trpml1  (Alomone Labs)


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    Alomone Labs trpml1
    Lycorine reduces TFEB nuclear translocation by attenuating oxidation of <t>TRPML1</t> 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
    Trpml1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    trpml1 - by Bioz Stars, 2022-06
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    1) 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

    2) 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

    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

    4) 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

    5) 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

    6) 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

    7) Product Images from "TRPML1 Channels Initiate Ca2+ Sparks in Vascular Smooth Muscle Cells"

    Article Title: TRPML1 Channels Initiate Ca2+ Sparks in Vascular Smooth Muscle Cells

    Journal: Science signaling

    doi: 10.1126/scisignal.aba1015

    TRPML1 channels, are present in vascular SMCs. A) RT-qPCR analysis of Mcoln1, Mcoln2, and Mcoln3 mRNA expression levels in homogeneous populations of native cerebral artery SMCs. Data are normalized to Actb , encoding β-actin (N = 3 animals/group). B) RT-qPCR analysis of Mcoln1, Mcoln2, and Mcoln3 mRNA expression levels in cerebral arteries from WT and Mcoln1 −/− mice. Data are normalized to Actb (N = 3 animals/group; *P ≤ 0.05). C) RT-qPCR analysis of Mcoln1, Mcoln2, and Mcoln3 mRNA expression levels in mesenteric arteries from WT and Mcoln1 −/− mice. Data are normalized to Actb (N= 3 animals/group; *P ≤ 0.05). D) Representative results of Wes protein analysis using cerebral artery lysates from WT and Mcoln1 −/− mice. Samples from WT animals probed with a primary antibody against TRPML1 generated a single band at the expected molecular weight of ~60 kDa that was absent in lysates from Mcoln1 −/− mice. Lysates were probed for β-actin as a loading control. Representative of 3 independent experiments using tissue from N = 3 mice. All data are shown as mean ± SEM.
    Figure Legend Snippet: TRPML1 channels, are present in vascular SMCs. A) RT-qPCR analysis of Mcoln1, Mcoln2, and Mcoln3 mRNA expression levels in homogeneous populations of native cerebral artery SMCs. Data are normalized to Actb , encoding β-actin (N = 3 animals/group). B) RT-qPCR analysis of Mcoln1, Mcoln2, and Mcoln3 mRNA expression levels in cerebral arteries from WT and Mcoln1 −/− mice. Data are normalized to Actb (N = 3 animals/group; *P ≤ 0.05). C) RT-qPCR analysis of Mcoln1, Mcoln2, and Mcoln3 mRNA expression levels in mesenteric arteries from WT and Mcoln1 −/− mice. Data are normalized to Actb (N= 3 animals/group; *P ≤ 0.05). D) Representative results of Wes protein analysis using cerebral artery lysates from WT and Mcoln1 −/− mice. Samples from WT animals probed with a primary antibody against TRPML1 generated a single band at the expected molecular weight of ~60 kDa that was absent in lysates from Mcoln1 −/− mice. Lysates were probed for β-actin as a loading control. Representative of 3 independent experiments using tissue from N = 3 mice. All data are shown as mean ± SEM.

    Techniques Used: Quantitative RT-PCR, Expressing, Mouse Assay, Generated, Molecular Weight

    SMCs from Mcoln1 −/− mice lack spontaneous Ca 2+ sparks. A) Representative time course images of a Ca 2+ spark site (seconds; scale bar = 10 µm) and trace showing changes in fractional fluorescence (F/F 0 ) as a function of time for a region of interest (white box) that includes an active Ca 2+ spark site recorded from native cerebral artery SMC isolated from a WT mouse. B) Representative time course images (seconds; scale bar = 10 µm) and trace recorded from native cerebral artery SMC isolated from a Mcoln1 −/− mouse showing lack of Ca 2+ sparks. C) Number of spontaneous Ca 2+ spark sites per native cerebral artery SMC from WT and Mcoln1 −/− mice (n = 13 cells from 4 WT animals, n = 19 cells from 4 Mcoln1 −/− animals; *P ≤ 0.05). D) Mean spontaneous Ca 2+ spark frequency in SMCs from WT and Mcoln1 −/− mice (n = 13 cells from 4 WT animals, n = 19 cells from 4 Mcoln1 −/− animals; *P ≤ 0.05). E) Total SR Ca 2+ store load in SMCs from WT and Mcoln1 −/− mice, assessed by imaging changes in global intracellular [Ca 2+ ] in response to administration of caffeine (10 mM) (n = 7 cells from 3 WT mice; n = 8 cells from 3 Mcoln1 −/− mice).
    Figure Legend Snippet: SMCs from Mcoln1 −/− mice lack spontaneous Ca 2+ sparks. A) Representative time course images of a Ca 2+ spark site (seconds; scale bar = 10 µm) and trace showing changes in fractional fluorescence (F/F 0 ) as a function of time for a region of interest (white box) that includes an active Ca 2+ spark site recorded from native cerebral artery SMC isolated from a WT mouse. B) Representative time course images (seconds; scale bar = 10 µm) and trace recorded from native cerebral artery SMC isolated from a Mcoln1 −/− mouse showing lack of Ca 2+ sparks. C) Number of spontaneous Ca 2+ spark sites per native cerebral artery SMC from WT and Mcoln1 −/− mice (n = 13 cells from 4 WT animals, n = 19 cells from 4 Mcoln1 −/− animals; *P ≤ 0.05). D) Mean spontaneous Ca 2+ spark frequency in SMCs from WT and Mcoln1 −/− mice (n = 13 cells from 4 WT animals, n = 19 cells from 4 Mcoln1 −/− animals; *P ≤ 0.05). E) Total SR Ca 2+ store load in SMCs from WT and Mcoln1 −/− mice, assessed by imaging changes in global intracellular [Ca 2+ ] in response to administration of caffeine (10 mM) (n = 7 cells from 3 WT mice; n = 8 cells from 3 Mcoln1 −/− mice).

    Techniques Used: Mouse Assay, Fluorescence, Isolation, Imaging

    SMCs from Mcoln1 −/− mice lack spontaneous Ca 2+ -activated BK channel activity A) Representative traces of STOCs recorded from voltage-clamped (−40 mV) native cerebral artery SMCs isolated from WT and Mcoln1 −/− mice. B and C) Summary of mean STOC frequency (B) and amplitude (C) over a range of holding potentials (−60 to −20 mV) (n = 9 cells from 3 WT animals, n = 11 cells from 4 Mcoln1 −/− animals; *P ≤ 0.05). D) Representative traces of STOCs recorded from voltage-clamped (−30 mV) SMCs isolated from WT and Mcoln1 −/− mice that were treated with the selective TRPML channel activator MK6–83 (1 μM). E) Summary of the effects of MK6–83 on STOC frequency (n = 5 cells from 3 animals for both groups; *P ≤ 0.05 compared to WT + Vehicle). F) Representative traces of STOCs recorded from voltage-clamped (−30 mV) SMCs isolated from WT and Mcoln1 −/− mice that were treated with the selective TRPV4 channel activator GSK1016790A (100 nM). G) Summary of the effects of GSK1016790A on STOC frequency (n = 7 cells from 3 WT mice, n = 8 cells from 3 Mcoln1 −/− mice; *P ≤ 0.05 compared to WT + Vehicle, # P ≤ 0.05 compared to Mcoln1 −/− + Vehicle). All data are shown as mean ± SEM.
    Figure Legend Snippet: SMCs from Mcoln1 −/− mice lack spontaneous Ca 2+ -activated BK channel activity A) Representative traces of STOCs recorded from voltage-clamped (−40 mV) native cerebral artery SMCs isolated from WT and Mcoln1 −/− mice. B and C) Summary of mean STOC frequency (B) and amplitude (C) over a range of holding potentials (−60 to −20 mV) (n = 9 cells from 3 WT animals, n = 11 cells from 4 Mcoln1 −/− animals; *P ≤ 0.05). D) Representative traces of STOCs recorded from voltage-clamped (−30 mV) SMCs isolated from WT and Mcoln1 −/− mice that were treated with the selective TRPML channel activator MK6–83 (1 μM). E) Summary of the effects of MK6–83 on STOC frequency (n = 5 cells from 3 animals for both groups; *P ≤ 0.05 compared to WT + Vehicle). F) Representative traces of STOCs recorded from voltage-clamped (−30 mV) SMCs isolated from WT and Mcoln1 −/− mice that were treated with the selective TRPV4 channel activator GSK1016790A (100 nM). G) Summary of the effects of GSK1016790A on STOC frequency (n = 7 cells from 3 WT mice, n = 8 cells from 3 Mcoln1 −/− mice; *P ≤ 0.05 compared to WT + Vehicle, # P ≤ 0.05 compared to Mcoln1 −/− + Vehicle). All data are shown as mean ± SEM.

    Techniques Used: Mouse Assay, Activity Assay, Isolation

    Super-resolution imaging demonstrates nanoscale colocalization of TRPML1 and RyR2 in native SMCs. A–C) Representative super-resolution localization maps of native, contractile cerebral artery SMCs co-immunolabeled for Lamp-1 and TRPML1 (A), Lamp-1 and RyR2 (B), or TRPML1 and RyR2 (C). Scale bars = 3 μm. Representative of n= 8–10 cells isolated from N = 3 animals. The second column of images shows a magnified view of the region enclosed in the white boxes. Scale bars = 1 μm. Insets show magnified views of the indicated regions of interest. Scale bars = 0.1 μm. D and E) Histograms showing the distribution of the surface areas of individual protein clusters for TRPML1 (D) and RyR2 (E) (TRPML1, n = 6143 clusters; RyR2, n = 35432 clusters). F) TRPML1 and RyR2 protein cluster density (n = 19 cells from N = 6 animals/group; *P ≤ 0.05) G) Histogram showing the area distribution of Lamp-1–positive LELs (n = 216 ovoids). H) Nearest neighbor analysis showing the distance between the center of RyR2 protein clusters and the edge of Lamp-1–positive LELs (n = 1409 RyR2 protein clusters). I) Object-based analysis comparing the fraction of TRPML1 and RyR2 co-localizing clusters with the fraction of clusters that co-localize in a simulated random distribution of RyR2 protein clusters (TRPML1-RyR2, 1.51 ± 0.12 %; Random, 0.44 ± 0.07 %; n = 10 cells from 3 animals; *P ≤ 0.05). All data are shown as mean ± SEM.
    Figure Legend Snippet: Super-resolution imaging demonstrates nanoscale colocalization of TRPML1 and RyR2 in native SMCs. A–C) Representative super-resolution localization maps of native, contractile cerebral artery SMCs co-immunolabeled for Lamp-1 and TRPML1 (A), Lamp-1 and RyR2 (B), or TRPML1 and RyR2 (C). Scale bars = 3 μm. Representative of n= 8–10 cells isolated from N = 3 animals. The second column of images shows a magnified view of the region enclosed in the white boxes. Scale bars = 1 μm. Insets show magnified views of the indicated regions of interest. Scale bars = 0.1 μm. D and E) Histograms showing the distribution of the surface areas of individual protein clusters for TRPML1 (D) and RyR2 (E) (TRPML1, n = 6143 clusters; RyR2, n = 35432 clusters). F) TRPML1 and RyR2 protein cluster density (n = 19 cells from N = 6 animals/group; *P ≤ 0.05) G) Histogram showing the area distribution of Lamp-1–positive LELs (n = 216 ovoids). H) Nearest neighbor analysis showing the distance between the center of RyR2 protein clusters and the edge of Lamp-1–positive LELs (n = 1409 RyR2 protein clusters). I) Object-based analysis comparing the fraction of TRPML1 and RyR2 co-localizing clusters with the fraction of clusters that co-localize in a simulated random distribution of RyR2 protein clusters (TRPML1-RyR2, 1.51 ± 0.12 %; Random, 0.44 ± 0.07 %; n = 10 cells from 3 animals; *P ≤ 0.05). All data are shown as mean ± SEM.

    Techniques Used: Imaging, Immunolabeling, Isolation

    Mcoln1 −/− mice are spontaneously hypertensive. A) Averaged systolic BP measurements over a 48-hour period in conscious radiotelemetered WT and Mcoln1 −/− mice. Shaded regions depict night cycles. B) Mean systolic BP of WT and Mcoln1 −/− mice during day and night cycles (n = 7 for WT, n = 5 for Mcoln1 −/− ; *P ≤ 0.05 compared to WT day, # P ≤ 0.05 compared to WT night). C) Averaged diastolic BP measurements over a 48-hour period in conscious radio telemetered WT and Mcoln1 −/− mice. D) Mean diastolic BP in WT and Mcoln1 −/− mice during day and night cycles (n = 7 for WT, n = 5 for Mcoln1 −/− ). E) Averaged MAP measurements over a 48-hour period in conscious radio telemetered WT and Mcoln1 −/− mice. F) Mean MAP in WT and Mcoln1 −/− mice during day and night cycles (n = 7 for WT, n = 5 for Mcoln1 −/− ; *P ≤ 0.05 compared to WT day, # P ≤ 0.05 compared to WT night). 48-hour recordings are shown as mean; bar graphs are shown as mean ± SEM.
    Figure Legend Snippet: Mcoln1 −/− mice are spontaneously hypertensive. A) Averaged systolic BP measurements over a 48-hour period in conscious radiotelemetered WT and Mcoln1 −/− mice. Shaded regions depict night cycles. B) Mean systolic BP of WT and Mcoln1 −/− mice during day and night cycles (n = 7 for WT, n = 5 for Mcoln1 −/− ; *P ≤ 0.05 compared to WT day, # P ≤ 0.05 compared to WT night). C) Averaged diastolic BP measurements over a 48-hour period in conscious radio telemetered WT and Mcoln1 −/− mice. D) Mean diastolic BP in WT and Mcoln1 −/− mice during day and night cycles (n = 7 for WT, n = 5 for Mcoln1 −/− ). E) Averaged MAP measurements over a 48-hour period in conscious radio telemetered WT and Mcoln1 −/− mice. F) Mean MAP in WT and Mcoln1 −/− mice during day and night cycles (n = 7 for WT, n = 5 for Mcoln1 −/− ; *P ≤ 0.05 compared to WT day, # P ≤ 0.05 compared to WT night). 48-hour recordings are shown as mean; bar graphs are shown as mean ± SEM.

    Techniques Used: Mouse Assay

    The majority of LELs in contractile SMCs are immobile. A) Representative image of a native contractile cerebral artery SMC isolated from a WT mouse stained with LysoTracker. Scale bar = 10 μm. B and C) Histogram of the total displacement (B) and particle speed (C) of individual LysoTracker-labeled particles within native contractile SMCs isolated from WT mice during a 25-minute recording period. A total of 298 particles (n = 12 cells) were tracked. D) Representative image of a proliferative cerebral artery SMC isolated from a WT mouse stained with LysoTracker. Scale bar = 10 µm. E and F) Histogram of the total displacement (E) and particle speed (F) of individual LysoTracker-labeled structures within proliferative SMCs isolated from WT mice during a 25-minute recording period. A total of 1953 particles (n = 15 cells) were tracked. G) Representative image of a native contractile cerebral artery SMC isolated from a Mcoln1 −/− mouse stained with LysoTracker. Scale bar = 10 μm. H and I) Histogram of the total displacement (H) and particle speed (I) of individual LysoTracker-labeled particles within native contractile SMCs isolated from Mcoln1 −/− mice during a 25-minute recording period. A total of 73 particles (n = 6 cells) were tracked. J) Representative image of a proliferative cerebral artery SMC isolated from a Mcoln1 −/− mouse stained with LysoTracker. Scale bar = 10 µm. K and L) Histogram of the total displacement (K) and particle speed (L) of individual LysoTracker-labeled structures within proliferative SMCs isolated from Mcoln1 −/− mice during a 25-minute recording period. A total of 748 particles (n = 7 cells) were tracked. M and N) Comparison of mean particle displacement (M) and speed (N) in contractile and proliferative SMCs isolated from WT and Mcoln1 −/− mice. A total of 298 particles (n = 12 cells) from contractile SMCs isolated from WT mice, 1953 particles (n = 15 cells) from proliferative SMCs isolated from WT mice, 73 particles (n = 6 cells) from contractile SMCs isolated from Mcoln1 −/− mice, and 748 particles (n = 7 cells) from proliferative SMCs isolated from Mcoln1 −/− mice were tracked (*P ≤ 0.05 between Contractile WT compared to Proliferative WT; # P ≤ 0.05 between Contractile WT compared to Contractile Mcoln1 −/− ; **P ≤ 0.05 between Contractile Mcoln1 −/− compared to Proliferative Mcoln1 −/− ; ## P ≤ 0.05 between Proliferative WT compared to Proliferative Mcoln1 −/− ). O) Total LysoTracker-positive particles per cell (n = 12 contractile SMCs from WT mice, n = 15 proliferative SMCs from WT mice, n = 6 contractile SMCs from Mcoln1 −/− mice, and n = 7 proliferative S MCs from Mcoln1 −/− mice; *P ≤ 0.05 between Contractile WT vs Proliferative WT; **P ≤ 0.05 between Contractile Mcoln1 −/− vs Proliferative Mcoln1 −/− ). All data are shown as mean ± SEM.
    Figure Legend Snippet: The majority of LELs in contractile SMCs are immobile. A) Representative image of a native contractile cerebral artery SMC isolated from a WT mouse stained with LysoTracker. Scale bar = 10 μm. B and C) Histogram of the total displacement (B) and particle speed (C) of individual LysoTracker-labeled particles within native contractile SMCs isolated from WT mice during a 25-minute recording period. A total of 298 particles (n = 12 cells) were tracked. D) Representative image of a proliferative cerebral artery SMC isolated from a WT mouse stained with LysoTracker. Scale bar = 10 µm. E and F) Histogram of the total displacement (E) and particle speed (F) of individual LysoTracker-labeled structures within proliferative SMCs isolated from WT mice during a 25-minute recording period. A total of 1953 particles (n = 15 cells) were tracked. G) Representative image of a native contractile cerebral artery SMC isolated from a Mcoln1 −/− mouse stained with LysoTracker. Scale bar = 10 μm. H and I) Histogram of the total displacement (H) and particle speed (I) of individual LysoTracker-labeled particles within native contractile SMCs isolated from Mcoln1 −/− mice during a 25-minute recording period. A total of 73 particles (n = 6 cells) were tracked. J) Representative image of a proliferative cerebral artery SMC isolated from a Mcoln1 −/− mouse stained with LysoTracker. Scale bar = 10 µm. K and L) Histogram of the total displacement (K) and particle speed (L) of individual LysoTracker-labeled structures within proliferative SMCs isolated from Mcoln1 −/− mice during a 25-minute recording period. A total of 748 particles (n = 7 cells) were tracked. M and N) Comparison of mean particle displacement (M) and speed (N) in contractile and proliferative SMCs isolated from WT and Mcoln1 −/− mice. A total of 298 particles (n = 12 cells) from contractile SMCs isolated from WT mice, 1953 particles (n = 15 cells) from proliferative SMCs isolated from WT mice, 73 particles (n = 6 cells) from contractile SMCs isolated from Mcoln1 −/− mice, and 748 particles (n = 7 cells) from proliferative SMCs isolated from Mcoln1 −/− mice were tracked (*P ≤ 0.05 between Contractile WT compared to Proliferative WT; # P ≤ 0.05 between Contractile WT compared to Contractile Mcoln1 −/− ; **P ≤ 0.05 between Contractile Mcoln1 −/− compared to Proliferative Mcoln1 −/− ; ## P ≤ 0.05 between Proliferative WT compared to Proliferative Mcoln1 −/− ). O) Total LysoTracker-positive particles per cell (n = 12 contractile SMCs from WT mice, n = 15 proliferative SMCs from WT mice, n = 6 contractile SMCs from Mcoln1 −/− mice, and n = 7 proliferative S MCs from Mcoln1 −/− mice; *P ≤ 0.05 between Contractile WT vs Proliferative WT; **P ≤ 0.05 between Contractile Mcoln1 −/− vs Proliferative Mcoln1 −/− ). All data are shown as mean ± SEM.

    Techniques Used: Isolation, Staining, Labeling, Mouse Assay

    Resistance arteries from Mcoln1 −/− mice are hypercontractile. A) Representative recordings of pressure-induced constriction of cerebral arteries from WT and Mcoln1 −/ − mice. Traces show luminal diameter as a function of time in response to stepwise changes in intraluminal pressure from 5 to 125 mmHg. The diameter of the same arteries superfused with Ca 2+ -containing and Ca 2+ -free physiological salt solution is shown to indicate the active and passive effects, respectively, of intraluminal pressure on vessel diameter. B) Summary data showing the myogenic tone of cerebral arteries from WT and Mcoln1 −/− mice as a function of intraluminal pressure (n = 9–11 arteries from 5–7 animals/group; *P ≤ 0.05). C) Representative recordings of the thromboxane A 2 receptor agonist U46619-induced constriction of cerebral arteries from WT and Mcoln1 −/ − mice. D) Summary data showing the contraction produced by U46619 in cerebral arteries from WT and Mcoln1 −/− mice (n = 8 arteries from 4–5 animals/group; *P ≤ 0.05). E) Representative recordings of pressure-induced constriction of mesenteric resistance arteries from WT and Mcoln1 −/ − mice. F) Summary data showing the myogenic tone of mesenteric arteries from WT and Mcoln1 −/− mice as a function of intraluminal pressure (n = 7–9 arteries from 4–6 animals/group; *P ≤ 0.05). G) Representative recordings of α 1 -adrenergic receptor agonist phenylephrine (PE)-induced constriction of mesenteric resistance arteries from WT and Mcoln1 −/ − mice. H) Summary data showing the contraction produced by PE in mesenteric arteries from WT and Mcoln1 −/− mice (n = 7–8 arteries from 3 animals/group; *P ≤ 0.05). All data are shown as mean ± SEM.
    Figure Legend Snippet: Resistance arteries from Mcoln1 −/− mice are hypercontractile. A) Representative recordings of pressure-induced constriction of cerebral arteries from WT and Mcoln1 −/ − mice. Traces show luminal diameter as a function of time in response to stepwise changes in intraluminal pressure from 5 to 125 mmHg. The diameter of the same arteries superfused with Ca 2+ -containing and Ca 2+ -free physiological salt solution is shown to indicate the active and passive effects, respectively, of intraluminal pressure on vessel diameter. B) Summary data showing the myogenic tone of cerebral arteries from WT and Mcoln1 −/− mice as a function of intraluminal pressure (n = 9–11 arteries from 5–7 animals/group; *P ≤ 0.05). C) Representative recordings of the thromboxane A 2 receptor agonist U46619-induced constriction of cerebral arteries from WT and Mcoln1 −/ − mice. D) Summary data showing the contraction produced by U46619 in cerebral arteries from WT and Mcoln1 −/− mice (n = 8 arteries from 4–5 animals/group; *P ≤ 0.05). E) Representative recordings of pressure-induced constriction of mesenteric resistance arteries from WT and Mcoln1 −/ − mice. F) Summary data showing the myogenic tone of mesenteric arteries from WT and Mcoln1 −/− mice as a function of intraluminal pressure (n = 7–9 arteries from 4–6 animals/group; *P ≤ 0.05). G) Representative recordings of α 1 -adrenergic receptor agonist phenylephrine (PE)-induced constriction of mesenteric resistance arteries from WT and Mcoln1 −/ − mice. H) Summary data showing the contraction produced by PE in mesenteric arteries from WT and Mcoln1 −/− mice (n = 7–8 arteries from 3 animals/group; *P ≤ 0.05). All data are shown as mean ± SEM.

    Techniques Used: Mouse Assay, Produced

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    16) 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

    17) 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

    18) 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

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    24) 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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    Lycorine reduces TFEB nuclear translocation by attenuating oxidation of <t>TRPML1</t> 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
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    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: To confirm the role of TRPML1 to exhibit the effect of lycorine on OCs, knockdown of TRPML1 was performed.

    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

    LAMTOR1 KD‐induced changes in lysosomal trafficking require TRMPL1‐mediated Ca 2+ release and dynein activation Quantification of the percent of lysosomes moving in the anterograde or retrograde direction in neurons infected with AAV expressing either LAMTOR1 shRNA (shLAMTOR1) or scrambled shRNA (shSc) and Accell lyspersin siRNA or control siRNA. Neurons were imaged with LysoTracker to visualize lysosomal trafficking in dendrites. N = 17–31 neurons from 3 to 6 independent experiments. Targets of pharmacological reagents and genetic manipulation used in this study. Blue text box, TRPML1 inhibition including TRPML1 inhibitor ML‐SI1, TRPML1 siRNA, and dynein inhibitor ciliobrevin D (CilioD); red text box, TRPML1 activation induced by TRPML1 activator ML‐SA1. Dash line separates the lysosome into lysosomes under control and LAMTOR1 (LT1) KD conditions. Quantification of the percent of lysosomes moving in the anterograde or retrograde direction. (C, E, F) Cultured hippocampal neurons were infected with AAV expressing either shLAMTOR1 or shSc; they were treated with vehicle control or ML‐SI1 (20 µM, C, n = 16–49 neurons from 3 to 10 independent experiments), or ML‐SA1 (20 µM, E, n = 13–49 neurons from 3 to 10 independent experiments), or CilioD (20 µM, F, n = 12–49 neurons from 3 to 10 independent experiments), and imaged with LysoTracker to visualize lysosomal trafficking in dendrites. Note that the data of shSc and shLAMTOR1 in (C, E, and F) are the same as shown in Fig 1C . (D) Neurons were infected with shLAMTOR1 or shSc AAV and Accell TRPML1 siRNA or control siRNA; they were imaged as described above. N = 7–31 neurons from 3 to 6 independent experiments. Note that the data of shSc/Accell siControl and shLAMTOR1/Accell siControl in (D) are the same as shown in (A). Data information: Data with error bars are represented as means ± SEM. *** P

    Journal: The EMBO Journal

    Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

    doi: 10.15252/embj.2021108119

    Figure Lengend Snippet: LAMTOR1 KD‐induced changes in lysosomal trafficking require TRMPL1‐mediated Ca 2+ release and dynein activation Quantification of the percent of lysosomes moving in the anterograde or retrograde direction in neurons infected with AAV expressing either LAMTOR1 shRNA (shLAMTOR1) or scrambled shRNA (shSc) and Accell lyspersin siRNA or control siRNA. Neurons were imaged with LysoTracker to visualize lysosomal trafficking in dendrites. N = 17–31 neurons from 3 to 6 independent experiments. Targets of pharmacological reagents and genetic manipulation used in this study. Blue text box, TRPML1 inhibition including TRPML1 inhibitor ML‐SI1, TRPML1 siRNA, and dynein inhibitor ciliobrevin D (CilioD); red text box, TRPML1 activation induced by TRPML1 activator ML‐SA1. Dash line separates the lysosome into lysosomes under control and LAMTOR1 (LT1) KD conditions. Quantification of the percent of lysosomes moving in the anterograde or retrograde direction. (C, E, F) Cultured hippocampal neurons were infected with AAV expressing either shLAMTOR1 or shSc; they were treated with vehicle control or ML‐SI1 (20 µM, C, n = 16–49 neurons from 3 to 10 independent experiments), or ML‐SA1 (20 µM, E, n = 13–49 neurons from 3 to 10 independent experiments), or CilioD (20 µM, F, n = 12–49 neurons from 3 to 10 independent experiments), and imaged with LysoTracker to visualize lysosomal trafficking in dendrites. Note that the data of shSc and shLAMTOR1 in (C, E, and F) are the same as shown in Fig 1C . (D) Neurons were infected with shLAMTOR1 or shSc AAV and Accell TRPML1 siRNA or control siRNA; they were imaged as described above. N = 7–31 neurons from 3 to 6 independent experiments. Note that the data of shSc/Accell siControl and shLAMTOR1/Accell siControl in (D) are the same as shown in (A). Data information: Data with error bars are represented as means ± SEM. *** P

    Article Snippet: Negative controls were incubated with anti‐LAMTOR1 only, anti‐TRPML1 only, or omitting primary antibodies.

    Techniques: Activation Assay, Infection, Expressing, shRNA, Inhibition, Cell Culture

    Characterization of the interaction between LAMTOR1 and TRPML1 Schematic structure of LAMTOR1 mutants. GCC are lipidation sites. Blue cans indicate α‐helices. Dashed lines indicate the locations of amino acid deletions. Arrowheads indicate the locations of amino acid substitutions. WT, wild‐type; ∆N, N‐terminal deletion; ∆C, C‐terminal deletion; ∆K1, K20R; ∆K2, K31R. Lysates from Hela cells cotransfected with LAMTOR1‐Flag or its mutants (∆N and ∆C in B, ∆N1, and ∆N2 in D, ∆K1, and ∆K2 in E) and TRPML1‐YFP were immunoprecipitated with anti‐GFP or control IgG antibodies and probed with the indicated antibodies. Left, input proteins; right, immunoprecipitated (IP) proteins. Note one group (Starved) of Hela cells transfected with LAMTOR1‐Flag and TRPML1‐YFP in (E) were incubated in medium without amino acids and serum for 2 h. (C) Lysates from Hela cells transfected as described in (B) were immunoprecipitated with anti‐Flag or control IgG antibodies and probed with Flag and GFP antibodies. See Appendix Fig S4 for negative controls. Quantification of co‐IP results. N = 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one‐way ANOVA with Dunnett’s post‐test (F). * P

    Journal: The EMBO Journal

    Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

    doi: 10.15252/embj.2021108119

    Figure Lengend Snippet: Characterization of the interaction between LAMTOR1 and TRPML1 Schematic structure of LAMTOR1 mutants. GCC are lipidation sites. Blue cans indicate α‐helices. Dashed lines indicate the locations of amino acid deletions. Arrowheads indicate the locations of amino acid substitutions. WT, wild‐type; ∆N, N‐terminal deletion; ∆C, C‐terminal deletion; ∆K1, K20R; ∆K2, K31R. Lysates from Hela cells cotransfected with LAMTOR1‐Flag or its mutants (∆N and ∆C in B, ∆N1, and ∆N2 in D, ∆K1, and ∆K2 in E) and TRPML1‐YFP were immunoprecipitated with anti‐GFP or control IgG antibodies and probed with the indicated antibodies. Left, input proteins; right, immunoprecipitated (IP) proteins. Note one group (Starved) of Hela cells transfected with LAMTOR1‐Flag and TRPML1‐YFP in (E) were incubated in medium without amino acids and serum for 2 h. (C) Lysates from Hela cells transfected as described in (B) were immunoprecipitated with anti‐Flag or control IgG antibodies and probed with Flag and GFP antibodies. See Appendix Fig S4 for negative controls. Quantification of co‐IP results. N = 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one‐way ANOVA with Dunnett’s post‐test (F). * P

    Article Snippet: Negative controls were incubated with anti‐LAMTOR1 only, anti‐TRPML1 only, or omitting primary antibodies.

    Techniques: Immunoprecipitation, Transfection, Incubation, Co-Immunoprecipitation Assay

    Effects of LAMTOR1 KD in field CA1 of hippocampus on levels of various lysosome proteins. Related to Fig 7 Representative images of CA1 pyramidal neurons stained with anti‐LAMTOR1 (red) and ‐GFP (green) antibodies in scrambled shRNA (shSc) or LAMTOR1 shRNA (shLAMTOR1)‐injected mice. Scale bar, 100 µm. Effects of LAMTOR1 knockdown in field CA1 of hippocampus on levels of LAMTOR1, LAMTOR2, TRPML1, LAMP2, cathepsin B, cathepsin D, and Rab7. (B) Representative Western blot images. (C) Quantitative analysis of blots in (B). N = 3 mice. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Student’s t ‐test. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

    doi: 10.15252/embj.2021108119

    Figure Lengend Snippet: Effects of LAMTOR1 KD in field CA1 of hippocampus on levels of various lysosome proteins. Related to Fig 7 Representative images of CA1 pyramidal neurons stained with anti‐LAMTOR1 (red) and ‐GFP (green) antibodies in scrambled shRNA (shSc) or LAMTOR1 shRNA (shLAMTOR1)‐injected mice. Scale bar, 100 µm. Effects of LAMTOR1 knockdown in field CA1 of hippocampus on levels of LAMTOR1, LAMTOR2, TRPML1, LAMP2, cathepsin B, cathepsin D, and Rab7. (B) Representative Western blot images. (C) Quantitative analysis of blots in (B). N = 3 mice. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Student’s t ‐test. Source data are available online for this figure.

    Article Snippet: Negative controls were incubated with anti‐LAMTOR1 only, anti‐TRPML1 only, or omitting primary antibodies.

    Techniques: Staining, shRNA, Injection, Mouse Assay, Western Blot

    Effects of TAT‐2031 on the interaction between LAMTOR1 and TRPML1 in vivo and lysosomal trafficking in dendrites of hippocampal neurons Interactions between LAMTOR1 and TRPML1 in mouse hippocampus. Binding of LAMTOR1 to TRPML1 in vivo was disrupted by systemic administration of the TAT‐2031 peptide. Wes protein analysis with anti‐LAMTOR1 and ‐TRPML1 antibodies of immunoprecipitation performed with anti‐LAMTOR1 antibodies or negative control anti‐HA antibodies using whole hippocampal homogenates from naïve, TAT or TAT‐2031‐treated mice. Quantification of the relative abundance of TPRML1 pulled down by LAMTOR1 in naïve, TAT or TAT‐2031‐treated mice. N = 3 mice for each group. Representative images from proximity ligation assay (PLA) performed on brain slices from naïve, TAT or TAT‐2031‐treated mice. Evidence of proximity between LAMTOR1 and TRPML1 is indicated by the appearance of red puncta. Nuclei are counterstained with DAPI (blue). Scale bar, 10 μm. See Appendix Fig S5E for negative controls. Quantification of the number of PLA signals in CA1 from naïve, TAT or TAT‐2031‐treated mice. N = 6 mice for each group. TAT‐2031 treatment increased lysosomal mobility. Neurons were infected with shLAMTOR1 or shSc AAV; they were treated with TAT or TAT‐2031 (10 µM) and imaged with LysoTracker to visualize lysosomal trafficking in dendrites. N = 17–22 neurons from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one‐way ANOVA with Dunnett's post‐test (B, D), or two‐way ANOVA with Tukey’s post‐test (E). ** P

    Journal: The EMBO Journal

    Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

    doi: 10.15252/embj.2021108119

    Figure Lengend Snippet: Effects of TAT‐2031 on the interaction between LAMTOR1 and TRPML1 in vivo and lysosomal trafficking in dendrites of hippocampal neurons Interactions between LAMTOR1 and TRPML1 in mouse hippocampus. Binding of LAMTOR1 to TRPML1 in vivo was disrupted by systemic administration of the TAT‐2031 peptide. Wes protein analysis with anti‐LAMTOR1 and ‐TRPML1 antibodies of immunoprecipitation performed with anti‐LAMTOR1 antibodies or negative control anti‐HA antibodies using whole hippocampal homogenates from naïve, TAT or TAT‐2031‐treated mice. Quantification of the relative abundance of TPRML1 pulled down by LAMTOR1 in naïve, TAT or TAT‐2031‐treated mice. N = 3 mice for each group. Representative images from proximity ligation assay (PLA) performed on brain slices from naïve, TAT or TAT‐2031‐treated mice. Evidence of proximity between LAMTOR1 and TRPML1 is indicated by the appearance of red puncta. Nuclei are counterstained with DAPI (blue). Scale bar, 10 μm. See Appendix Fig S5E for negative controls. Quantification of the number of PLA signals in CA1 from naïve, TAT or TAT‐2031‐treated mice. N = 6 mice for each group. TAT‐2031 treatment increased lysosomal mobility. Neurons were infected with shLAMTOR1 or shSc AAV; they were treated with TAT or TAT‐2031 (10 µM) and imaged with LysoTracker to visualize lysosomal trafficking in dendrites. N = 17–22 neurons from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one‐way ANOVA with Dunnett's post‐test (B, D), or two‐way ANOVA with Tukey’s post‐test (E). ** P

    Article Snippet: Negative controls were incubated with anti‐LAMTOR1 only, anti‐TRPML1 only, or omitting primary antibodies.

    Techniques: In Vivo, Binding Assay, Immunoprecipitation, Negative Control, Mouse Assay, Proximity Ligation Assay, Infection

    Colocalization of LAMTOR1‐Flag or its mutants with LAMP2 or TRPML1‐YFP in Hela cells, and effects of TAT‐2031 on Ragulator integrity and mTORC1 activation. Related to Figs 4 and 5 Colocalization of LAMTOR1‐Flag with LAMP2 in Hela cells. Hela cells were transfected with LAMTOR1‐Flag or its mutants before being processed for LAMP2 (red) and Flag (green) immunofluorescence assay and imaging. Scale bar, 10 µm. (B) Quantification of LAMTOR1‐Flag and LAMP2 colocalization shown in (A). N = 8–17 cells from 3 independent experiments. Co‐localization of LAMTOR1‐Flag with TRPML1‐YFP in Hela cells. Hela cells were transfected with LAMTOR1‐Flag or its mutants and TRPML1‐YFP (green) before processing for Flag (red) immunofluorescence assay and imaging. Scale bar, 10 µm. (D) Quantification of LAMTOR1‐Flag and LAMP2 colocalization shown in (C). N = 8–20 cells from 3 independent experiments. Effects of TAT‐2031 on the interaction between LAMTOR1 and other members of the Ragulator (E) and mTORC1 signaling (F) in mouse hippocampus. E Lysates from fresh hippocampal tissue were immunoprecipitated with anti‐LAMTOR1 antibodies or negative control anti‐HA antibodies and probed with the indicated antibodies. Top, representative Western blot images; bottom, quantification of the relative abundance of LAMTOR2‐5 pulled down by LAMTOR1 in naïve, TAT or TAT‐2031‐treated mice. F Protein lysates of hippocampal tissue from TAT or TAT‐2031‐treated mice were prepared for Western blot analysis. Top, representative Western blot images; bottom, quantitative analysis. N = 3 mice. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one‐way ANOVA with Dunnett’s post‐test (B, D) and Student’s t ‐test (E, F). n.s., not significant. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

    doi: 10.15252/embj.2021108119

    Figure Lengend Snippet: Colocalization of LAMTOR1‐Flag or its mutants with LAMP2 or TRPML1‐YFP in Hela cells, and effects of TAT‐2031 on Ragulator integrity and mTORC1 activation. Related to Figs 4 and 5 Colocalization of LAMTOR1‐Flag with LAMP2 in Hela cells. Hela cells were transfected with LAMTOR1‐Flag or its mutants before being processed for LAMP2 (red) and Flag (green) immunofluorescence assay and imaging. Scale bar, 10 µm. (B) Quantification of LAMTOR1‐Flag and LAMP2 colocalization shown in (A). N = 8–17 cells from 3 independent experiments. Co‐localization of LAMTOR1‐Flag with TRPML1‐YFP in Hela cells. Hela cells were transfected with LAMTOR1‐Flag or its mutants and TRPML1‐YFP (green) before processing for Flag (red) immunofluorescence assay and imaging. Scale bar, 10 µm. (D) Quantification of LAMTOR1‐Flag and LAMP2 colocalization shown in (C). N = 8–20 cells from 3 independent experiments. Effects of TAT‐2031 on the interaction between LAMTOR1 and other members of the Ragulator (E) and mTORC1 signaling (F) in mouse hippocampus. E Lysates from fresh hippocampal tissue were immunoprecipitated with anti‐LAMTOR1 antibodies or negative control anti‐HA antibodies and probed with the indicated antibodies. Top, representative Western blot images; bottom, quantification of the relative abundance of LAMTOR2‐5 pulled down by LAMTOR1 in naïve, TAT or TAT‐2031‐treated mice. F Protein lysates of hippocampal tissue from TAT or TAT‐2031‐treated mice were prepared for Western blot analysis. Top, representative Western blot images; bottom, quantitative analysis. N = 3 mice. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one‐way ANOVA with Dunnett’s post‐test (B, D) and Student’s t ‐test (E, F). n.s., not significant. Source data are available online for this figure.

    Article Snippet: Negative controls were incubated with anti‐LAMTOR1 only, anti‐TRPML1 only, or omitting primary antibodies.

    Techniques: Activation Assay, Transfection, Immunofluorescence, Imaging, Immunoprecipitation, Negative Control, Western Blot, Mouse Assay

    LAMTOR1 KD‐induced learning and memory impairment in mice is ameliorated by TRPML1 inhibition Mice received 10 min of training in an environment with two identical objects and received a retention test 24 h later in which one object was replaced with a novel one. LAMTOR1 shRNA‐injected mice exhibited a significant deficit 24 h after training, which was reversed by ML‐SI1 treatment ( N = 7–18 mice). % freezing for different experimental groups in context memory ( N = 7–19 mice). Model illustrating the proposed role of LAMTOR1‐mediated inhibition of lysosomal Ca 2+ release via TRPML1 in the regulation of lysosome motility in dendrites and synaptic plasticity. More lysosomes with lower pH move faster and longer distances in LAMTOR1 KD neurons, as compared to control neurons. LAMTOR1 interaction with TRPML1 inhibits its Ca 2+ release and LAMTOR1 KD increased its Ca 2+ release and dynein‐dependent dendritic lysosome trafficking (①). LAMTOR1 KD‐induced TRPML1 Ca 2+ release also activates CaN, efficiently dephosphorylating GluA1 and targeting internalized AMPARs to lysosomes for degradation, thereby regulating synaptic plasticity (②). Figs 3B and 5A , and 8C were created with BioRender.com. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two‐way ANOVA with Tukey’s post‐test. * P

    Journal: The EMBO Journal

    Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

    doi: 10.15252/embj.2021108119

    Figure Lengend Snippet: LAMTOR1 KD‐induced learning and memory impairment in mice is ameliorated by TRPML1 inhibition Mice received 10 min of training in an environment with two identical objects and received a retention test 24 h later in which one object was replaced with a novel one. LAMTOR1 shRNA‐injected mice exhibited a significant deficit 24 h after training, which was reversed by ML‐SI1 treatment ( N = 7–18 mice). % freezing for different experimental groups in context memory ( N = 7–19 mice). Model illustrating the proposed role of LAMTOR1‐mediated inhibition of lysosomal Ca 2+ release via TRPML1 in the regulation of lysosome motility in dendrites and synaptic plasticity. More lysosomes with lower pH move faster and longer distances in LAMTOR1 KD neurons, as compared to control neurons. LAMTOR1 interaction with TRPML1 inhibits its Ca 2+ release and LAMTOR1 KD increased its Ca 2+ release and dynein‐dependent dendritic lysosome trafficking (①). LAMTOR1 KD‐induced TRPML1 Ca 2+ release also activates CaN, efficiently dephosphorylating GluA1 and targeting internalized AMPARs to lysosomes for degradation, thereby regulating synaptic plasticity (②). Figs 3B and 5A , and 8C were created with BioRender.com. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two‐way ANOVA with Tukey’s post‐test. * P

    Article Snippet: Negative controls were incubated with anti‐LAMTOR1 only, anti‐TRPML1 only, or omitting primary antibodies.

    Techniques: Mouse Assay, Inhibition, shRNA, Injection

    Detection of TRPML1‐mediated lysosomal Ca 2+ release and its regulation by LAMTOR1 Structure of the TRPML1‐GCaMP6m‐based Ca 2+ sensor. Colocalization of TRPML1‐GCaMP6m (green) with LysoTracker (red) in Hela cells. Scale bar, 5 µm. ML‐SA1 (20 µM)‐induced peak GCaMP6m responses (ΔF/F 0 ) were increased in CRISPR‐Cas9‐mediated LAMTOR1 KD cells. N = 9–10 cells from 4 independent experiments. TAT‐2031 treatment (10 µM) increased ML‐SA1‐induced TRPML1 Ca 2+ release in both control and LAMTOR1 KD cells. Quantification of peak responses as shown in (D). N = 11–19 cells from 5 independent experiments. Colocalization of TRPML1‐GCaMP6m (green) with LysoTracker (red) in hippocampal neurons. Scale bar, 10 µm. Treatment with GPN (200 µM), BAPTA‐AM (20 µM), or ML‐SI1 (20 µM) blocked ML‐SA1‐induced TRPML1‐GCaMP6m responses in neurons. Quantification of responses in (G). N = 5–8 cells from 3 independent experiments. Both LAMTOR1 KD and TAT‐2031 treatment (10 µM) increased ML‐SA1‐induced TRPML1‐GCaMP6m responses in neurons. Quantification of peak responses as shown in I. N = 6–13 cells from 3 independent experiments. LAMTOR1 KD increased PI(3,5)P2 (0.5 µM)‐induced TRPML1‐GCaMP6m responses and the blocking effect of ML‐SI1 treatment (20 µM). Quantification of peak TRPML1‐GCaMP6m responses as shown in (K). N = 6–16 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Student’s t ‐test (C), two‐way ANOVA with Tukey’s post‐hoc analysis (E, J, L), and one‐way ANOVA with Dunnett’s post‐test (H). * P

    Journal: The EMBO Journal

    Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

    doi: 10.15252/embj.2021108119

    Figure Lengend Snippet: Detection of TRPML1‐mediated lysosomal Ca 2+ release and its regulation by LAMTOR1 Structure of the TRPML1‐GCaMP6m‐based Ca 2+ sensor. Colocalization of TRPML1‐GCaMP6m (green) with LysoTracker (red) in Hela cells. Scale bar, 5 µm. ML‐SA1 (20 µM)‐induced peak GCaMP6m responses (ΔF/F 0 ) were increased in CRISPR‐Cas9‐mediated LAMTOR1 KD cells. N = 9–10 cells from 4 independent experiments. TAT‐2031 treatment (10 µM) increased ML‐SA1‐induced TRPML1 Ca 2+ release in both control and LAMTOR1 KD cells. Quantification of peak responses as shown in (D). N = 11–19 cells from 5 independent experiments. Colocalization of TRPML1‐GCaMP6m (green) with LysoTracker (red) in hippocampal neurons. Scale bar, 10 µm. Treatment with GPN (200 µM), BAPTA‐AM (20 µM), or ML‐SI1 (20 µM) blocked ML‐SA1‐induced TRPML1‐GCaMP6m responses in neurons. Quantification of responses in (G). N = 5–8 cells from 3 independent experiments. Both LAMTOR1 KD and TAT‐2031 treatment (10 µM) increased ML‐SA1‐induced TRPML1‐GCaMP6m responses in neurons. Quantification of peak responses as shown in I. N = 6–13 cells from 3 independent experiments. LAMTOR1 KD increased PI(3,5)P2 (0.5 µM)‐induced TRPML1‐GCaMP6m responses and the blocking effect of ML‐SI1 treatment (20 µM). Quantification of peak TRPML1‐GCaMP6m responses as shown in (K). N = 6–16 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Student’s t ‐test (C), two‐way ANOVA with Tukey’s post‐hoc analysis (E, J, L), and one‐way ANOVA with Dunnett’s post‐test (H). * P

    Article Snippet: Negative controls were incubated with anti‐LAMTOR1 only, anti‐TRPML1 only, or omitting primary antibodies.

    Techniques: CRISPR, Blocking Assay

    LAMTOR1 KD reduces LTP and enhances LTD in field CA1 of hippocampus by increasing TRPML1‐mediated Ca 2+ release and calcineurin activation Effects of LAMTOR1 KD and ML‐SI1 treatment on TBS‐induced LTP (A) or LFS‐induced LTD (C) in CA1. (B, D) Means ± SEM of fEPSPs measured 40 min after TBS (B, n = 4–5 slices from 4 to 5 mice) or LFS (D, n = 4–8 slices from 4 to 8 mice) in different groups. Effects of ML‐SI1 treatment on LFS‐induced LTD in CA1 from 2‐ to 3‐weeks‐old mice. Means ± SEM of fEPSPs measured 45 min after LFS in different groups. N = 6–8 slices from 6 to 8 mice. Effects of TAT‐2031 treatment on TBS‐induced LTP (G) or LFS‐induced LTD (I) in CA1. H, J Means ± SEM of fEPSPs measured 40 min after TBS (H, n = 6 slices from 6 mice) or LFS (J, n = 3–4 slices from 3 to 4 mice) in different groups. Effects of LAMTOR1 KD and treatment with a calcineurin inhibitor, FK506, on LFS‐induced LTD in CA1. Means ± SEM of fEPSPs measured 45 min after LFS in different groups ( n = 3–4 slices from 3 to 4 mice). Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10‐min baseline (A, C, E, G, I, K). Statistical significance was assessed by two‐way ANOVA with Tukey’s post‐test (B, D, L) and Student’s t ‐test (F, H, J). * P

    Journal: The EMBO Journal

    Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

    doi: 10.15252/embj.2021108119

    Figure Lengend Snippet: LAMTOR1 KD reduces LTP and enhances LTD in field CA1 of hippocampus by increasing TRPML1‐mediated Ca 2+ release and calcineurin activation Effects of LAMTOR1 KD and ML‐SI1 treatment on TBS‐induced LTP (A) or LFS‐induced LTD (C) in CA1. (B, D) Means ± SEM of fEPSPs measured 40 min after TBS (B, n = 4–5 slices from 4 to 5 mice) or LFS (D, n = 4–8 slices from 4 to 8 mice) in different groups. Effects of ML‐SI1 treatment on LFS‐induced LTD in CA1 from 2‐ to 3‐weeks‐old mice. Means ± SEM of fEPSPs measured 45 min after LFS in different groups. N = 6–8 slices from 6 to 8 mice. Effects of TAT‐2031 treatment on TBS‐induced LTP (G) or LFS‐induced LTD (I) in CA1. H, J Means ± SEM of fEPSPs measured 40 min after TBS (H, n = 6 slices from 6 mice) or LFS (J, n = 3–4 slices from 3 to 4 mice) in different groups. Effects of LAMTOR1 KD and treatment with a calcineurin inhibitor, FK506, on LFS‐induced LTD in CA1. Means ± SEM of fEPSPs measured 45 min after LFS in different groups ( n = 3–4 slices from 3 to 4 mice). Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10‐min baseline (A, C, E, G, I, K). Statistical significance was assessed by two‐way ANOVA with Tukey’s post‐test (B, D, L) and Student’s t ‐test (F, H, J). * P

    Article Snippet: Negative controls were incubated with anti‐LAMTOR1 only, anti‐TRPML1 only, or omitting primary antibodies.

    Techniques: Activation Assay, Mouse Assay

    Characterization of a lysosome‐targeted genetically encoded Ca 2+ indicator TRPML1‐GCaMP6m in Hela cells and cultured neurons. Related to Fig 6 GPN (50 µM, A) and ML‐SA1 (20 µM, B)‐induced lysosomal Ca 2+ release in TRPML1‐GCaMP6m‐transfected Hela cells. Increased cytosolic calcium triggered by thapsigargin (2 μM) was not detected by TRPML1‐GCaMP6m, but by BioTracker 609 Red Ca 2+ AM Dye in Hela cells. Increased cytosolic calcium triggered by thapsigargin (2 μM) was not detected by TRPML1‐GCaMP6m in control and LAMTOR1 KD Hela cells. Images of Hela cells immunostained for LAMTOR1 (green). Hela cells were transfected with CRISPR‐Cas9 plasmids (with mCherry reporter, indicated by asterisks) with (LAMTOR1 KD) or without (control) sgRNA targeting LAMTOR1 . Scale bar, 10 µm. Representative images of Hela cells transfected with TRPML1‐GCaMP6m (green) and CRISPR‐Cas9 plasmids as described in E. Scale bar, 5 µm. ML‐SA1 (20 µM)‐induced peak GCaMP6m responses (ΔF/F 0 ) were increased in Hela cells transfected with the LAMTOR1 mutant ΔN1 (lacking amino acids 20–31). Right, Quantification of peak responses as shown in the left panel. N = 14 cells from 3 independent experiments. Increased cytosolic calcium triggered by thapsigargin (2 μM), under low (

    Journal: The EMBO Journal

    Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

    doi: 10.15252/embj.2021108119

    Figure Lengend Snippet: Characterization of a lysosome‐targeted genetically encoded Ca 2+ indicator TRPML1‐GCaMP6m in Hela cells and cultured neurons. Related to Fig 6 GPN (50 µM, A) and ML‐SA1 (20 µM, B)‐induced lysosomal Ca 2+ release in TRPML1‐GCaMP6m‐transfected Hela cells. Increased cytosolic calcium triggered by thapsigargin (2 μM) was not detected by TRPML1‐GCaMP6m, but by BioTracker 609 Red Ca 2+ AM Dye in Hela cells. Increased cytosolic calcium triggered by thapsigargin (2 μM) was not detected by TRPML1‐GCaMP6m in control and LAMTOR1 KD Hela cells. Images of Hela cells immunostained for LAMTOR1 (green). Hela cells were transfected with CRISPR‐Cas9 plasmids (with mCherry reporter, indicated by asterisks) with (LAMTOR1 KD) or without (control) sgRNA targeting LAMTOR1 . Scale bar, 10 µm. Representative images of Hela cells transfected with TRPML1‐GCaMP6m (green) and CRISPR‐Cas9 plasmids as described in E. Scale bar, 5 µm. ML‐SA1 (20 µM)‐induced peak GCaMP6m responses (ΔF/F 0 ) were increased in Hela cells transfected with the LAMTOR1 mutant ΔN1 (lacking amino acids 20–31). Right, Quantification of peak responses as shown in the left panel. N = 14 cells from 3 independent experiments. Increased cytosolic calcium triggered by thapsigargin (2 μM), under low (

    Article Snippet: Negative controls were incubated with anti‐LAMTOR1 only, anti‐TRPML1 only, or omitting primary antibodies.

    Techniques: Cell Culture, Transfection, CRISPR, Mutagenesis