Journal: International Journal of Molecular Sciences

Article Title: Spreading of Isolated Ptch Mutant Basal Cell Carcinoma Precursors Is Physiologically Suppressed and Counteracts Tumor Formation in Mice

doi: 10.3390/ijms21239295

Figure Lengend Snippet: Primary and secondary antibodies used for immunohistochemical or immunofluorescent stainings of individual isolated hair follicles, in vitro cultured cells and paraffine or cryotome sections.

Article Snippet: K5 , rb anti-K5 , m/h , Bio Legend, San Diego, CA, USA (Poly19055) , citric acid, pH6 , 1:1000 , d anti-rb-Alexa488 (1:200; Jackson ImmunoResearch; #711-545-152) d anti-rb-Cy3 (1:400; Jackson ImmunoResearch; #711-165-152).

Techniques: Immunohistochemical staining, Isolation, In Vitro, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α

doi: 10.1016/j.jbc.2022.102531

Figure Lengend Snippet: p38MAPK inhibition by SB203580 increases αSyn turnover and lowers αSyn secretion and toxicity in cells expressing p25α. A , NGF-differentiated PC12-αSyn/p25α cells were treated with SB203580 as indicated with doxycycline (to induce αSyn and p25α transgenes) for 2 days before Western blot analysis of αSyn in TCA-precipitated conditioned medium and actin and αSyn in the lysate fraction (ordinary one-way ANOVA, N = 4–6). B , PC12-αSyn/p25α cell cultures received SB203580 either concurrently with doxycycline treatment (concurrently) or 24 h after doxycycline induction (post). After 2 days, conditioned medium was analyzed for lactate dehydrogenase (LDH) to assess cell death (Kruskal–Wallis test, N = 4). C , αSyn and p25α expression was induced in PC12-αSyn/p25α cells by doxycycline treatment for 24 h with/without concurrent SB203580 administration followed by doxycycline washout. Cells were chased for a further 4 days with/without SB203580 with sampling of replicate wells each day for analysis of intracellular αSyn by Western blotting. The Western blot shows a representative experiment, whereas graphs represent αSyn normalized to actin Western blot band absorbance relative to D1 values (two-way ANOVA, N = 3). D , NGF-differentiated PC12 cells expressing αSyn or αSyn/p25α were treated or not with SB203580 at 1 or 2 μM for 2 days of transgene expression and then lysates were analyzed by Western blotting for total and phosphorylated p38MAPK and actin. The graphs show mean integrated absorbance of Western blot bands for the ( D ) ratio of phosphorylated to total p38MAPK ( i.e. , specific activity) for a SB203580 concentration of 2 μM, the ratio being derived from the absorbances of ( E ) total p38MAPK and ( F ) p-p38MAPK bands (Kruskal–Wallis test, N = 4). All graphs show mean ± SEM. αSyn, α-synuclein; NGF, nerve growth factor; TCA, trichloroacetic acid.

Article Snippet: G , differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α and then analyzed as aforementioned except anti-p38MAPK-α antibody (StressMarq Biosciences; SMC-152D) was used.

Techniques: Inhibition, Expressing, Western Blot, Sampling, Activity Assay, Concentration Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α

doi: 10.1016/j.jbc.2022.102531

Figure Lengend Snippet: shRNA knockdown of p38MAPK-α reproduces the effects of SB203580 treatment. A , differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α (one shRNA) and p38MAPK-γ isoforms (two shRNAs), incubated with doxycycline for 2 days, and subsequently lysed and Western blotted for expression of p38MAPK isoforms (using anti-p38MAPKα antibody 9212 from Cell Signaling) or phosphorylated p-p38MAPK. Cropped lanes are derived from the same membrane. B , the same cells were analyzed by Western blotting for expression of αSyn in the conditioned medium, and αSyn, LC3, and p62 in the lysate. C – E , quantitation of Western blot band absorbances from aforementioned experiment for double p38MAPK-α and p38MAPK-γ knockdown for ( C ) αSyn in conditioned medium, or ( D ) αSyn, and ( E ) LC3-II in the lysate fraction. (Kruskal–Wallis test, N = 4–6). F , LDH release from the same cells (ordinary one-way ANOVA, N = 4). G , differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α and then analyzed as aforementioned except anti-p38MAPK-α antibody (StressMarq Biosciences; SMC-152D) was used. Cropped lanes are derived from the same membrane. H and I , the graphs show quantitation of levels of p38MAPK-α isoform (unpaired t test, N = 4) and active (phosphorylated) p38MAPK (one-sample t test, N = 4). J , secretion of αSyn to the medium (unpaired t test, N = 3) as performed in ( G ). K , LDH release to the medium (unpaired t test, N = 4). All graphs show mean ± SEM. αSyn, α-synuclein; LDH, lactate dehydrogenase.

Article Snippet: G , differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α and then analyzed as aforementioned except anti-p38MAPK-α antibody (StressMarq Biosciences; SMC-152D) was used.

Techniques: shRNA, Knockdown, Transduction, Expressing, Incubation, Western Blot, Derivative Assay, Membrane, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α

doi: 10.1016/j.jbc.2022.102531

Figure Lengend Snippet: SB203580 decreases p-ser129 αSyn staining in primary neurons. A , primary mouse cortical neurons expressing transgene human αSyn were treated with 1 μg/ml αSyn fibrils ±1 or 5 μM SB203580 at DIV5 in the further presence or not of lysosomal inhibitors leupeptin, pepstatin, and E64 (LPE) and harvested DIV7. B and C , neuronal cultures were fixed and stained with primary antibodies for ( B ) p-Ser129 αSyn and ( C ) Hoechst (to assess live nuclei) for quantitation by Cellomics Array Scanner analysis (ordinary one-way ANOVA, N = 3). D , representative spinning disc confocal microscopy images of neuronal cultures stained for p-Ser129 ( green ), Map2 ( red ), and Hoechst ( gray ) from a Cellomics experiment as aforementioned; bars represent 32 μm. E , cell lysates prepared from DIV7 neuronal cultures, treated with SB203580 and/or LPE in the absence of αSyn fibrils as indicated, were Western blotted with anti-LC3, anti-p62, anti-p-p38MAPK, or anti-actin antibodies as shown. F – H , quantitation of aforementioned immunoblots was performed for ( F ) LC3-II, ( G ) p62/SQSTM1, and ( H ) p-p38MAPK (without αSyn fibrils) (ordinary one-way ANOVA for F and G and Kruskal–Wallis test for H , N = 3). All graphs show mean ± SEM. αSyn, α-synuclein; DIV, days in vitro .

Article Snippet: G , differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α and then analyzed as aforementioned except anti-p38MAPK-α antibody (StressMarq Biosciences; SMC-152D) was used.

Techniques: Staining, Expressing, Quantitation Assay, Confocal Microscopy, Western Blot, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α

doi: 10.1016/j.jbc.2022.102531

Figure Lengend Snippet: αSyn degradation under basal conditions and p38MAPK inhibition. Under basal conditions αSyn is degraded by multiple mechanisms first and foremost by macroautophagy and Ndfip1-mediated internalization of αSyn; however, chaperone-mediated autophagy (LAMP2a and Hsc70) also contributes. When p25α-mediated p38MAPK activation is opposed pharmacologically or genetically, the majority of αSyn is turned over in an ESCRT-dependent process relying on Hsc70 and TSG101, and αSyn degradation commences in late endosomes. Under basal conditions, lysosome fusion with autophagosomes and amphisomes (the fusion product of an autophagosome with a late endosome) is partially blocked by p25α, which results in their exocytosis and release of αSyn. In contrast, under conditions of p38MAPK inhibition, ESCRT-dependent αSyn import and degradation begins in the late endosome, and the endosomal pathway runs to completion to deliver αSyn to lysosomes for degradation. αSyn, α-synuclein; ESCRT, endosomal sorting complex required for transport; LAMP, lysosome-associated membrane protein.

Article Snippet: G , differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing shRNA to p38MAPK-α and then analyzed as aforementioned except anti-p38MAPK-α antibody (StressMarq Biosciences; SMC-152D) was used.

Techniques: Inhibition, Activation Assay, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Spreading of Isolated Ptch Mutant Basal Cell Carcinoma Precursors Is Physiologically Suppressed and Counteracts Tumor Formation in Mice

doi: 10.3390/ijms21239295

Figure Lengend Snippet: Primary and secondary antibodies used for immunohistochemical or immunofluorescent stainings of individual isolated hair follicles, in vitro cultured cells and paraffine or cryotome sections.

Article Snippet: RFP * , rb anti-RFP , , Rockland Immunochemicals, Inc., Gilbertsville, PA, USA (600-401-379S) , boric acid or citric acid, pH6 , 1:500 , d anti-rb-Cy3 (1:400; Jackson ImmunoResearch; #711-165-152) g anti-rb-Cy3 (1:400; Jackson ImmunoResearch; #111-165-003).

Techniques: Immunohistochemical staining, Isolation, In Vitro, Cell Culture