trpml1  (Alomone Labs)


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    Alomone Labs trpml1
    Autophagy enhancement and apoptosis inhibition by <t>TRPML1</t> activation. (a–d) Representative immunoblots of beclin-1, LC3, and p62 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (a, n = 6) or transfected with TRPML1 lentivirus (c, n = 6) 1 hr after OGD. (b) or (d) was the statistics for (a) or (c). (e–h) Representative immunoblots of cleaved caspase3 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (e, n = 6) or transfected with TRPML1 lentivirus (g, n = 6) 1 hr after OGD. (f) or (h) was the statistics for (e) or (g). β -Actin serves as a loading control. All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗ P
    Trpml1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trpml1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trpml1 - by Bioz Stars, 2022-01
    93/100 stars

    Images

    1) Product Images from "Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia"

    Article Title: Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2018/4612727

    Autophagy enhancement and apoptosis inhibition by TRPML1 activation. (a–d) Representative immunoblots of beclin-1, LC3, and p62 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (a, n = 6) or transfected with TRPML1 lentivirus (c, n = 6) 1 hr after OGD. (b) or (d) was the statistics for (a) or (c). (e–h) Representative immunoblots of cleaved caspase3 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (e, n = 6) or transfected with TRPML1 lentivirus (g, n = 6) 1 hr after OGD. (f) or (h) was the statistics for (e) or (g). β -Actin serves as a loading control. All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗ P
    Figure Legend Snippet: Autophagy enhancement and apoptosis inhibition by TRPML1 activation. (a–d) Representative immunoblots of beclin-1, LC3, and p62 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (a, n = 6) or transfected with TRPML1 lentivirus (c, n = 6) 1 hr after OGD. (b) or (d) was the statistics for (a) or (c). (e–h) Representative immunoblots of cleaved caspase3 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (e, n = 6) or transfected with TRPML1 lentivirus (g, n = 6) 1 hr after OGD. (f) or (h) was the statistics for (e) or (g). β -Actin serves as a loading control. All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗ P

    Techniques Used: Inhibition, Activation Assay, Western Blot, Cell Culture, Transfection

    Reduction of the infarct volumes and mortality by TRPML1 activation. (a, b) Infarct volumes of mice infusion with or without 5 μ M ML-SA1after 12 hrs reperfusion ( n = 6). (b) was the statistics for (a). (c) Evaluation of neurological deficit after 24 hrs reperfusion was evaluated in mice infusion with or without 5 μ M ML-SA1 ( n = 7). (d) Evaluation of the survival rate in mice infusion with or without 5 μ M ML-SA1 after tBCCAO ( n = 8). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗∗ P
    Figure Legend Snippet: Reduction of the infarct volumes and mortality by TRPML1 activation. (a, b) Infarct volumes of mice infusion with or without 5 μ M ML-SA1after 12 hrs reperfusion ( n = 6). (b) was the statistics for (a). (c) Evaluation of neurological deficit after 24 hrs reperfusion was evaluated in mice infusion with or without 5 μ M ML-SA1 ( n = 7). (d) Evaluation of the survival rate in mice infusion with or without 5 μ M ML-SA1 after tBCCAO ( n = 8). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗∗ P

    Techniques Used: Activation Assay, Mouse Assay

    Proteasome-mediated degradation of TRPML1 of hippocampal neurons in ischemia. (a–d) Representative immunoblots of TRPML1 from cultured hippocampal neurons (a, n = 4) or astrocytes (c, n = 4) 0.5 or 1 hrs after OGD. (b) or (d) was the statistics for (a) or (c). (e, f) Immunoblots of analysis of the indicated marker expression in sorted neurons (e) and astrocytes (f) (GFAP: astrocyte marker, NeuN: neuron marker). (g–j) Representative immunoblots of TRPML1 from sorted neurons (g, n = 9) or astrocytes (i, n = 9) 40 min after tBCCAo. (h) or (j) was the statistics for (g) or (i). (k) Quantitative PCR analysis of TRPML1 mRNA levels in hippocampal neurons 1 hr after OGD ( n = 5). (l, m) Representative immunoblots of TRPML1 from cultured hippocampal neurons ( n = 5) incubated with/without calpeptin (10 μ M), DEVD (10 μ M), or MG132 (5 μ M). (m) was the statistics for (l). β -Actin serves as a loading control. All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test (b, d, and k), Student's t -test, two tailed (h, j), or two-way ANOVA with Bonferroni post hoc test (m). ∗ P
    Figure Legend Snippet: Proteasome-mediated degradation of TRPML1 of hippocampal neurons in ischemia. (a–d) Representative immunoblots of TRPML1 from cultured hippocampal neurons (a, n = 4) or astrocytes (c, n = 4) 0.5 or 1 hrs after OGD. (b) or (d) was the statistics for (a) or (c). (e, f) Immunoblots of analysis of the indicated marker expression in sorted neurons (e) and astrocytes (f) (GFAP: astrocyte marker, NeuN: neuron marker). (g–j) Representative immunoblots of TRPML1 from sorted neurons (g, n = 9) or astrocytes (i, n = 9) 40 min after tBCCAo. (h) or (j) was the statistics for (g) or (i). (k) Quantitative PCR analysis of TRPML1 mRNA levels in hippocampal neurons 1 hr after OGD ( n = 5). (l, m) Representative immunoblots of TRPML1 from cultured hippocampal neurons ( n = 5) incubated with/without calpeptin (10 μ M), DEVD (10 μ M), or MG132 (5 μ M). (m) was the statistics for (l). β -Actin serves as a loading control. All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test (b, d, and k), Student's t -test, two tailed (h, j), or two-way ANOVA with Bonferroni post hoc test (m). ∗ P

    Techniques Used: Western Blot, Cell Culture, Marker, Expressing, Real-time Polymerase Chain Reaction, Incubation, Two Tailed Test

    Reduction of neuron survival in OGD by TRPML1 degradation. (a) Hippocampal neuron survival rate determined by MTT assay after 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (b) Immunoblot analysis of HA-TRPML1 expression in hippocampal neuron. (c) The survival rate of hippocampal neuron transfected with TRPML1 lentivirus determined by MTT assay after 3 hrs OGD ( n = 5). β -Actin serves as a loading control. (d, f) Hippocampal neuron apoptosis was analyzed by flow cytometry 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (e, g) The apoptosis of hippocampal neuron transfected with TRPML1 lentivirus was determined by flow cytometry after 3 hrs OGD ( n = 4). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗ P
    Figure Legend Snippet: Reduction of neuron survival in OGD by TRPML1 degradation. (a) Hippocampal neuron survival rate determined by MTT assay after 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (b) Immunoblot analysis of HA-TRPML1 expression in hippocampal neuron. (c) The survival rate of hippocampal neuron transfected with TRPML1 lentivirus determined by MTT assay after 3 hrs OGD ( n = 5). β -Actin serves as a loading control. (d, f) Hippocampal neuron apoptosis was analyzed by flow cytometry 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (e, g) The apoptosis of hippocampal neuron transfected with TRPML1 lentivirus was determined by flow cytometry after 3 hrs OGD ( n = 4). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗ P

    Techniques Used: MTT Assay, Expressing, Transfection, Flow Cytometry, Cytometry

    2) Product Images from "Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia"

    Article Title: Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2018/4612727

    Autophagy enhancement and apoptosis inhibition by TRPML1 activation. (a–d) Representative immunoblots of beclin-1, LC3, and p62 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (a, n = 6) or transfected with TRPML1 lentivirus (c, n = 6) 1 hr after OGD. (b) or (d) was the statistics for (a) or (c). (e–h) Representative immunoblots of cleaved caspase3 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (e, n = 6) or transfected with TRPML1 lentivirus (g, n = 6) 1 hr after OGD. (f) or (h) was the statistics for (e) or (g). β -Actin serves as a loading control. All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗ P
    Figure Legend Snippet: Autophagy enhancement and apoptosis inhibition by TRPML1 activation. (a–d) Representative immunoblots of beclin-1, LC3, and p62 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (a, n = 6) or transfected with TRPML1 lentivirus (c, n = 6) 1 hr after OGD. (b) or (d) was the statistics for (a) or (c). (e–h) Representative immunoblots of cleaved caspase3 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (e, n = 6) or transfected with TRPML1 lentivirus (g, n = 6) 1 hr after OGD. (f) or (h) was the statistics for (e) or (g). β -Actin serves as a loading control. All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗ P

    Techniques Used: Inhibition, Activation Assay, Western Blot, Cell Culture, Transfection

    Reduction of the infarct volumes and mortality by TRPML1 activation. (a, b) Infarct volumes of mice infusion with or without 5 μ M ML-SA1after 12 hrs reperfusion ( n = 6). (b) was the statistics for (a). (c) Evaluation of neurological deficit after 24 hrs reperfusion was evaluated in mice infusion with or without 5 μ M ML-SA1 ( n = 7). (d) Evaluation of the survival rate in mice infusion with or without 5 μ M ML-SA1 after tBCCAO ( n = 8). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗∗ P
    Figure Legend Snippet: Reduction of the infarct volumes and mortality by TRPML1 activation. (a, b) Infarct volumes of mice infusion with or without 5 μ M ML-SA1after 12 hrs reperfusion ( n = 6). (b) was the statistics for (a). (c) Evaluation of neurological deficit after 24 hrs reperfusion was evaluated in mice infusion with or without 5 μ M ML-SA1 ( n = 7). (d) Evaluation of the survival rate in mice infusion with or without 5 μ M ML-SA1 after tBCCAO ( n = 8). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗∗ P

    Techniques Used: Activation Assay, Mouse Assay

    Proteasome-mediated degradation of TRPML1 of hippocampal neurons in ischemia. (a–d) Representative immunoblots of TRPML1 from cultured hippocampal neurons (a, n = 4) or astrocytes (c, n = 4) 0.5 or 1 hrs after OGD. (b) or (d) was the statistics for (a) or (c). (e, f) Immunoblots of analysis of the indicated marker expression in sorted neurons (e) and astrocytes (f) (GFAP: astrocyte marker, NeuN: neuron marker). (g–j) Representative immunoblots of TRPML1 from sorted neurons (g, n = 9) or astrocytes (i, n = 9) 40 min after tBCCAo. (h) or (j) was the statistics for (g) or (i). (k) Quantitative PCR analysis of TRPML1 mRNA levels in hippocampal neurons 1 hr after OGD ( n = 5). (l, m) Representative immunoblots of TRPML1 from cultured hippocampal neurons ( n = 5) incubated with/without calpeptin (10 μ M), DEVD (10 μ M), or MG132 (5 μ M). (m) was the statistics for (l). β -Actin serves as a loading control. All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test (b, d, and k), Student's t -test, two tailed (h, j), or two-way ANOVA with Bonferroni post hoc test (m). ∗ P
    Figure Legend Snippet: Proteasome-mediated degradation of TRPML1 of hippocampal neurons in ischemia. (a–d) Representative immunoblots of TRPML1 from cultured hippocampal neurons (a, n = 4) or astrocytes (c, n = 4) 0.5 or 1 hrs after OGD. (b) or (d) was the statistics for (a) or (c). (e, f) Immunoblots of analysis of the indicated marker expression in sorted neurons (e) and astrocytes (f) (GFAP: astrocyte marker, NeuN: neuron marker). (g–j) Representative immunoblots of TRPML1 from sorted neurons (g, n = 9) or astrocytes (i, n = 9) 40 min after tBCCAo. (h) or (j) was the statistics for (g) or (i). (k) Quantitative PCR analysis of TRPML1 mRNA levels in hippocampal neurons 1 hr after OGD ( n = 5). (l, m) Representative immunoblots of TRPML1 from cultured hippocampal neurons ( n = 5) incubated with/without calpeptin (10 μ M), DEVD (10 μ M), or MG132 (5 μ M). (m) was the statistics for (l). β -Actin serves as a loading control. All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test (b, d, and k), Student's t -test, two tailed (h, j), or two-way ANOVA with Bonferroni post hoc test (m). ∗ P

    Techniques Used: Western Blot, Cell Culture, Marker, Expressing, Real-time Polymerase Chain Reaction, Incubation, Two Tailed Test

    Reduction of neuron survival in OGD by TRPML1 degradation. (a) Hippocampal neuron survival rate determined by MTT assay after 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (b) Immunoblot analysis of HA-TRPML1 expression in hippocampal neuron. (c) The survival rate of hippocampal neuron transfected with TRPML1 lentivirus determined by MTT assay after 3 hrs OGD ( n = 5). β -Actin serves as a loading control. (d, f) Hippocampal neuron apoptosis was analyzed by flow cytometry 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (e, g) The apoptosis of hippocampal neuron transfected with TRPML1 lentivirus was determined by flow cytometry after 3 hrs OGD ( n = 4). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗ P
    Figure Legend Snippet: Reduction of neuron survival in OGD by TRPML1 degradation. (a) Hippocampal neuron survival rate determined by MTT assay after 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (b) Immunoblot analysis of HA-TRPML1 expression in hippocampal neuron. (c) The survival rate of hippocampal neuron transfected with TRPML1 lentivirus determined by MTT assay after 3 hrs OGD ( n = 5). β -Actin serves as a loading control. (d, f) Hippocampal neuron apoptosis was analyzed by flow cytometry 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (e, g) The apoptosis of hippocampal neuron transfected with TRPML1 lentivirus was determined by flow cytometry after 3 hrs OGD ( n = 4). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗ P

    Techniques Used: MTT Assay, Expressing, Transfection, Flow Cytometry, Cytometry

    3) Product Images from "Lycorine Attenuates Autophagy in Osteoclasts via an Axis of mROS/TRPML1/TFEB to Reduce LPS-Induced Bone Loss"

    Article Title: Lycorine Attenuates Autophagy in Osteoclasts via an Axis of mROS/TRPML1/TFEB to Reduce LPS-Induced Bone Loss

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2019/8982147

    Lycorine reduces TFEB nuclear translocation by attenuating oxidation of TRPML1 via decreased mitochondrial ROS upon LPS stimulation in OC. BMMs were prepared, incubated with RANKL (40 ng/ml) in the presence of M-CSF (30 ng/ml) for 40 h, washed, and then incubated further with the indicated conditions (DPI, 5 nM; Mito-TEMPO, 100 nM; lycorine, 1.6 μ M) in the presence of LPS (50 ng/ml) and M-CSF (30 ng/ml). Mitochondrial ROS and cytosolic ROS were determined by flow cytometry using MitoSOX Red after 16 h and using H 2 DCF-DA after 24 h, respectively (a, b). After 48 h, cell lysates were prepared (c) or fixed (d). Cell lysates were subjected to Western blot to determine p62 and LC3II with the addition of bafilomycin A1 (25 nM) for 4 h. The quantified levels of p62 and LC3II are shown normalized to β -actin (c). Cell lysates were labeled with N -(biotinoyl)- N ′-(iodoacetyl) ethylenediamine, and TRPML1 was immunoprecipitated (IP) from each sample. HRP-streptavidin immunoblotting was performed to evaluate the reduced form of TRPML1 (e). Cells were transfected with 50 nM of scRNA or siTRPML1 and incubated further for 48 h with LPS and M-CSF. siRNA-mediated silencing of TRPML1 was confirmed by RT-PCR and qPCR (f). After fixation, more than 70 TRAP-positive MNCs in each culture were randomly selected to determine the area and fusion activity of the formed OCs (d, f). Whole cell extracts, cytoplasmic fractions, and nuclear fractions were harvested from cultured cells and subjected to Western blot analysis with anti-TFEB Ab. Abs for β -actin and lamin B1 were used for the normalization of cytoplasmic and nuclear extracts, respectively. Quantification of TFEB normalized to β -actin or lamin B1 was plotted (g). ∗ p
    Figure Legend Snippet: Lycorine reduces TFEB nuclear translocation by attenuating oxidation of TRPML1 via decreased mitochondrial ROS upon LPS stimulation in OC. BMMs were prepared, incubated with RANKL (40 ng/ml) in the presence of M-CSF (30 ng/ml) for 40 h, washed, and then incubated further with the indicated conditions (DPI, 5 nM; Mito-TEMPO, 100 nM; lycorine, 1.6 μ M) in the presence of LPS (50 ng/ml) and M-CSF (30 ng/ml). Mitochondrial ROS and cytosolic ROS were determined by flow cytometry using MitoSOX Red after 16 h and using H 2 DCF-DA after 24 h, respectively (a, b). After 48 h, cell lysates were prepared (c) or fixed (d). Cell lysates were subjected to Western blot to determine p62 and LC3II with the addition of bafilomycin A1 (25 nM) for 4 h. The quantified levels of p62 and LC3II are shown normalized to β -actin (c). Cell lysates were labeled with N -(biotinoyl)- N ′-(iodoacetyl) ethylenediamine, and TRPML1 was immunoprecipitated (IP) from each sample. HRP-streptavidin immunoblotting was performed to evaluate the reduced form of TRPML1 (e). Cells were transfected with 50 nM of scRNA or siTRPML1 and incubated further for 48 h with LPS and M-CSF. siRNA-mediated silencing of TRPML1 was confirmed by RT-PCR and qPCR (f). After fixation, more than 70 TRAP-positive MNCs in each culture were randomly selected to determine the area and fusion activity of the formed OCs (d, f). Whole cell extracts, cytoplasmic fractions, and nuclear fractions were harvested from cultured cells and subjected to Western blot analysis with anti-TFEB Ab. Abs for β -actin and lamin B1 were used for the normalization of cytoplasmic and nuclear extracts, respectively. Quantification of TFEB normalized to β -actin or lamin B1 was plotted (g). ∗ p

    Techniques Used: Translocation Assay, Incubation, Flow Cytometry, Cytometry, Western Blot, Labeling, Immunoprecipitation, Transfection, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Activity Assay, Cell Culture

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    Alomone Labs trpml1
    Autophagy enhancement and apoptosis inhibition by <t>TRPML1</t> activation. (a–d) Representative immunoblots of beclin-1, LC3, and p62 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (a, n = 6) or transfected with TRPML1 lentivirus (c, n = 6) 1 hr after OGD. (b) or (d) was the statistics for (a) or (c). (e–h) Representative immunoblots of cleaved caspase3 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (e, n = 6) or transfected with TRPML1 lentivirus (g, n = 6) 1 hr after OGD. (f) or (h) was the statistics for (e) or (g). β -Actin serves as a loading control. All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗ P
    Trpml1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trpml1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trpml1 - by Bioz Stars, 2022-01
    93/100 stars
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    Autophagy enhancement and apoptosis inhibition by TRPML1 activation. (a–d) Representative immunoblots of beclin-1, LC3, and p62 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (a, n = 6) or transfected with TRPML1 lentivirus (c, n = 6) 1 hr after OGD. (b) or (d) was the statistics for (a) or (c). (e–h) Representative immunoblots of cleaved caspase3 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (e, n = 6) or transfected with TRPML1 lentivirus (g, n = 6) 1 hr after OGD. (f) or (h) was the statistics for (e) or (g). β -Actin serves as a loading control. All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia

    doi: 10.1155/2018/4612727

    Figure Lengend Snippet: Autophagy enhancement and apoptosis inhibition by TRPML1 activation. (a–d) Representative immunoblots of beclin-1, LC3, and p62 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (a, n = 6) or transfected with TRPML1 lentivirus (c, n = 6) 1 hr after OGD. (b) or (d) was the statistics for (a) or (c). (e–h) Representative immunoblots of cleaved caspase3 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (e, n = 6) or transfected with TRPML1 lentivirus (g, n = 6) 1 hr after OGD. (f) or (h) was the statistics for (e) or (g). β -Actin serves as a loading control. All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗ P

    Article Snippet: Antibodies were used as follows: TRPML1 from Alomone Labs (acc-081), GFAP from Sigma (G3893), NeuN from Abcam (ab177487), HA from Sigma (H6908), LC3 from Abcam (ab192890), beclin-1 from Cell Signaling Technology (#3495), p62 from Proteintech (18420-1-AP), cleaved caspase3 from Cell Signaling Technology (#9661), and β -actin from Abcam (ab8227).

    Techniques: Inhibition, Activation Assay, Western Blot, Cell Culture, Transfection

    Reduction of the infarct volumes and mortality by TRPML1 activation. (a, b) Infarct volumes of mice infusion with or without 5 μ M ML-SA1after 12 hrs reperfusion ( n = 6). (b) was the statistics for (a). (c) Evaluation of neurological deficit after 24 hrs reperfusion was evaluated in mice infusion with or without 5 μ M ML-SA1 ( n = 7). (d) Evaluation of the survival rate in mice infusion with or without 5 μ M ML-SA1 after tBCCAO ( n = 8). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia

    doi: 10.1155/2018/4612727

    Figure Lengend Snippet: Reduction of the infarct volumes and mortality by TRPML1 activation. (a, b) Infarct volumes of mice infusion with or without 5 μ M ML-SA1after 12 hrs reperfusion ( n = 6). (b) was the statistics for (a). (c) Evaluation of neurological deficit after 24 hrs reperfusion was evaluated in mice infusion with or without 5 μ M ML-SA1 ( n = 7). (d) Evaluation of the survival rate in mice infusion with or without 5 μ M ML-SA1 after tBCCAO ( n = 8). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗∗ P

    Article Snippet: Antibodies were used as follows: TRPML1 from Alomone Labs (acc-081), GFAP from Sigma (G3893), NeuN from Abcam (ab177487), HA from Sigma (H6908), LC3 from Abcam (ab192890), beclin-1 from Cell Signaling Technology (#3495), p62 from Proteintech (18420-1-AP), cleaved caspase3 from Cell Signaling Technology (#9661), and β -actin from Abcam (ab8227).

    Techniques: Activation Assay, Mouse Assay

    Proteasome-mediated degradation of TRPML1 of hippocampal neurons in ischemia. (a–d) Representative immunoblots of TRPML1 from cultured hippocampal neurons (a, n = 4) or astrocytes (c, n = 4) 0.5 or 1 hrs after OGD. (b) or (d) was the statistics for (a) or (c). (e, f) Immunoblots of analysis of the indicated marker expression in sorted neurons (e) and astrocytes (f) (GFAP: astrocyte marker, NeuN: neuron marker). (g–j) Representative immunoblots of TRPML1 from sorted neurons (g, n = 9) or astrocytes (i, n = 9) 40 min after tBCCAo. (h) or (j) was the statistics for (g) or (i). (k) Quantitative PCR analysis of TRPML1 mRNA levels in hippocampal neurons 1 hr after OGD ( n = 5). (l, m) Representative immunoblots of TRPML1 from cultured hippocampal neurons ( n = 5) incubated with/without calpeptin (10 μ M), DEVD (10 μ M), or MG132 (5 μ M). (m) was the statistics for (l). β -Actin serves as a loading control. All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test (b, d, and k), Student's t -test, two tailed (h, j), or two-way ANOVA with Bonferroni post hoc test (m). ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia

    doi: 10.1155/2018/4612727

    Figure Lengend Snippet: Proteasome-mediated degradation of TRPML1 of hippocampal neurons in ischemia. (a–d) Representative immunoblots of TRPML1 from cultured hippocampal neurons (a, n = 4) or astrocytes (c, n = 4) 0.5 or 1 hrs after OGD. (b) or (d) was the statistics for (a) or (c). (e, f) Immunoblots of analysis of the indicated marker expression in sorted neurons (e) and astrocytes (f) (GFAP: astrocyte marker, NeuN: neuron marker). (g–j) Representative immunoblots of TRPML1 from sorted neurons (g, n = 9) or astrocytes (i, n = 9) 40 min after tBCCAo. (h) or (j) was the statistics for (g) or (i). (k) Quantitative PCR analysis of TRPML1 mRNA levels in hippocampal neurons 1 hr after OGD ( n = 5). (l, m) Representative immunoblots of TRPML1 from cultured hippocampal neurons ( n = 5) incubated with/without calpeptin (10 μ M), DEVD (10 μ M), or MG132 (5 μ M). (m) was the statistics for (l). β -Actin serves as a loading control. All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test (b, d, and k), Student's t -test, two tailed (h, j), or two-way ANOVA with Bonferroni post hoc test (m). ∗ P

    Article Snippet: Antibodies were used as follows: TRPML1 from Alomone Labs (acc-081), GFAP from Sigma (G3893), NeuN from Abcam (ab177487), HA from Sigma (H6908), LC3 from Abcam (ab192890), beclin-1 from Cell Signaling Technology (#3495), p62 from Proteintech (18420-1-AP), cleaved caspase3 from Cell Signaling Technology (#9661), and β -actin from Abcam (ab8227).

    Techniques: Western Blot, Cell Culture, Marker, Expressing, Real-time Polymerase Chain Reaction, Incubation, Two Tailed Test

    Reduction of neuron survival in OGD by TRPML1 degradation. (a) Hippocampal neuron survival rate determined by MTT assay after 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (b) Immunoblot analysis of HA-TRPML1 expression in hippocampal neuron. (c) The survival rate of hippocampal neuron transfected with TRPML1 lentivirus determined by MTT assay after 3 hrs OGD ( n = 5). β -Actin serves as a loading control. (d, f) Hippocampal neuron apoptosis was analyzed by flow cytometry 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (e, g) The apoptosis of hippocampal neuron transfected with TRPML1 lentivirus was determined by flow cytometry after 3 hrs OGD ( n = 4). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia

    doi: 10.1155/2018/4612727

    Figure Lengend Snippet: Reduction of neuron survival in OGD by TRPML1 degradation. (a) Hippocampal neuron survival rate determined by MTT assay after 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (b) Immunoblot analysis of HA-TRPML1 expression in hippocampal neuron. (c) The survival rate of hippocampal neuron transfected with TRPML1 lentivirus determined by MTT assay after 3 hrs OGD ( n = 5). β -Actin serves as a loading control. (d, f) Hippocampal neuron apoptosis was analyzed by flow cytometry 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (e, g) The apoptosis of hippocampal neuron transfected with TRPML1 lentivirus was determined by flow cytometry after 3 hrs OGD ( n = 4). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗ P

    Article Snippet: Antibodies were used as follows: TRPML1 from Alomone Labs (acc-081), GFAP from Sigma (G3893), NeuN from Abcam (ab177487), HA from Sigma (H6908), LC3 from Abcam (ab192890), beclin-1 from Cell Signaling Technology (#3495), p62 from Proteintech (18420-1-AP), cleaved caspase3 from Cell Signaling Technology (#9661), and β -actin from Abcam (ab8227).

    Techniques: MTT Assay, Expressing, Transfection, Flow Cytometry, Cytometry

    Phagolysosomal dysfunction in the RPE of miR‐211 −/− mice Confocal images of representative eye cryosections immunostained with anti‐Lamp1 antibody from 2‐month‐old miR‐211 −/− and control mice sacrificed 3 h after light on, at 10 AM (diurnal condition). Enlarged boxes highlight Lamp1‐positive structures (white arrowheads) in the RPE. Autofluorescence from lipofuscin granules is visible in all cryosections of miR‐211 −/− compared to control littermates. Nuclei were counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 10 μm. RPE was isolated at 10 AM (diurnal condition) from 2‐month‐old WT and miR‐211 −/− mice. Representative Western blot analysis of the Lamp1, Cln5, Trpml1, Sqstm1/p62, Cathepsin D (CtsD), and LC3 proteins from WT and miR‐211 −/− mice. Note a decrease of both pro‐CtsD and its maturation CtsD heavy chain (hc). The plots show the quantification of the indicated proteins normalized to the Gapdh loading control. Bar graphs represent mean values ± SEM of independent experiments ( n = 6 mice). Mann and Whitney test (miR‐211 −/− versus WT), * P ≤ 0.05, ** P ≤ 0.01. Cathepsin B (CtsB) activity in RPE lysates from 2‐month‐old miR‐211 −/− and control mice sacrificed 3 h after light on at 10 AM (diurnal condition). CtsB was reduced in miR‐211 −/− compared to control mice. Bar graphs represent percentage of CtsB activity ± SEM of independent experiments ( n = 3 mice). Mann and Whitney test (miR‐211 −/− versus WT), * P ≤ 0.05. Representative images of conventional TEM analysis of RPE of both 2‐month‐old and 3‐month‐old WT and miR‐211 −/− mice. miR‐211 −/− mice show accumulation of phagolysosomes (red arrows) in the RPE. Scale bar 2 μm. Enlarged boxes highlight phagolysosome‐like structures containing poorly processed POS. Scale bar 1.5 μm. Representative images of conventional TEM analysis of RPE shown at higher magnification also highlighted accumulation of lipofuscin‐like granules (blue arrows) in 3‐month‐old miR‐211 −/− compared to control WT mice. Melanosomes are indicated by white asterisks. Scale bar 500 nm ( n = 3 mice). Graphs showing number of lipofuscin‐like granules ( n /100 μm 2 ) from the RPE of 3‐month‐old miR‐211 −/− mice as in (D). Bar graphs represent mean values ± SEM. Mann and Whitney test (miR‐211 −/− versus WT) *** P ≤ 0.005 ( n = 6 mice). Data information: (RPE) retinal pigment epithelium; (OS) outer segment; (ONL) outer nuclear layer. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Light‐responsive microRNA miR‐211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance

    doi: 10.15252/embj.2019102468

    Figure Lengend Snippet: Phagolysosomal dysfunction in the RPE of miR‐211 −/− mice Confocal images of representative eye cryosections immunostained with anti‐Lamp1 antibody from 2‐month‐old miR‐211 −/− and control mice sacrificed 3 h after light on, at 10 AM (diurnal condition). Enlarged boxes highlight Lamp1‐positive structures (white arrowheads) in the RPE. Autofluorescence from lipofuscin granules is visible in all cryosections of miR‐211 −/− compared to control littermates. Nuclei were counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 10 μm. RPE was isolated at 10 AM (diurnal condition) from 2‐month‐old WT and miR‐211 −/− mice. Representative Western blot analysis of the Lamp1, Cln5, Trpml1, Sqstm1/p62, Cathepsin D (CtsD), and LC3 proteins from WT and miR‐211 −/− mice. Note a decrease of both pro‐CtsD and its maturation CtsD heavy chain (hc). The plots show the quantification of the indicated proteins normalized to the Gapdh loading control. Bar graphs represent mean values ± SEM of independent experiments ( n = 6 mice). Mann and Whitney test (miR‐211 −/− versus WT), * P ≤ 0.05, ** P ≤ 0.01. Cathepsin B (CtsB) activity in RPE lysates from 2‐month‐old miR‐211 −/− and control mice sacrificed 3 h after light on at 10 AM (diurnal condition). CtsB was reduced in miR‐211 −/− compared to control mice. Bar graphs represent percentage of CtsB activity ± SEM of independent experiments ( n = 3 mice). Mann and Whitney test (miR‐211 −/− versus WT), * P ≤ 0.05. Representative images of conventional TEM analysis of RPE of both 2‐month‐old and 3‐month‐old WT and miR‐211 −/− mice. miR‐211 −/− mice show accumulation of phagolysosomes (red arrows) in the RPE. Scale bar 2 μm. Enlarged boxes highlight phagolysosome‐like structures containing poorly processed POS. Scale bar 1.5 μm. Representative images of conventional TEM analysis of RPE shown at higher magnification also highlighted accumulation of lipofuscin‐like granules (blue arrows) in 3‐month‐old miR‐211 −/− compared to control WT mice. Melanosomes are indicated by white asterisks. Scale bar 500 nm ( n = 3 mice). Graphs showing number of lipofuscin‐like granules ( n /100 μm 2 ) from the RPE of 3‐month‐old miR‐211 −/− mice as in (D). Bar graphs represent mean values ± SEM. Mann and Whitney test (miR‐211 −/− versus WT) *** P ≤ 0.005 ( n = 6 mice). Data information: (RPE) retinal pigment epithelium; (OS) outer segment; (ONL) outer nuclear layer. Source data are available online for this figure.

    Article Snippet: For Western blot analysis, the following antibodies were used: mouse anti‐Lamp1 (1:500, Sigma‐Aldrich, L1418), rabbit anti‐Cln5 (1:1,000, Abcam ), rabbit anti‐Trpml1 (1:1,000, ALOMONE Lab ACC‐081), rabbit anti‐LC3 (1:1,000, Novus LC3B/MAP1LC3B), rabbit anti‐SQSTM1/p62 (1:1,000, Sigma‐Aldrich, P0067), mouse anti‐CtsD (1:1,000, Santa Cruz SC‐377124), mouse anti‐Ezrin (1:1,000, Novex, 357300), rabbit anti‐phospho‐Ezrin (Th567) that detects endogenous levels of Ezrin only when phosphorylated at threonine 567 (1:700 Sigma‐Aldrich, PA5‐37763), mouse anti‐Gapdh (1:1,000, Santa Cruz, SC‐32233), mouse anti‐β‐actin (1:700, Sigma‐Aldrich, A5441), anti‐TFEB (1:1,000 Cell Signaling, 4240), mouse anti‐FLAG (1:1,000, Sigma, F3165).

    Techniques: Mouse Assay, Isolation, Western Blot, Activity Assay, Transmission Electron Microscopy

    MiR‐211 participates in lysosomal biogenesis in vitro A Immuno‐electron microscopy (IEM) analysis for LAMP1 of ARPE‐19 cells transfected with anti‐miR‐ctrl, anti‐miR‐211, miR‐ctrl, and miR‐211. Black arrows indicate LAMP1‐positive structures in the lysosome‐like vesicles. Scale bar 500 nm. B, C Quantifications of the number of lysosome‐like structures and their size. Bar graphs indicate the means values ± SEM. Mann and Whitney test (anti‐miR‐ctrl versus anti‐miR‐211 and miR‐ctrl versus miR‐211) *** P ≤ 0.005. D Quantification of LAMP1 surface density. Bar graphs indicate the means values ± SEM. Mann and Whitney test (anti‐miR‐ctrl versus anti‐miR‐211 and miR‐ctrl versus miR‐211) *** P ≤ 0.005. E Representative Western blot analysis of the LAMP1, EZRIN, CLN5, TRPML1, SQSTM1/p62, CTSD, and LC3 proteins from anti‐miR‐ctrl, anti‐miR‐211, miR‐ctrl, and miR‐211 transfected cells. Note changes of both pro‐CTSD and its maturation CTSD heavy chain (hc) in blot assays. The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control. Bar graphs represent mean values ± SEM of at least 4 independent experiments. Mann and Whitney test (miR‐211 versus miR‐ctrl and anti‐miR‐211 versus anti‐miR‐ctrl) * P ≤ 0.05, *** P ≤ 0.005. F qRT‐PCR assay from transfected ARPE‐19 cells. The plot shows the expression level of miR‐211 normalized to the U6 snRNA control. Bar graphs represent mean values ± SEM of at least 4 independent experiments. Mann and Whitney test, ** P ≤ 0.01. G The plot shows the expression level of EZRIN normalized to the HPRT control. Bar graphs represent mean values ± SEM of at least 4 independent experiments. Mann and Whitney test, * P ≤ 0.05, *** P ≤ 0.005. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Light‐responsive microRNA miR‐211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance

    doi: 10.15252/embj.2019102468

    Figure Lengend Snippet: MiR‐211 participates in lysosomal biogenesis in vitro A Immuno‐electron microscopy (IEM) analysis for LAMP1 of ARPE‐19 cells transfected with anti‐miR‐ctrl, anti‐miR‐211, miR‐ctrl, and miR‐211. Black arrows indicate LAMP1‐positive structures in the lysosome‐like vesicles. Scale bar 500 nm. B, C Quantifications of the number of lysosome‐like structures and their size. Bar graphs indicate the means values ± SEM. Mann and Whitney test (anti‐miR‐ctrl versus anti‐miR‐211 and miR‐ctrl versus miR‐211) *** P ≤ 0.005. D Quantification of LAMP1 surface density. Bar graphs indicate the means values ± SEM. Mann and Whitney test (anti‐miR‐ctrl versus anti‐miR‐211 and miR‐ctrl versus miR‐211) *** P ≤ 0.005. E Representative Western blot analysis of the LAMP1, EZRIN, CLN5, TRPML1, SQSTM1/p62, CTSD, and LC3 proteins from anti‐miR‐ctrl, anti‐miR‐211, miR‐ctrl, and miR‐211 transfected cells. Note changes of both pro‐CTSD and its maturation CTSD heavy chain (hc) in blot assays. The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control. Bar graphs represent mean values ± SEM of at least 4 independent experiments. Mann and Whitney test (miR‐211 versus miR‐ctrl and anti‐miR‐211 versus anti‐miR‐ctrl) * P ≤ 0.05, *** P ≤ 0.005. F qRT‐PCR assay from transfected ARPE‐19 cells. The plot shows the expression level of miR‐211 normalized to the U6 snRNA control. Bar graphs represent mean values ± SEM of at least 4 independent experiments. Mann and Whitney test, ** P ≤ 0.01. G The plot shows the expression level of EZRIN normalized to the HPRT control. Bar graphs represent mean values ± SEM of at least 4 independent experiments. Mann and Whitney test, * P ≤ 0.05, *** P ≤ 0.005. Source data are available online for this figure.

    Article Snippet: For Western blot analysis, the following antibodies were used: mouse anti‐Lamp1 (1:500, Sigma‐Aldrich, L1418), rabbit anti‐Cln5 (1:1,000, Abcam ), rabbit anti‐Trpml1 (1:1,000, ALOMONE Lab ACC‐081), rabbit anti‐LC3 (1:1,000, Novus LC3B/MAP1LC3B), rabbit anti‐SQSTM1/p62 (1:1,000, Sigma‐Aldrich, P0067), mouse anti‐CtsD (1:1,000, Santa Cruz SC‐377124), mouse anti‐Ezrin (1:1,000, Novex, 357300), rabbit anti‐phospho‐Ezrin (Th567) that detects endogenous levels of Ezrin only when phosphorylated at threonine 567 (1:700 Sigma‐Aldrich, PA5‐37763), mouse anti‐Gapdh (1:1,000, Santa Cruz, SC‐32233), mouse anti‐β‐actin (1:700, Sigma‐Aldrich, A5441), anti‐TFEB (1:1,000 Cell Signaling, 4240), mouse anti‐FLAG (1:1,000, Sigma, F3165).

    Techniques: In Vitro, Immuno-Electron Microscopy, Transfection, Western Blot, Quantitative RT-PCR, Expressing

    Model of miR‐211‐mediated regulation of lysosomal biogenesis and function Under light‐phase conditions, Ezrin is repressed by miR‐211. The inhibition of Ezrin releases a Ca 2+ microdomain flux into cells thorough the TRPML1 channel. This leads to calcineurin (Cn) activation and lysosomal biogenesis via TFEB nuclear translocation. Under night‐phase conditions, miR‐211 is down‐regulated, this in turn induces Ezrin upregulation and repression of TFEB‐mediated lysosomal biogenesis. Both miR‐211 and Ezrin may converge on and integrate into the mTOR pathway.

    Journal: The EMBO Journal

    Article Title: Light‐responsive microRNA miR‐211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance

    doi: 10.15252/embj.2019102468

    Figure Lengend Snippet: Model of miR‐211‐mediated regulation of lysosomal biogenesis and function Under light‐phase conditions, Ezrin is repressed by miR‐211. The inhibition of Ezrin releases a Ca 2+ microdomain flux into cells thorough the TRPML1 channel. This leads to calcineurin (Cn) activation and lysosomal biogenesis via TFEB nuclear translocation. Under night‐phase conditions, miR‐211 is down‐regulated, this in turn induces Ezrin upregulation and repression of TFEB‐mediated lysosomal biogenesis. Both miR‐211 and Ezrin may converge on and integrate into the mTOR pathway.

    Article Snippet: For Western blot analysis, the following antibodies were used: mouse anti‐Lamp1 (1:500, Sigma‐Aldrich, L1418), rabbit anti‐Cln5 (1:1,000, Abcam ), rabbit anti‐Trpml1 (1:1,000, ALOMONE Lab ACC‐081), rabbit anti‐LC3 (1:1,000, Novus LC3B/MAP1LC3B), rabbit anti‐SQSTM1/p62 (1:1,000, Sigma‐Aldrich, P0067), mouse anti‐CtsD (1:1,000, Santa Cruz SC‐377124), mouse anti‐Ezrin (1:1,000, Novex, 357300), rabbit anti‐phospho‐Ezrin (Th567) that detects endogenous levels of Ezrin only when phosphorylated at threonine 567 (1:700 Sigma‐Aldrich, PA5‐37763), mouse anti‐Gapdh (1:1,000, Santa Cruz, SC‐32233), mouse anti‐β‐actin (1:700, Sigma‐Aldrich, A5441), anti‐TFEB (1:1,000 Cell Signaling, 4240), mouse anti‐FLAG (1:1,000, Sigma, F3165).

    Techniques: Inhibition, Activation Assay, Translocation Assay

    Pharmacological inhibition of Ezrin results in autophagy induction in ARPE‐19 cells Representative images from ARPE‐19 cells treated with DMSO and NSC668394 . All cells were fixed and stained with anti‐LC3 (green) and anti‐LAMP1 (red) antibodies and nuclei were counterstained with DAPI (blue). Scale bar 5 μm. The plot shows the quantification of the numbers of LC3/LAMP1‐positive vesicles per cell ( n ≥ 100). Values represent means ± SEM from three independent experiments. Student's t ‐test ( NSC668394 versus DMSO) *** P ≤ 0.005. Representative Western blots of LAMP1, EZRIN, EZRIN‐pT567, CLN5, TRPML1, SQSTM1/p62, CTSD, and LC3 proteins from DMSO or NSC668394 treated cells. Note an increase of both pro‐CTSD and its maturation CTSD heavy chain (hc). The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control. Bar graphs represent mean values ± SEM of at least n = 6 independent experiments. Mann and Whitney test ( NSC668394 versus DMSO) * P ≤ 0.05, *** P ≤ 0.005. Representative images of RFP‐GFP‐LC3 assay in ARPE‐19 cells transiently transfected with RFP‐GFP‐LC3 and treated with DMSO or NSC668394 . Nuclei were counterstained with DAPI (blue). Scale bars: 5 μm. Box plots showing quantitative analysis of RFP + GFP + puncta (Autophagosome) and RFP + GFP − puncta (autolysosome) ( n ≥ 100 cells) from three independent experiments. Box limits represent 25 th percentile and 75 th percentile; horizontal lines represent medians; whiskers display min. to max. values. Student's t ‐test ( NSC668394 versus DMSO) * P ≤ 0.05. Endolysosomal Cathepsin activity was revealed by incubation with Cathepsin B (CTSB) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis. Scale bar 10 μm. Graph showing the mean values of intensity of cresyl violet fluorescence ± SEM of representative data ( n ≥ 100 cells) from three independent experiments. Mann and Whitney test (NSC668394 versus DMSO) * P ≤ 0.05. Cathepsin B (CTSB) activity in lysates from NSC668394 ‐treated and DMSO‐control ARPE‐19 cells. Graph showing the percentage of values ± SEM of CTSB activity from 4 independent experiments. Mann and Whitney test ( NSC668394 versus DMSO) * P ≤ 0.05. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Light‐responsive microRNA miR‐211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance

    doi: 10.15252/embj.2019102468

    Figure Lengend Snippet: Pharmacological inhibition of Ezrin results in autophagy induction in ARPE‐19 cells Representative images from ARPE‐19 cells treated with DMSO and NSC668394 . All cells were fixed and stained with anti‐LC3 (green) and anti‐LAMP1 (red) antibodies and nuclei were counterstained with DAPI (blue). Scale bar 5 μm. The plot shows the quantification of the numbers of LC3/LAMP1‐positive vesicles per cell ( n ≥ 100). Values represent means ± SEM from three independent experiments. Student's t ‐test ( NSC668394 versus DMSO) *** P ≤ 0.005. Representative Western blots of LAMP1, EZRIN, EZRIN‐pT567, CLN5, TRPML1, SQSTM1/p62, CTSD, and LC3 proteins from DMSO or NSC668394 treated cells. Note an increase of both pro‐CTSD and its maturation CTSD heavy chain (hc). The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control. Bar graphs represent mean values ± SEM of at least n = 6 independent experiments. Mann and Whitney test ( NSC668394 versus DMSO) * P ≤ 0.05, *** P ≤ 0.005. Representative images of RFP‐GFP‐LC3 assay in ARPE‐19 cells transiently transfected with RFP‐GFP‐LC3 and treated with DMSO or NSC668394 . Nuclei were counterstained with DAPI (blue). Scale bars: 5 μm. Box plots showing quantitative analysis of RFP + GFP + puncta (Autophagosome) and RFP + GFP − puncta (autolysosome) ( n ≥ 100 cells) from three independent experiments. Box limits represent 25 th percentile and 75 th percentile; horizontal lines represent medians; whiskers display min. to max. values. Student's t ‐test ( NSC668394 versus DMSO) * P ≤ 0.05. Endolysosomal Cathepsin activity was revealed by incubation with Cathepsin B (CTSB) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis. Scale bar 10 μm. Graph showing the mean values of intensity of cresyl violet fluorescence ± SEM of representative data ( n ≥ 100 cells) from three independent experiments. Mann and Whitney test (NSC668394 versus DMSO) * P ≤ 0.05. Cathepsin B (CTSB) activity in lysates from NSC668394 ‐treated and DMSO‐control ARPE‐19 cells. Graph showing the percentage of values ± SEM of CTSB activity from 4 independent experiments. Mann and Whitney test ( NSC668394 versus DMSO) * P ≤ 0.05. Source data are available online for this figure.

    Article Snippet: For Western blot analysis, the following antibodies were used: mouse anti‐Lamp1 (1:500, Sigma‐Aldrich, L1418), rabbit anti‐Cln5 (1:1,000, Abcam ), rabbit anti‐Trpml1 (1:1,000, ALOMONE Lab ACC‐081), rabbit anti‐LC3 (1:1,000, Novus LC3B/MAP1LC3B), rabbit anti‐SQSTM1/p62 (1:1,000, Sigma‐Aldrich, P0067), mouse anti‐CtsD (1:1,000, Santa Cruz SC‐377124), mouse anti‐Ezrin (1:1,000, Novex, 357300), rabbit anti‐phospho‐Ezrin (Th567) that detects endogenous levels of Ezrin only when phosphorylated at threonine 567 (1:700 Sigma‐Aldrich, PA5‐37763), mouse anti‐Gapdh (1:1,000, Santa Cruz, SC‐32233), mouse anti‐β‐actin (1:700, Sigma‐Aldrich, A5441), anti‐TFEB (1:1,000 Cell Signaling, 4240), mouse anti‐FLAG (1:1,000, Sigma, F3165).

    Techniques: Inhibition, Staining, Western Blot, Transfection, Activity Assay, Incubation, Fluorescence

    Autophagy enhancement and apoptosis inhibition by TRPML1 activation. (a–d) Representative immunoblots of beclin-1, LC3, and p62 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (a, n = 6) or transfected with TRPML1 lentivirus (c, n = 6) 1 hr after OGD. (b) or (d) was the statistics for (a) or (c). (e–h) Representative immunoblots of cleaved caspase3 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (e, n = 6) or transfected with TRPML1 lentivirus (g, n = 6) 1 hr after OGD. (f) or (h) was the statistics for (e) or (g). β -Actin serves as a loading control. All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia

    doi: 10.1155/2018/4612727

    Figure Lengend Snippet: Autophagy enhancement and apoptosis inhibition by TRPML1 activation. (a–d) Representative immunoblots of beclin-1, LC3, and p62 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (a, n = 6) or transfected with TRPML1 lentivirus (c, n = 6) 1 hr after OGD. (b) or (d) was the statistics for (a) or (c). (e–h) Representative immunoblots of cleaved caspase3 from cultured hippocampal neurons preincubated with 10 μ M ML-SA1 (e, n = 6) or transfected with TRPML1 lentivirus (g, n = 6) 1 hr after OGD. (f) or (h) was the statistics for (e) or (g). β -Actin serves as a loading control. All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗ P

    Article Snippet: Drugs and Antibodies Antibodies were used as follows: TRPML1 from Alomone Labs (acc-081), GFAP from Sigma (G3893), NeuN from Abcam (ab177487), HA from Sigma (H6908), LC3 from Abcam (ab192890), beclin-1 from Cell Signaling Technology (#3495), p62 from Proteintech (18420-1-AP), cleaved caspase3 from Cell Signaling Technology (#9661), and β -actin from Abcam (ab8227).

    Techniques: Inhibition, Activation Assay, Western Blot, Cell Culture, Transfection

    Reduction of the infarct volumes and mortality by TRPML1 activation. (a, b) Infarct volumes of mice infusion with or without 5 μ M ML-SA1after 12 hrs reperfusion ( n = 6). (b) was the statistics for (a). (c) Evaluation of neurological deficit after 24 hrs reperfusion was evaluated in mice infusion with or without 5 μ M ML-SA1 ( n = 7). (d) Evaluation of the survival rate in mice infusion with or without 5 μ M ML-SA1 after tBCCAO ( n = 8). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia

    doi: 10.1155/2018/4612727

    Figure Lengend Snippet: Reduction of the infarct volumes and mortality by TRPML1 activation. (a, b) Infarct volumes of mice infusion with or without 5 μ M ML-SA1after 12 hrs reperfusion ( n = 6). (b) was the statistics for (a). (c) Evaluation of neurological deficit after 24 hrs reperfusion was evaluated in mice infusion with or without 5 μ M ML-SA1 ( n = 7). (d) Evaluation of the survival rate in mice infusion with or without 5 μ M ML-SA1 after tBCCAO ( n = 8). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗∗ P

    Article Snippet: Drugs and Antibodies Antibodies were used as follows: TRPML1 from Alomone Labs (acc-081), GFAP from Sigma (G3893), NeuN from Abcam (ab177487), HA from Sigma (H6908), LC3 from Abcam (ab192890), beclin-1 from Cell Signaling Technology (#3495), p62 from Proteintech (18420-1-AP), cleaved caspase3 from Cell Signaling Technology (#9661), and β -actin from Abcam (ab8227).

    Techniques: Activation Assay, Mouse Assay

    Proteasome-mediated degradation of TRPML1 of hippocampal neurons in ischemia. (a–d) Representative immunoblots of TRPML1 from cultured hippocampal neurons (a, n = 4) or astrocytes (c, n = 4) 0.5 or 1 hrs after OGD. (b) or (d) was the statistics for (a) or (c). (e, f) Immunoblots of analysis of the indicated marker expression in sorted neurons (e) and astrocytes (f) (GFAP: astrocyte marker, NeuN: neuron marker). (g–j) Representative immunoblots of TRPML1 from sorted neurons (g, n = 9) or astrocytes (i, n = 9) 40 min after tBCCAo. (h) or (j) was the statistics for (g) or (i). (k) Quantitative PCR analysis of TRPML1 mRNA levels in hippocampal neurons 1 hr after OGD ( n = 5). (l, m) Representative immunoblots of TRPML1 from cultured hippocampal neurons ( n = 5) incubated with/without calpeptin (10 μ M), DEVD (10 μ M), or MG132 (5 μ M). (m) was the statistics for (l). β -Actin serves as a loading control. All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test (b, d, and k), Student's t -test, two tailed (h, j), or two-way ANOVA with Bonferroni post hoc test (m). ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia

    doi: 10.1155/2018/4612727

    Figure Lengend Snippet: Proteasome-mediated degradation of TRPML1 of hippocampal neurons in ischemia. (a–d) Representative immunoblots of TRPML1 from cultured hippocampal neurons (a, n = 4) or astrocytes (c, n = 4) 0.5 or 1 hrs after OGD. (b) or (d) was the statistics for (a) or (c). (e, f) Immunoblots of analysis of the indicated marker expression in sorted neurons (e) and astrocytes (f) (GFAP: astrocyte marker, NeuN: neuron marker). (g–j) Representative immunoblots of TRPML1 from sorted neurons (g, n = 9) or astrocytes (i, n = 9) 40 min after tBCCAo. (h) or (j) was the statistics for (g) or (i). (k) Quantitative PCR analysis of TRPML1 mRNA levels in hippocampal neurons 1 hr after OGD ( n = 5). (l, m) Representative immunoblots of TRPML1 from cultured hippocampal neurons ( n = 5) incubated with/without calpeptin (10 μ M), DEVD (10 μ M), or MG132 (5 μ M). (m) was the statistics for (l). β -Actin serves as a loading control. All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test (b, d, and k), Student's t -test, two tailed (h, j), or two-way ANOVA with Bonferroni post hoc test (m). ∗ P

    Article Snippet: Drugs and Antibodies Antibodies were used as follows: TRPML1 from Alomone Labs (acc-081), GFAP from Sigma (G3893), NeuN from Abcam (ab177487), HA from Sigma (H6908), LC3 from Abcam (ab192890), beclin-1 from Cell Signaling Technology (#3495), p62 from Proteintech (18420-1-AP), cleaved caspase3 from Cell Signaling Technology (#9661), and β -actin from Abcam (ab8227).

    Techniques: Western Blot, Cell Culture, Marker, Expressing, Real-time Polymerase Chain Reaction, Incubation, Two Tailed Test

    Reduction of neuron survival in OGD by TRPML1 degradation. (a) Hippocampal neuron survival rate determined by MTT assay after 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (b) Immunoblot analysis of HA-TRPML1 expression in hippocampal neuron. (c) The survival rate of hippocampal neuron transfected with TRPML1 lentivirus determined by MTT assay after 3 hrs OGD ( n = 5). β -Actin serves as a loading control. (d, f) Hippocampal neuron apoptosis was analyzed by flow cytometry 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (e, g) The apoptosis of hippocampal neuron transfected with TRPML1 lentivirus was determined by flow cytometry after 3 hrs OGD ( n = 4). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia

    doi: 10.1155/2018/4612727

    Figure Lengend Snippet: Reduction of neuron survival in OGD by TRPML1 degradation. (a) Hippocampal neuron survival rate determined by MTT assay after 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (b) Immunoblot analysis of HA-TRPML1 expression in hippocampal neuron. (c) The survival rate of hippocampal neuron transfected with TRPML1 lentivirus determined by MTT assay after 3 hrs OGD ( n = 5). β -Actin serves as a loading control. (d, f) Hippocampal neuron apoptosis was analyzed by flow cytometry 3 hrs OGD in the preincubation with ML-SA1 (10 μ M, n = 4). (e, g) The apoptosis of hippocampal neuron transfected with TRPML1 lentivirus was determined by flow cytometry after 3 hrs OGD ( n = 4). All data were presented as mean ± SEM. All comparisons between groups for statistical significance were performed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. ∗∗ P

    Article Snippet: Drugs and Antibodies Antibodies were used as follows: TRPML1 from Alomone Labs (acc-081), GFAP from Sigma (G3893), NeuN from Abcam (ab177487), HA from Sigma (H6908), LC3 from Abcam (ab192890), beclin-1 from Cell Signaling Technology (#3495), p62 from Proteintech (18420-1-AP), cleaved caspase3 from Cell Signaling Technology (#9661), and β -actin from Abcam (ab8227).

    Techniques: MTT Assay, Expressing, Transfection, Flow Cytometry, Cytometry

    Lycorine reduces TFEB nuclear translocation by attenuating oxidation of TRPML1 via decreased mitochondrial ROS upon LPS stimulation in OC. BMMs were prepared, incubated with RANKL (40 ng/ml) in the presence of M-CSF (30 ng/ml) for 40 h, washed, and then incubated further with the indicated conditions (DPI, 5 nM; Mito-TEMPO, 100 nM; lycorine, 1.6 μ M) in the presence of LPS (50 ng/ml) and M-CSF (30 ng/ml). Mitochondrial ROS and cytosolic ROS were determined by flow cytometry using MitoSOX Red after 16 h and using H 2 DCF-DA after 24 h, respectively (a, b). After 48 h, cell lysates were prepared (c) or fixed (d). Cell lysates were subjected to Western blot to determine p62 and LC3II with the addition of bafilomycin A1 (25 nM) for 4 h. The quantified levels of p62 and LC3II are shown normalized to β -actin (c). Cell lysates were labeled with N -(biotinoyl)- N ′-(iodoacetyl) ethylenediamine, and TRPML1 was immunoprecipitated (IP) from each sample. HRP-streptavidin immunoblotting was performed to evaluate the reduced form of TRPML1 (e). Cells were transfected with 50 nM of scRNA or siTRPML1 and incubated further for 48 h with LPS and M-CSF. siRNA-mediated silencing of TRPML1 was confirmed by RT-PCR and qPCR (f). After fixation, more than 70 TRAP-positive MNCs in each culture were randomly selected to determine the area and fusion activity of the formed OCs (d, f). Whole cell extracts, cytoplasmic fractions, and nuclear fractions were harvested from cultured cells and subjected to Western blot analysis with anti-TFEB Ab. Abs for β -actin and lamin B1 were used for the normalization of cytoplasmic and nuclear extracts, respectively. Quantification of TFEB normalized to β -actin or lamin B1 was plotted (g). ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Lycorine Attenuates Autophagy in Osteoclasts via an Axis of mROS/TRPML1/TFEB to Reduce LPS-Induced Bone Loss

    doi: 10.1155/2019/8982147

    Figure Lengend Snippet: Lycorine reduces TFEB nuclear translocation by attenuating oxidation of TRPML1 via decreased mitochondrial ROS upon LPS stimulation in OC. BMMs were prepared, incubated with RANKL (40 ng/ml) in the presence of M-CSF (30 ng/ml) for 40 h, washed, and then incubated further with the indicated conditions (DPI, 5 nM; Mito-TEMPO, 100 nM; lycorine, 1.6 μ M) in the presence of LPS (50 ng/ml) and M-CSF (30 ng/ml). Mitochondrial ROS and cytosolic ROS were determined by flow cytometry using MitoSOX Red after 16 h and using H 2 DCF-DA after 24 h, respectively (a, b). After 48 h, cell lysates were prepared (c) or fixed (d). Cell lysates were subjected to Western blot to determine p62 and LC3II with the addition of bafilomycin A1 (25 nM) for 4 h. The quantified levels of p62 and LC3II are shown normalized to β -actin (c). Cell lysates were labeled with N -(biotinoyl)- N ′-(iodoacetyl) ethylenediamine, and TRPML1 was immunoprecipitated (IP) from each sample. HRP-streptavidin immunoblotting was performed to evaluate the reduced form of TRPML1 (e). Cells were transfected with 50 nM of scRNA or siTRPML1 and incubated further for 48 h with LPS and M-CSF. siRNA-mediated silencing of TRPML1 was confirmed by RT-PCR and qPCR (f). After fixation, more than 70 TRAP-positive MNCs in each culture were randomly selected to determine the area and fusion activity of the formed OCs (d, f). Whole cell extracts, cytoplasmic fractions, and nuclear fractions were harvested from cultured cells and subjected to Western blot analysis with anti-TFEB Ab. Abs for β -actin and lamin B1 were used for the normalization of cytoplasmic and nuclear extracts, respectively. Quantification of TFEB normalized to β -actin or lamin B1 was plotted (g). ∗ p

    Article Snippet: Transfection of siRNA After treatment with M-CSF and RANKL for 40 h, BMMs were transfected with small interfering RNA (siRNA) against TRPML1 or with scrambled siRNA (scRNA) using Lipofectamine 3000.

    Techniques: Translocation Assay, Incubation, Flow Cytometry, Cytometry, Western Blot, Labeling, Immunoprecipitation, Transfection, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Activity Assay, Cell Culture