 1/1000, formic acid and microwave pretreatment), α-synuclein (KM51, Leica Biosystems—Novocastra, Newcastle, UK, 1/40, formic acid pretreatment), amyloid β-protein (Aβ) (4G8, Covance, Dedham, MA, USA, 1/5000, formic acid pretreatment), p62 (clone number 3/p62 LCK ligand, BD Transduction Laboratories, Mountain View, CA, USA, 1:500), TDP43 (clone 2E2-D3, Novus Biologicals, Littleton, CO, USA, 1:2000, formic acid and microwave pretreatment), paraplegin (polyclonal rabbit, Acris Antibodies, San Diego, CA, USA, 1/100), and the 68 kDa subunit of neurofilaments (NF-L, SPM 204, Zytomed, 1/100, microwave pretreatment; 24 h at 22 °C) was performed.

Techniques: Staining, Silver Staining, Labeling