Journal: Cellular and Molecular Bioengineering

Article Title: Hyperosmotic Stress Promotes the Nuclear Translocation of TFEB in Tubular Epithelial Cells Depending on Intracellular Ca 2+ Signals via TRPML Channels

doi: 10.1007/s12195-024-00839-6

Figure Lengend Snippet: The effects of ML-SI3 on the mannitol-mediated hyperosmotic stress-induced nuclear translocation of TFEB in NRK-52E cells. a Representative fluorescence images of Hoechst 33342 (blue) and TFEB (green) staining of NRK-52E cells cotreated with mannitol (200 mM) and ML-SI3 (1 μM or 10 μM) for 1 h. Scale bar: 25 μm. b A summary of the ratios between the nuclear and cytosolic TFEB fluorescence intensities of NRK-52E cells. The data relating to the 200 mM mannitol alone group are identical to those in Fig. c (at 1 h) and are shown for comparison. Data are presented as box and whisker plots, with average (×), median, IQR, minimum value, and maximum value (200 mM mannitol + 1 μM ML-SI3: n = 29; 200 mM mannitol + 10 μM ML-SI3: n = 20). “n” represents the number of images analyzed. ** P < 0.01 vs. 200 mM mannitol treatment (Dunnett’s test). c Representative fluorescence images of Hoechst 33342 (blue) and NFAT (red) staining of NRK-52E cells cotreated with mannitol (200 mM) and ML-SI3 (1 μM or 10 μM) for 1 h. Scale bar: 25 μm. d A summary of the ratios between the nuclear and cytosolic NFAT fluorescence intensities of NRK-52E cells. Data are presented as box and whisker plots, with average (×), median, IQR, minimum value, and maximum value (200 mM mannitol + 1 μM ML-SI3: n = 28; 200 mM mannitol + 10 μM ML-SI3: n = 30). “n” represents the number of images analyzed. ** P < 0.01 vs. 200 mM mannitol treatment (Dunnett’s test)

Article Snippet: For the inhibitor experiments, cells were pre-treated for 15 minutes with ethylene glycol tetraacetic acid (EGTA, 4 mM; FUJIFILM Wako Pure Chemical), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, 50 μM; Tokyo Kasei Kogyo, Tokyo, Japan), FK-506 (10 μM or 50 μM; Cayman Chemical, Ann Arbor, MI), ML-SI3 (1 μM or 10 μM; a TRPML antagonist, MedChemExpress, Monmouth Junction, NJ), or rapamycin (10 μM; FUJIFILM Wako Pure Chemical).

Techniques: Translocation Assay, Fluorescence, Staining, Comparison, Whisker Assay

Journal: Cellular and Molecular Bioengineering

Article Title: Hyperosmotic Stress Promotes the Nuclear Translocation of TFEB in Tubular Epithelial Cells Depending on Intracellular Ca 2+ Signals via TRPML Channels

doi: 10.1007/s12195-024-00839-6

Figure Lengend Snippet: The effects of ML-SI3 on the mannitol-mediated hyperosmotic stress-induced LC3-II upregulation in NRK-52E cells. a , b NRK-52E cells were treated with 200 mM mannitol or cotreated with ML-SI3 (10 μM) for 0, 1, 2, 4, 8, and 12 h. a Representative blot images of LC3-I and LC3-II in NRK-52E cells treated with 200 mM mannitol alone (top) or cotreated with 10 μM ML-SI3 (bottom), where GAPDH served as the loading control. b Relative LC3-II expression level was calculated by normalizing to the level at 0 h, or with no treatment, and plotted as mean ± SEM (mannitol: n = 3; mannitol + ML-SI3: n = 4). “n” represents the number of independent cultures. ** P < 0.01 vs. 0-h or no treatment (Steel test). # P < 0.05 and ## P < 0.01 vs. 200 mM mannitol treatment (Student’s t -test)

Article Snippet: For the inhibitor experiments, cells were pre-treated for 15 minutes with ethylene glycol tetraacetic acid (EGTA, 4 mM; FUJIFILM Wako Pure Chemical), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, 50 μM; Tokyo Kasei Kogyo, Tokyo, Japan), FK-506 (10 μM or 50 μM; Cayman Chemical, Ann Arbor, MI), ML-SI3 (1 μM or 10 μM; a TRPML antagonist, MedChemExpress, Monmouth Junction, NJ), or rapamycin (10 μM; FUJIFILM Wako Pure Chemical).

Techniques: Control, Expressing

Journal: Scientific Reports

Article Title: Obinutuzumab induces lysosomal destabilization via sphingomyelin-dependent inhibition of TRPML2

doi: 10.1038/s41598-026-38087-5

Figure Lengend Snippet: Lysosomal stress and TRPML inhibition enhance obinutuzumab-mediated direct cell death. ( a ) LMP and DCD were measured in Raji cells treated with hypertonic medium (200 mM sucrose) or hypotonic medium (60% distilled water in complete culture medium) for 2 h in the presence of OBI (Hereafter, the concentration of OBI is 10 μg/ml unless otherwise stated). LMP and DCD were evaluated by LysoTracker Green and propidium iodide (PI) staining. One-way ANOVA with Dunnett’s multiple-comparisons test (vs. control). ( b ) OBI-induced LMP and DCD were assessed in Raji cells co-incubated with increasing concentrations of the TRPML inhibitor (1R,2R)-ML-SI3 (10 µM). In parallel, cells pretreated overnight with the PIKfyve inhibitor Apilimod (50 nM) showed enhanced OBI responses, but this effect was abolished when TRPMLs were simultaneously inhibited. One-way ANOVA with Dunnett’s multiple-comparisons test (vs. control). ( c ) Dose-dependent enhancement of OBI-induced DCD was observed following 2-h co-incubation with a fixed concentration of the (1R,2R)-ML-SI3 (10 µM, over 2 h incubation, this (1R,2R)-ML-SI3 treatment condition was used unless otherwise stated). ( d , e ) Time-dependent enhancement of OBI-induced LMP ( d ) and DCD ( e ) were assessed in TRPMLs-targeting siRNA cell lines. ( f ) Lysosome enlargement and fluorescence intensity were indicated by LT Deep Red staining, then imaged by confocal microscopy. (1R, 2R)-ML-SI3, hypertonic, and hypotonic medium were incubated 2 h. Scale bars in all images represent 5 µM. ( g , h ) Quantification of cathepsin B release induced by OBI and (1R,2R)-ML-SI3 treatment. ( g ) Raji cells were treated with (1R,2R)-ML-SI3, hypertonic medium (200 mM sucrose), or hypotonic medium (60% distilled water in complete medium) for 2 h, and with OBI for 4 h. Cathepsin B releases were visualized by confocal microscopy by immunostaining. ( h ) The summary of the release data shows significant cathepsin B secretion in response to OBI treatment, which was further modulated by (1R,2R)-ML-SI3 and osmotic stress conditions. Data are presented as mean ± SEM. *p < 0.05, ***p < 0.001 compared to obi-treated group.

Article Snippet: For drug treatments, (1R,2R)-ML-SI3 (HY-134819A; MCE), ML2-SA1 (axon Medchem), and 3- O -methyl Estradiol (#42995, Cayman Chemical) were administered for 2 h at 37 °C, followed by OBI (0.3 μg/ml) treatment for an additional 2 h. Endocytosis inhibition was performed using filipin (HY-N6716, MCE), dynasore (S8047, Sellekchem), pitstop2 (HY-115605, MCE), Sphingomyelinase (S7651-10UN, Sigma), or heat-inactivated Sphingomyelinase for 30 min at 37 °C before antibodies treatment (10 μg/ml, IgG, RTX, OBI).

Techniques: Inhibition, Concentration Assay, Staining, Control, Incubation, Fluorescence, Confocal Microscopy, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: Activated Endolysosomal Cation Channel TRPML1 Facilitates Maturation of α-Synuclein-Containing Autophagosomes

doi: 10.3389/fncel.2022.861202

Figure Lengend Snippet: Effect of TRPML1-agonist ML-SA1 and TRPML1-antagonist ML-SI3 on autophagy and α-synuclein particles in human dopaminergic neurons. (A) Example images of LUHMES-derived dopaminergic neurons transduced with α-synuclein-T2A-GFP-lentivirus. Six days after transduction neurons were treated for 24 h either with DMSO or 10 μM ML-SA1. Neurons were further stained against human α-synuclein, lysosomal marker Lamp1 and neuronal marker using MAP2. The scale bar represents 20 μm. (B) Close up of (A) . Closed arrowhead marks a α-synuclein-positive/Lamp1-negative particle, open arrowhead marks a α-synuclein-positive and Lamp1-positive particle, asterisk marks a α-synuclein-negative/Lamp1-positive lysosome. (C) Percentage of Lamp1-positive synuclein particles per neuron. Results of 30 cells per condition. Unpaired t -test depicted with respect to DMSO ( p < 0.001). (D) Representative immunoblot of lysates from transduced LUHMES-derived neurons on day 9, treated with DMSO, ML-SA1 10 μM, or ML-SI3 10 μM for 2 h. Full blot shown in . (E) Quantification of the LC3II band relative to LC3I band in n = 3 independent experiments as in (D) . One-way ANOVA showed no significance ( p = 0.8). (F) Representative immunoblot of lysates from transduced LUHMES-derived neurons on day 9, treated with DMSO, ML-SA1 10 μM or ML-SI3 10 μM for 24 h. Full blot shown in . (G) Quantification of the p62 band relative to beta-Tubulin in n = 3 independent experiments as in (F) . One-way ANOVA showed significance ( p < 0.01). Dunnet’s multiple comparisons test is depicted with respect to DMSO. (H) Quantification of Synuclein load relative to beta-Tubulin in n = 3 independent experiments as in (F) . One-way ANOVA showed no significance ( p = 0.2). ** p < 0.01, *** p < 0.001, non-significant differences are depicted as n.s.

Article Snippet: ML-SI3 (MedKoo Biosciences, Inc., Morrisville, USA) was dissolved in DMSO to a 25 mM stock solution using a final concentration on neurons as depicted.

Techniques: Derivative Assay, Transduction, Staining, Marker, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Activated Endolysosomal Cation Channel TRPML1 Facilitates Maturation of α-Synuclein-Containing Autophagosomes

doi: 10.3389/fncel.2022.861202

Figure Lengend Snippet: Effect of TRPML1-agonist ML-SA1 and TRPML1-antagonist ML-SI3 on α-synuclein particles in iPSC-derived mature neurons. (A) Example images of NPC-derived neurons transduced with α-synuclein-T2A-GFP using lentivirus. Twenty-one days after transduction, cells were treated for 24 h either with 20 μM ML-SA1 or DMSO. Cells were further stained against human α-synuclein, lysosomal marker Lamp1 and neuronal marker MAP2. The scale bar represents 20 μm. (B) Close up of (A) . Closed arrowhead marks a α-synuclein-positive/Lamp1-negative particle, open arrowhead marks a α-synuclein-positive and Lamp1-positive particle, asterisk marks a α-synuclein-negative/Lamp1-positive lysosome. (C) Percentage of Lamp1-positive synuclein particles per cell. Results of at least 30 neurons per condition. Unpaired t -test depicted with respect to DMSO ( p = 0.01). (D) Representative immunoblot of lysates from transduced neurons on day 21, treated with DMSO, ML-SA1 10 μM, or ML-SI3 10 μM and additionally DMSO or 100 nM BafA1 for 2 h. Full blot shown in . (E) Quantification of the Synuclein-T2A-dimer band relative to total synuclein load in n = 3 independent experiments as in (D) . One-way ANOVA showed significance ( p = 0.03). Results of Dunnet’s multiple comparisons test are depicted with respect to DMSO ( p = 0.03). (F) Quantification of Synuclein load relative to beta-Tubulin in n = 3 independent experiments as in (D) . Two-way-ANOVA showed significant interaction ( p = 0.03), Sidak’s multiple comparisons test is depicted with respect to DMSO. * p < 0.05, ** p < 0.01, non-significant differences are depicted as n.s.

Article Snippet: ML-SI3 (MedKoo Biosciences, Inc., Morrisville, USA) was dissolved in DMSO to a 25 mM stock solution using a final concentration on neurons as depicted.

Techniques: Derivative Assay, Transduction, Staining, Marker, Western Blot