Journal: Frontiers in Cell and Developmental Biology

Article Title: Identification of a novel aromatic-turmerone analog that activates chaperone-mediated autophagy through the persistent activation of p38

doi: 10.3389/fcell.2024.1418296

Figure Lengend Snippet: Primer sequences used in RT-qPCR analyses.

Article Snippet: The anti-human LAMP2A rabbit polyclonal antibody (ab125068), anti-TSG101 rabbit polyclonal antibody (ab125011), and anti-α-synuclein rabbit monoclonal antibody (ab138501) were obtained from AbCam (Cambridge, United Kingdom).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Identification of a novel aromatic-turmerone analog that activates chaperone-mediated autophagy through the persistent activation of p38

doi: 10.3389/fcell.2024.1418296

Figure Lengend Snippet: Effects of ar-turmerone analogs on CMA/mA activity in SH-SY5Y cells stably expressing GAPDH-HT. (A) Representative fluorescence image of SaraFluor 650T attached to GAPDH-HT in cells treated with vehicle, MPA (5 μM), or ar-turmerone analogs (20 μM) for 24 h. (C) Representative fluorescence image of GFP and SaraFluor 650T attached to GAPDH-HT and merged images of GFP (green) and SaraFluor (magenta) in cells treated with vehicle or A4 (20 μM) for 24 h. Cells expressing GFP means cells expressing miRNA (negative control miRNA, LAMP2A miRNA, or TSG101 miRNA). Scale bars = 10 μm. (B, D) Quantitative analyses of GAPDH-HT puncta per cell shown in A and C, respectively. Data are represented as the percentage of GAPDH-HT puncta in cells treated with vehicle (B) and in cells transfected with control miRNA and treated with vehicle (D) . Numbers in the columns represent the number of observed cells. n.s., not significant, *** p < 0.001 (Kruskal–Wallis test, followed by a post hoc Dunn’s test).

Article Snippet: The anti-human LAMP2A rabbit polyclonal antibody (ab125068), anti-TSG101 rabbit polyclonal antibody (ab125011), and anti-α-synuclein rabbit monoclonal antibody (ab138501) were obtained from AbCam (Cambridge, United Kingdom).

Techniques: Activity Assay, Stable Transfection, Expressing, Fluorescence, Negative Control, Transfection, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Identification of a novel aromatic-turmerone analog that activates chaperone-mediated autophagy through the persistent activation of p38

doi: 10.3389/fcell.2024.1418296

Figure Lengend Snippet: Effects of ar-turmerone analogs on the expression of CMA-related proteins and mRNAs in SH-SY5Y cells stably expressing GAPDH-HT. (A) Representative immunoblot images of LAMP2A, Hsc70, MEF2D, and β-tubulin in cells treated with vehicle, 5 μM MPA, or ar-turmerone analogs (20 μM) for 24 h. (B) Quantitative analyses of the immunoreactive bands of LAMP2A, Hsc70, and MEF2D. The band intensities were normalized with the bands of β-tubulin as an internal control. Data are presented as the mean ± SEM of four different samples. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. vehicle (one-way ANOVA, followed by a post hoc Tukey test). (C) The mRNA amounts of LAMP2A, LAMP2B, LAMP1, HSPA8 (Hsc70), TFEB, and MEF2D were quantified with RT-qPCR. Cells were treated with vehicle, 20 μM A2, or 20 μM A4 for 24 h. The data are presented as the mean ± SEM of four independent samples. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. vehicle (one-way ANOVA, followed by a post hoc Tukey test).

Article Snippet: The anti-human LAMP2A rabbit polyclonal antibody (ab125068), anti-TSG101 rabbit polyclonal antibody (ab125011), and anti-α-synuclein rabbit monoclonal antibody (ab138501) were obtained from AbCam (Cambridge, United Kingdom).

Techniques: Expressing, Stable Transfection, Western Blot, Control, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: Identification of a novel aromatic-turmerone analog that activates chaperone-mediated autophagy through the persistent activation of p38

doi: 10.3389/fcell.2024.1418296

Figure Lengend Snippet: Effects of ar-turmerone analogs on the cytotoxicity of rotenone in SH-SY5Y cells stably expressing GAPDH-HT. (A) Survival of cells treated with vehicle and rotenone (10 μM) and co-treated with rotenone and A2 (2, 20 μM) or A4 (2, 20 μM). (B) Survival of cells treated with vehicle, A4 (20 μM), and/or SB203580 (SB, 10 μM) in the absence or presence of rotenone (10 μM). Data are represented as the percentage of cell survival in cells treated with vehicle alone and as the mean ± SEM of twelve (A) and fourteen (B) different samples. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. vehicle alone, # p < 0.05, ## p < 0.01 (Kruskal–Wallis test, followed by a post hoc Dunn’s test). (C) Representative fluorescence image of GFP and DAPI and merged images of GFP (green) and DAPI (blue) in cells transfected with plasmids to express GFP and control or LAMP2A miRNA. Cells were treated with vehicle (Veh, 0.2% DMSO), 10 μM rotenone (Rot), or rotenone and 20 μM A4 (Rot + A4) for 24 h. Yellow arrows indicate the GFP-positive cells with condensed nuclei. Scale bars = 50 μm. (D) Quantitative analyses of cytotoxicity indicated by the nuclear condensation. We calculated the percentage of cells with condensed nuclei in all GFP-positive cells. Data are presented as the mean ± SEM of four different wells. n.s.: not significant, * p < 0.05, *** p < 0.001 (one way ANOVA, followed by a post hoc Tukey test).

Article Snippet: The anti-human LAMP2A rabbit polyclonal antibody (ab125068), anti-TSG101 rabbit polyclonal antibody (ab125011), and anti-α-synuclein rabbit monoclonal antibody (ab138501) were obtained from AbCam (Cambridge, United Kingdom).

Techniques: Stable Transfection, Expressing, Fluorescence, Transfection, Control

Journal: iScience

Article Title: Modification of lysine-260 2-hydroxyisobutyrylation destabilizes ALDH1A1 expression to regulate bladder cancer progression

doi: 10.1016/j.isci.2023.108142

Figure Lengend Snippet: 2-Hydroxyisobutyrylation at lys-260 promoting ALDH1A1 degradation through chaperone-mediated autophagy (A) T24 cells were treated with CQ, and the ALDH1A1 protein levels were detected by immunoblotting at the indicated time points. (B) MG132 or CQ was cotreated with TSA in T24 cells to observe the protein levels of ALDH1A1 by Western blotting. (C) LAMP2A was knocked down in T24 cells by siRNA, and the knockdown efficiency of LAMP2A and ALDH1A1 protein levels were examined by Western blotting. (D) Endogenous LAMP2A was IPed from T24 cells. Rabbit IgG was used as a control. (E) Colocalization of ALDH1A1 and LAMP2A in T24 cells was observed by confocal microscopy. Scale bar = 20 μm. (F) TSA treatment decreased the level of ALDH1A1 wild-type, but not K260R mutant ALDH1A1 in T24 cells. (G) TSA treatment decreased the level of ALDH1A1 wild-type, but not K260R mutant ALDH1A1 in 293T cells. (H) HA-tagged HSC70 was co-transfected with vector, flag-tagged ALDH1A1 WT , or flag-tagged ALDH1A1 K260T into T24cells, the ALDH1A1-HSC70 binding was determined by immunoprecipitation-Western blotting analysis. (I) The cell lysates were collected with ALDH1A1 WT , ALDH1A1 K260T , or ALDH1A1 K260R overexpressed in T24 cells followed treatment with 1% glutaraldehyde, while the polymerizations of ALDH1A1 were examined by immunoblotting. (J and K) The crystal structures of ALDH1A1 WT and ALDH1A1 K260T were analyzed by AlphaFold 2.0 software. For cell experiments, each experiment was performed at least three times.

Article Snippet: Rabbit monoclonal anti-LAMP2A , Abcam , Cat#:ab125068; RRID:AB_10971511.

Techniques: Western Blot, Confocal Microscopy, Mutagenesis, Transfection, Plasmid Preparation, Binding Assay, Immunoprecipitation, Software

Journal: iScience

Article Title: Modification of lysine-260 2-hydroxyisobutyrylation destabilizes ALDH1A1 expression to regulate bladder cancer progression

doi: 10.1016/j.isci.2023.108142

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-LAMP2A , Abcam , Cat#:ab125068; RRID:AB_10971511.

Techniques: Virus, Recombinant, Activity Assay, SYBR Green Assay, Software