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Addgene inc er gfp addgene plasmid
Er Gfp Addgene Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 49 article reviews

er gfp addgene plasmid - by Bioz Stars, 2026-05

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(A) Schematic representation of the YM201636 treatment and washout used for synchronization of endocytic lysosomal reformation (ELR) events. (B) Live-cell imaging of cells expressing LAMP1-GFP (green) and stained with LysoTracker Deep Red (magenta), following washout of YM201636 and recovery in drug-free media for 3 h. Images were acquired every 15 min to monitor the timeline and progression of ELR events. (C) Quantification of LAMP1-positive structures from (B). The line plot depicts the number of LAMP1-positive compartments per cell over the recovery period. Data represent measurements from 30 cells. The central line indicates the mean; shaded regions or lines above and below represent the standard deviation (SD). (D) Quantification of LysoTracker-positive structures from (B). The line plot shows the number of LysoTracker-positive compartments per cell over the recovery period. Data represent measurements from 30 cells. The central line indicates the mean; shaded regions or lines above and below represent the SD. (E) Super-resolution imaging of endolysosomes undergoing reformation using spinning disk confocal microscopy equipped with SoRa Disk. Cells expressing LAMP1-GFP (green) were treated with YM201636 for 2 h and imaged immediately after washout in drug-free media to capture ELR events. Images were acquired over 2 min at 2 sec intervals. White arrowheads indicate sites of fission and ELR events. (F) Quantification of data from (E). Table summarizing the percentage occurence of three distinct types of reformation events observed out of the first 100 events from 30 cells.

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Schematic representation of the YM201636 treatment and washout used for synchronization of endocytic lysosomal reformation (ELR) events. (B) Live-cell imaging of cells expressing LAMP1-GFP (green) and stained with LysoTracker Deep Red (magenta), following washout of YM201636 and recovery in drug-free media for 3 h. Images were acquired every 15 min to monitor the timeline and progression of ELR events. (C) Quantification of LAMP1-positive structures from (B). The line plot depicts the number of LAMP1-positive compartments per cell over the recovery period. Data represent measurements from 30 cells. The central line indicates the mean; shaded regions or lines above and below represent the standard deviation (SD). (D) Quantification of LysoTracker-positive structures from (B). The line plot shows the number of LysoTracker-positive compartments per cell over the recovery period. Data represent measurements from 30 cells. The central line indicates the mean; shaded regions or lines above and below represent the SD. (E) Super-resolution imaging of endolysosomes undergoing reformation using spinning disk confocal microscopy equipped with SoRa Disk. Cells expressing LAMP1-GFP (green) were treated with YM201636 for 2 h and imaged immediately after washout in drug-free media to capture ELR events. Images were acquired over 2 min at 2 sec intervals. White arrowheads indicate sites of fission and ELR events. (F) Quantification of data from (E). Table summarizing the percentage occurence of three distinct types of reformation events observed out of the first 100 events from 30 cells.

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Live Cell Imaging, Expressing, Staining, Standard Deviation, Imaging, Confocal Microscopy

(A) Live-cell time-lapse imaging of cells expressing LAMP1-GFP (green) and stained with Lysotracker Deep Red (magenta). Cells were treated with Nigericin to enlarge endocytic compartments. Nigericin was washed out and imaging was performed after 20 min of recovery. Each cell was imaged for 2 min at an interval of 2 sec. (B) Live-cell time-lapse imaging of cells expressing LAMP1-GFP (green) and stained with Lysotracker Deep Red (magenta) treated with sucrose and invertase. Cells were fed with sucrose overnight and treated with invertase for 2 h before imaging to initiate ELR. Each cell was imaged for 2 min at an interval of 2 sec. (C) Immunofluorescent image of cells stained for LC3. Cells treated with YM20163 and recovery conditioned were fixed using 4%PFA while keeping starved and untreated cells as positive and negative control respectively. After fixation, coverslips were stained with anti-LC3 antibodies for imaging. (D) Immunoblot analysis to check the autophagic flux in cells treated with YM2016136 and recovery condition. Starvation with HBSS was used as a positive control for LC3 II lipidation. Tubulin was used as a loading control. (E) Fold change of LC3 II/LC3 I was assessed and plotted for bafilomycin positive conditions. Plot represents the mean fold change across n=3 biological replicates; One way ANOVA with Dunn’s multiple comparison; ***P= 0.0005 (untreated vs HBSS), ns P = 0.8087 (untreated vs 2 h YM201636), ns P = 0.2845 untreated vs 2 h YM201636 + 1h recovery)

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Live-cell time-lapse imaging of cells expressing LAMP1-GFP (green) and stained with Lysotracker Deep Red (magenta). Cells were treated with Nigericin to enlarge endocytic compartments. Nigericin was washed out and imaging was performed after 20 min of recovery. Each cell was imaged for 2 min at an interval of 2 sec. (B) Live-cell time-lapse imaging of cells expressing LAMP1-GFP (green) and stained with Lysotracker Deep Red (magenta) treated with sucrose and invertase. Cells were fed with sucrose overnight and treated with invertase for 2 h before imaging to initiate ELR. Each cell was imaged for 2 min at an interval of 2 sec. (C) Immunofluorescent image of cells stained for LC3. Cells treated with YM20163 and recovery conditioned were fixed using 4%PFA while keeping starved and untreated cells as positive and negative control respectively. After fixation, coverslips were stained with anti-LC3 antibodies for imaging. (D) Immunoblot analysis to check the autophagic flux in cells treated with YM2016136 and recovery condition. Starvation with HBSS was used as a positive control for LC3 II lipidation. Tubulin was used as a loading control. (E) Fold change of LC3 II/LC3 I was assessed and plotted for bafilomycin positive conditions. Plot represents the mean fold change across n=3 biological replicates; One way ANOVA with Dunn’s multiple comparison; ***P= 0.0005 (untreated vs HBSS), ns P = 0.8087 (untreated vs 2 h YM201636), ns P = 0.2845 untreated vs 2 h YM201636 + 1h recovery)

Techniques: Imaging, Expressing, Staining, Negative Control, Western Blot, Positive Control, Control, Comparison

(A) Immunoblot analysis of LAMP1 and Cathepsin D during reformation, using tubulin as a loading control. (B) Quantification of (A). Protein levels of LAMP1 and Cathepsin D at different maturation stages were analyzed for untreated, during YM201636 treatment, and post 1 h recovery. Fold change was determined by comparison to untreated control. Data represent mean of n=3 replicates; one-way ANOVA using Tukey’s multiple comparison, ns P = 0.8081 (Untreated vs 2 h YM201636), ns P= 0.4754 (Untreated vs 1 h recovery), ns P=0.2265 (2 h YM201636 vs 1 h recovery). For CTSD, ns P= 0.9871 (Untreated vs 2 h YM201636), ns P= 0.6745 (Untreated vs 1 h recovery), ns P=0.5877 (2 h YM201636 vs 1 h recovery) (C) Schematic representation of the analysis for cargo transport in tubules. (D) Super-resolution imaging of a tubulating endolysosome from a cell expressing LAMP1-GFP (green) and stained with Lysotracker Deep Red (magenta) using a spinning disk confocal with SoRa disk. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate the ELR. (E) Intensity plot for (D), showing intensity values of green and magenta channels over distance (μm). (F) Super-resolution image of a tubulating endolysosome from a cell expressing LAMP1-GFP (green) and stained with Magic Red (magenta) using a spinning disk confocal with SoRa disk. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate the ELR. (G) Intensity plot for (F), showing intensity values of green and magenta channels over distance (μm). (H) Super-resolution image of a tubulating endolysosome from a cell expressing LAMP1-GFP (green) and Cathepsin D-RFP (magenta) using a spinning disk confocal with SoRa disk. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate the ELR. (I) Intensity plot for (H), showing intensity values of green and magenta channels over distance (μm). (J) Super-resolution image of a tubulating endolysosome from a cell expressing LAMP1-GFP (green) and EGFR-mCherry (magenta) using a spinning disk confocal with SoRa disk. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate the ELR. Cells were also stimulated with EGF for 30 min to increase EGFR uptake. (K) Intensity plot for (J), showing intensity values of green and magenta channels over distance (μm).

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Immunoblot analysis of LAMP1 and Cathepsin D during reformation, using tubulin as a loading control. (B) Quantification of (A). Protein levels of LAMP1 and Cathepsin D at different maturation stages were analyzed for untreated, during YM201636 treatment, and post 1 h recovery. Fold change was determined by comparison to untreated control. Data represent mean of n=3 replicates; one-way ANOVA using Tukey’s multiple comparison, ns P = 0.8081 (Untreated vs 2 h YM201636), ns P= 0.4754 (Untreated vs 1 h recovery), ns P=0.2265 (2 h YM201636 vs 1 h recovery). For CTSD, ns P= 0.9871 (Untreated vs 2 h YM201636), ns P= 0.6745 (Untreated vs 1 h recovery), ns P=0.5877 (2 h YM201636 vs 1 h recovery) (C) Schematic representation of the analysis for cargo transport in tubules. (D) Super-resolution imaging of a tubulating endolysosome from a cell expressing LAMP1-GFP (green) and stained with Lysotracker Deep Red (magenta) using a spinning disk confocal with SoRa disk. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate the ELR. (E) Intensity plot for (D), showing intensity values of green and magenta channels over distance (μm). (F) Super-resolution image of a tubulating endolysosome from a cell expressing LAMP1-GFP (green) and stained with Magic Red (magenta) using a spinning disk confocal with SoRa disk. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate the ELR. (G) Intensity plot for (F), showing intensity values of green and magenta channels over distance (μm). (H) Super-resolution image of a tubulating endolysosome from a cell expressing LAMP1-GFP (green) and Cathepsin D-RFP (magenta) using a spinning disk confocal with SoRa disk. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate the ELR. (I) Intensity plot for (H), showing intensity values of green and magenta channels over distance (μm). (J) Super-resolution image of a tubulating endolysosome from a cell expressing LAMP1-GFP (green) and EGFR-mCherry (magenta) using a spinning disk confocal with SoRa disk. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate the ELR. Cells were also stimulated with EGF for 30 min to increase EGFR uptake. (K) Intensity plot for (J), showing intensity values of green and magenta channels over distance (μm).

Techniques: Western Blot, Control, Comparison, Imaging, Expressing, Staining

(A) Live-cell time-lapse imaging of cells expressing LAMP-GFP to assess the effect of translation inhibition on ELR kinetics. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate ELR. During recovery, cycloheximide was introduced in the recovery media while keeping media with ethanol as a vehicle control. Cells were imaged for 2 h at an interval of 15 min. (B) Quantification of LAMP1-positive structures from (A). The line plot shows the number of LAMP1-positive compartments per cell over the recovery period. Data represent measurements from 30 cells. The central line indicates the mean; error bars represent the standard deviation (SD). C) Live-cell super resolution imaging of tubulating endolysosome from a cell expressing LAMP1-GFP (green) and stained with Lysotracker Deep Red (magenta). Cells were imaged for 2 min at an interval of 2 sec. Enlarged ROI shows the fission events of LAMP1-positive compartments (green) and corresponding LysoTracker signal (magenta). (D) Live-cell super confocal imaging of tubulating endolysosome from a cell expressing LAMP1-GFP (green) and stained with Magic Red (magenta). Cells were imaged for 2 min at an interval of 2 sec. Enlarged ROI shows the fission events of LAMP1-positive compartments (green) and corresponding Magic Red signal (magenta). E) Live-cell super resolution imaging of tubulating endolysosome from a cell co-expressing LAMP1-GFP (green) and EGFR-mCherry. Cells were stimulated with EGF for 30 min to increase EGFR uptake and imaged for 2 min at an interval of 2 sec. Enlarged ROI shows the fission events of LAMP1 positive compartments (green) and corresponding EGFR-mCherry signal (magenta). (F) Live-cell super confocal imaging of tubulating endolysosome from a cell co-expressing LAMP1-GFP (green) and cathepsin D-RFP (magenta). Cells were imaged for 2 min at an interval of 2 sec. Enlarged ROI shows the fission events of LAMP1-positive compartments (green) and corresponding cathepsin D signal (magenta).

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Live-cell time-lapse imaging of cells expressing LAMP-GFP to assess the effect of translation inhibition on ELR kinetics. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate ELR. During recovery, cycloheximide was introduced in the recovery media while keeping media with ethanol as a vehicle control. Cells were imaged for 2 h at an interval of 15 min. (B) Quantification of LAMP1-positive structures from (A). The line plot shows the number of LAMP1-positive compartments per cell over the recovery period. Data represent measurements from 30 cells. The central line indicates the mean; error bars represent the standard deviation (SD). C) Live-cell super resolution imaging of tubulating endolysosome from a cell expressing LAMP1-GFP (green) and stained with Lysotracker Deep Red (magenta). Cells were imaged for 2 min at an interval of 2 sec. Enlarged ROI shows the fission events of LAMP1-positive compartments (green) and corresponding LysoTracker signal (magenta). (D) Live-cell super confocal imaging of tubulating endolysosome from a cell expressing LAMP1-GFP (green) and stained with Magic Red (magenta). Cells were imaged for 2 min at an interval of 2 sec. Enlarged ROI shows the fission events of LAMP1-positive compartments (green) and corresponding Magic Red signal (magenta). E) Live-cell super resolution imaging of tubulating endolysosome from a cell co-expressing LAMP1-GFP (green) and EGFR-mCherry. Cells were stimulated with EGF for 30 min to increase EGFR uptake and imaged for 2 min at an interval of 2 sec. Enlarged ROI shows the fission events of LAMP1 positive compartments (green) and corresponding EGFR-mCherry signal (magenta). (F) Live-cell super confocal imaging of tubulating endolysosome from a cell co-expressing LAMP1-GFP (green) and cathepsin D-RFP (magenta). Cells were imaged for 2 min at an interval of 2 sec. Enlarged ROI shows the fission events of LAMP1-positive compartments (green) and corresponding cathepsin D signal (magenta).

Techniques: Imaging, Expressing, Inhibition, Control, Standard Deviation, Staining

(A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 (K44A)-GFP to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 (K44A)-GFP to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.

Techniques: Western Blot, Phospho-proteomics, Positive Control, Inhibition, Control, Comparison, Live Cell Imaging, Expressing, Staining, Mutagenesis

(A ) Live-cell time-lapse imaging of cells expressing LAMP1–GFP to assess the effect of mTOR inhibition on ELR kinetics. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate ELR. During recovery, the mTOR inhibitor torin was added to the medium, with DMSO as vehicle control. Cells were imaged for 1 h at 15-min intervals. (B) Quantification of LAMP1-positive structures from (A). Line plot shows the number of LAMP1-positive compartments per cell over the recovery period. Data represent measurements from 30 cells. The central line indicates the mean; error bars indicate the standard deviation . ( C) Live-cell imaging of control knockout and dynamin-2 knockout cells expressing LAMP1-GFP to assess the role of dynamin 2 in endolysosomal tubule fission. Cells were imaged after 2 h YM201636 treatment followed by 30-min washout and recovery. Untreated cells were used as controls.

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A ) Live-cell time-lapse imaging of cells expressing LAMP1–GFP to assess the effect of mTOR inhibition on ELR kinetics. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate ELR. During recovery, the mTOR inhibitor torin was added to the medium, with DMSO as vehicle control. Cells were imaged for 1 h at 15-min intervals. (B) Quantification of LAMP1-positive structures from (A). Line plot shows the number of LAMP1-positive compartments per cell over the recovery period. Data represent measurements from 30 cells. The central line indicates the mean; error bars indicate the standard deviation . ( C) Live-cell imaging of control knockout and dynamin-2 knockout cells expressing LAMP1-GFP to assess the role of dynamin 2 in endolysosomal tubule fission. Cells were imaged after 2 h YM201636 treatment followed by 30-min washout and recovery. Untreated cells were used as controls.

Techniques: Imaging, Expressing, Inhibition, Control, Standard Deviation, Live Cell Imaging, Knock-Out

(A) Live-cell imaging of cells co transfected with LAMP1-GFP (green) and DRP1-mCherry (magenta) to assess the localization of DRP1 on lysosomes during reformation. Cells were imaged after treating with YM201636 for 2 h to inhibit the ELR and also after washout and 30 min of recovery to visualize the DRP1 on tubulating structures. (B) Co-localization of DRP1 with LAMP1 was measured in both YM201636 and recovery condition using Mander’s coefficient of DRP1 on LAMP1. Each dot represents the average Mander’s coefficient value of 100 μm 2 ROI. The plot shows mean Mander’s coefficient of 45 ROIs obtained from 15 cells in n=3 biological replicates; Unpaired t -test: ****P < 0.1 ×10 −14 (YM201636 vs 30 min recovery). (C) Live-cell imaging of cells co transfected with LAMP1-GFP (green) and DRP1-mCherry (magenta) to assess the localization of DRP1 on lysosomes in untreated condition. (D) DRP1 (magenta) localized on the LAMP1-positive compartments (green) are highlighted in enlarged ROI using white arrowheads. The localization was also plotted using line plots measuring the gray values of LAMP1-GFP (green) and DRP1-mCherry (magenta) channels over the distance in µm. (E) Live-cell imaging of cells co-expressing LAMP-GFP and wild-type DRP1 or the DRP1 K38A mutant. (F) Quantification of (E). Number of tubular lysosomes were quantified and plotted. Each dot represents the number of LAMP1-positive tubules per 100 μm 2 ROI. The plot shows the mean number of tubular structures of 90 ROIs measure across 30 cells in n=3 replicate; Unpaired t -test: ****P = 0.4659 ×10 −6 (DRP1 WT vs DRP1 K38A). (G) Live-cell imaging of cells co-expressing LAMP-GFP and wild type DRP1 or the DRP1 K38A mutant. Cells were imaged after 2 h YM201636 treatment followed by washout and recovery of 30 min. (H) Quantification of LAMP1-positive tubule length from (G). Each data point represents the length of a single tubule. The plot shows the mean length of tubules from 30 cells across n=3 biological replicates; Unpaired t -test: ****P < 0.1 ×10 −14 (DRP1 WT vs DRP1 K38A).

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Live-cell imaging of cells co transfected with LAMP1-GFP (green) and DRP1-mCherry (magenta) to assess the localization of DRP1 on lysosomes during reformation. Cells were imaged after treating with YM201636 for 2 h to inhibit the ELR and also after washout and 30 min of recovery to visualize the DRP1 on tubulating structures. (B) Co-localization of DRP1 with LAMP1 was measured in both YM201636 and recovery condition using Mander’s coefficient of DRP1 on LAMP1. Each dot represents the average Mander’s coefficient value of 100 μm 2 ROI. The plot shows mean Mander’s coefficient of 45 ROIs obtained from 15 cells in n=3 biological replicates; Unpaired t -test: ****P < 0.1 ×10 −14 (YM201636 vs 30 min recovery). (C) Live-cell imaging of cells co transfected with LAMP1-GFP (green) and DRP1-mCherry (magenta) to assess the localization of DRP1 on lysosomes in untreated condition. (D) DRP1 (magenta) localized on the LAMP1-positive compartments (green) are highlighted in enlarged ROI using white arrowheads. The localization was also plotted using line plots measuring the gray values of LAMP1-GFP (green) and DRP1-mCherry (magenta) channels over the distance in µm. (E) Live-cell imaging of cells co-expressing LAMP-GFP and wild-type DRP1 or the DRP1 K38A mutant. (F) Quantification of (E). Number of tubular lysosomes were quantified and plotted. Each dot represents the number of LAMP1-positive tubules per 100 μm 2 ROI. The plot shows the mean number of tubular structures of 90 ROIs measure across 30 cells in n=3 replicate; Unpaired t -test: ****P = 0.4659 ×10 −6 (DRP1 WT vs DRP1 K38A). (G) Live-cell imaging of cells co-expressing LAMP-GFP and wild type DRP1 or the DRP1 K38A mutant. Cells were imaged after 2 h YM201636 treatment followed by washout and recovery of 30 min. (H) Quantification of LAMP1-positive tubule length from (G). Each data point represents the length of a single tubule. The plot shows the mean length of tubules from 30 cells across n=3 biological replicates; Unpaired t -test: ****P < 0.1 ×10 −14 (DRP1 WT vs DRP1 K38A).

Techniques: Live Cell Imaging, Transfection, Expressing, Mutagenesis

(A) Live-cell confocal microscopy of cells expressing LAMP1-GFP (green) and DRP1-mCherry treated with YM201636 for 2 h, followed by washout and recovery in drug-free imaging buffer. Images were captured every 2 sec. White arrows indicate a LAMP1-positive tubule undergoing fission and DRP1 recruitment at the fission site. (B) Intensity plot for (A), showing green and magenta channel intensities across distance (μm) at three time points during fission events. (C) Imaging of LAMP1-positive tubules in wild-type and DRP1 knockout (KO) cells, as well as DRP1 KO cells overexpressing wild-type DRP1 or the DRP1 K38A mutant. Cells were treated with YM201636 for 2 h, followed by 30 min washout and recovery. White arrows indicate LAMP1-positive tubules and correspond to DRP1 localization. (D) Quantification of LAMP1-positive tubule length from (C). Each data point represents the length of a single tubule. Data from n=3 replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ****P < 0.1×10 −14 (Control KO vs. DRP1 KO; DRP1 KO vs. DRP1 KO + WT DRP1; rescue with WT DRP1 vs. rescue with DRP1 K38A; Control KO vs. DRP1 KO + DRP1 K38A); ns, P > 0.9 ×10 −14 (Control KO vs. DRP1 KO + WT DRP1; DRP1 KO vs. DRP1 KO + DRP1 K38A) (E) Immunoblot analysis confirming absence of DRP1 in DRP1 KO cells and overexpression of DRP1 in rescue experiments. Tubulin was used as a loading control.

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Live-cell confocal microscopy of cells expressing LAMP1-GFP (green) and DRP1-mCherry treated with YM201636 for 2 h, followed by washout and recovery in drug-free imaging buffer. Images were captured every 2 sec. White arrows indicate a LAMP1-positive tubule undergoing fission and DRP1 recruitment at the fission site. (B) Intensity plot for (A), showing green and magenta channel intensities across distance (μm) at three time points during fission events. (C) Imaging of LAMP1-positive tubules in wild-type and DRP1 knockout (KO) cells, as well as DRP1 KO cells overexpressing wild-type DRP1 or the DRP1 K38A mutant. Cells were treated with YM201636 for 2 h, followed by 30 min washout and recovery. White arrows indicate LAMP1-positive tubules and correspond to DRP1 localization. (D) Quantification of LAMP1-positive tubule length from (C). Each data point represents the length of a single tubule. Data from n=3 replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ****P < 0.1×10 −14 (Control KO vs. DRP1 KO; DRP1 KO vs. DRP1 KO + WT DRP1; rescue with WT DRP1 vs. rescue with DRP1 K38A; Control KO vs. DRP1 KO + DRP1 K38A); ns, P > 0.9 ×10 −14 (Control KO vs. DRP1 KO + WT DRP1; DRP1 KO vs. DRP1 KO + DRP1 K38A) (E) Immunoblot analysis confirming absence of DRP1 in DRP1 KO cells and overexpression of DRP1 in rescue experiments. Tubulin was used as a loading control.

Techniques: Confocal Microscopy, Expressing, Imaging, Knock-Out, Mutagenesis, Control, Western Blot, Over Expression

(A) Super-resolution imaging of endolysosomal tubules in cells expressing LAMP1-GFP (yellow), DRP1-mCherry (magenta), and stained with mitotracker deep red (cyan). Endolysosomal tubules were enriched using YM201636 and washout condition. (B) Intensity plot for data (A) obtained by plotting intensity values of LAMP1-GFP, DRP1-mCherry, and mitotracker channels over the distance in µm. (C) Super resolution imaging of endolysosomal tubules in cells expressing LAMP1-GFP (yellow), DRP1-mCherry (magenta), and KDEL-BFP (cyan). Endolysosomal tubules were enriched using YM201636 and washout condition. (D) Intensity plot for data (C) obtained by plotting intensity values of LAMP1-GFP, DRP1-mCherry, and KDEL-BFP over the distance in µm. (E) Super-resolution imaging of cells transfected with LAMP1-GFP (yellow), KDEL-BFP (magenta), and stained with mitotracker deep red (cyan). Endolysosomal tubules were enriched by YM201636 treatment for 2 h followed by washout and recovery in drug-free imaging buffer. White arrow indicates the contact site between ER, mitochondria, and endolysosomal tubule. (F) Intensity plots for data (E), showing intensities of all three channels along the line. Two intensity plots represent the proximity of three channels in two selected regions of interest (ROIs). (G) Live-cell super resolution microscopy shows contact sites exactly at the fission site. Cells were transfected with LAMP1-GFP (yellow), KDEL-BFP (magenta), and stained with mitotracker deep red (cyan). Fission events were enriched using YM201636 treatment for 2 h followed by washout and recovery. Cells were recorded for 2 min at 2 second intervals. The white arrowhead indicates the fission site. (H) Quantification of endolysosomal tubule fission events based on proximity to ER, mitochondria, both, or none. 100 fission events were counted from n=3 biological replicates.

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Super-resolution imaging of endolysosomal tubules in cells expressing LAMP1-GFP (yellow), DRP1-mCherry (magenta), and stained with mitotracker deep red (cyan). Endolysosomal tubules were enriched using YM201636 and washout condition. (B) Intensity plot for data (A) obtained by plotting intensity values of LAMP1-GFP, DRP1-mCherry, and mitotracker channels over the distance in µm. (C) Super resolution imaging of endolysosomal tubules in cells expressing LAMP1-GFP (yellow), DRP1-mCherry (magenta), and KDEL-BFP (cyan). Endolysosomal tubules were enriched using YM201636 and washout condition. (D) Intensity plot for data (C) obtained by plotting intensity values of LAMP1-GFP, DRP1-mCherry, and KDEL-BFP over the distance in µm. (E) Super-resolution imaging of cells transfected with LAMP1-GFP (yellow), KDEL-BFP (magenta), and stained with mitotracker deep red (cyan). Endolysosomal tubules were enriched by YM201636 treatment for 2 h followed by washout and recovery in drug-free imaging buffer. White arrow indicates the contact site between ER, mitochondria, and endolysosomal tubule. (F) Intensity plots for data (E), showing intensities of all three channels along the line. Two intensity plots represent the proximity of three channels in two selected regions of interest (ROIs). (G) Live-cell super resolution microscopy shows contact sites exactly at the fission site. Cells were transfected with LAMP1-GFP (yellow), KDEL-BFP (magenta), and stained with mitotracker deep red (cyan). Fission events were enriched using YM201636 treatment for 2 h followed by washout and recovery. Cells were recorded for 2 min at 2 second intervals. The white arrowhead indicates the fission site. (H) Quantification of endolysosomal tubule fission events based on proximity to ER, mitochondria, both, or none. 100 fission events were counted from n=3 biological replicates.

Techniques: Imaging, Expressing, Staining, Transfection, Super-Resolution Microscopy

(A) Disruption of ER contact by overexpressing CLIMP63. Cells were transfected with LAMP1-GFP (green) and KDEL-mCherry or CLIMP63-mCherry. Imaging was performed after 2 h of YM201636 treatment, washout, and 30 min of recovery. White arrows depict the LAMP1-positive compartment with respective ER morphology. (B) Schematic representation of CLIMP63 overexpression and its implication on ELR. (C) Mitochondrial morphology was disturbed by treating cells with 20 μM CCCP for 30 min. Cells expressing LAMP1-GFP (green) were treated with YM201636 for two h followed by washout and 30 min recovery. Twenty μM CCCP was introduced in the recovery media. Mitochondria were stained using MitoTracker Deep Red (magenta). Images were taken after 30 min of recovery to observe the effect. White arrow shows the morphology of the LAMP1-positive compartment in DMSO versus CCCP-treated cells. (D) Schematic representation of CCCP treatment on mitochondria and its implication on ELR. (E) Live-cell imaging of LAMP1-positive lysosomes (green) in control KO and MFF knockout (KO) cells. Cells were imaged after staining with MitoTracker Deep Red (magenta). White arrowheads highlight the LAMP-1positive compartments in the ROIs. (F) Quantification of tubules length of endolysosomes from data (A). Each dot represents the length of individual tubules and the plot shows the mean of 90 tubules measured in 30 cells across n=3 biological replicates; Unpaired t -test: ****P < 0.1 ×10 −14 (KDEL OE vs CLIMP OE). (G) Quantification of the average area of LAMP1-positive structures for (C). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n= 3 biological replicates; Unpaired t -test: ****P < 0.1 ×10 −14 (DMSO vs CCCP). (H) Quantification of the average area of LAMP1-positive structures for (E). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n = 3 biolog ical replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ****P = 0.5356 ×10 −4 (Control KO vs MFF KO C1), ****P = 0.2273 ×10 −4 (Control KO vs MFF KO C 2), ns P > 0.9 ×10 −14 (MFF KO C1 vs MFF KO C2).

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Disruption of ER contact by overexpressing CLIMP63. Cells were transfected with LAMP1-GFP (green) and KDEL-mCherry or CLIMP63-mCherry. Imaging was performed after 2 h of YM201636 treatment, washout, and 30 min of recovery. White arrows depict the LAMP1-positive compartment with respective ER morphology. (B) Schematic representation of CLIMP63 overexpression and its implication on ELR. (C) Mitochondrial morphology was disturbed by treating cells with 20 μM CCCP for 30 min. Cells expressing LAMP1-GFP (green) were treated with YM201636 for two h followed by washout and 30 min recovery. Twenty μM CCCP was introduced in the recovery media. Mitochondria were stained using MitoTracker Deep Red (magenta). Images were taken after 30 min of recovery to observe the effect. White arrow shows the morphology of the LAMP1-positive compartment in DMSO versus CCCP-treated cells. (D) Schematic representation of CCCP treatment on mitochondria and its implication on ELR. (E) Live-cell imaging of LAMP1-positive lysosomes (green) in control KO and MFF knockout (KO) cells. Cells were imaged after staining with MitoTracker Deep Red (magenta). White arrowheads highlight the LAMP-1positive compartments in the ROIs. (F) Quantification of tubules length of endolysosomes from data (A). Each dot represents the length of individual tubules and the plot shows the mean of 90 tubules measured in 30 cells across n=3 biological replicates; Unpaired t -test: ****P < 0.1 ×10 −14 (KDEL OE vs CLIMP OE). (G) Quantification of the average area of LAMP1-positive structures for (C). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n= 3 biological replicates; Unpaired t -test: ****P < 0.1 ×10 −14 (DMSO vs CCCP). (H) Quantification of the average area of LAMP1-positive structures for (E). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n = 3 biolog ical replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ****P = 0.5356 ×10 −4 (Control KO vs MFF KO C1), ****P = 0.2273 ×10 −4 (Control KO vs MFF KO C 2), ns P > 0.9 ×10 −14 (MFF KO C1 vs MFF KO C2).

Techniques: Disruption, Transfection, Imaging, Over Expression, Expressing, Staining, Live Cell Imaging, Control, Knock-Out

(A) Schematic representation of calcium transport during ELR. (B) Schematic representation of the assay used to inhibit the lysosomal calcium exporter (TRPML1) or the mitochondrial calcium importer (VDAC) to assess its effect on ELR. (C) Live-cell imaging of cells co-transfected with LAMP1-GFP (green) and the mitochondrial calcium sensor mt-RCAMP1h (magenta). ELR was initiated with 2 h YM201636 treatment and washout. Cells were imaged for 30 min at 15-min intervals. D) Mean fluorescence intensity of mt-RCAMP1h (magenta) was quantified and plotted for 0 min, 15 min and 30 min recovery timepoints. Each dot represents the gray value of an individual cell. The plot shows the mean fluorescence intensity of 30 cells from n = 3 biological replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ****P = 0.5257 × 10 −4 (YM201636 vs. 15 min recovery), ****P = 0.03522 × 10 −6 (YM201636 vs. 30 min recovery), ns P > 0.9473 (15 min recovery vs. 30 min recovery). E) Live-cell imaging of cells expressing LAMP1-GFP and treated with YM201636 together with the TRPML1 inhibitor ML-SI3. Cells were subsequently washed and recovered in media containing only ML-SI3 to test the effect of ML-SI3 alone, as described in (B). ROIs show the LAMP1-positive compartments in representative parts of cells. (F) Quantification of the average area of LAMP1-positive structures for (E). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n = 3 biological replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ns P > 0.9 ×10 −14 ( 2 h YM201636 + 30 min recovery vs. 2 h YM201636 and ML-SI3 + 30 min recovery), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 + 30 min recovery with ML-SI3), ****P = 0.691 × 10 −12 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and ML-SI3 + 15 min recovery), ns P > 0.9 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and ML-SI3 + 30 min recovery), ****P = 0.4885 ×10 −4 (2 h YM201636 + 30 min recovery with ML-SI3 vs. 2 h YM201636 and ML-SI3 + 15 min recovery), ****P = < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery with ML-SI3 vs. 2 h YM201636 and ML-SI3 + 30 min recovery). (G) Live-cell imaging of cells expressing LAMP1-GFP and treated with YM201636 together with the VDAC2/3 inhibitor erastin. Cells were subsequently washed and recovered in media containing only erastin to test the effect of erastin alone, as described in (B). ROIs show the LAMP1-positive compartments in representative parts of cells. (H) Quantification of the average area of LAMP1-positive structures for (G). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n = 3 biological replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ns P > 0.9 ×10 −14 ( 2 h YM201636 + 30 min recovery vs. 2 h YM201636 and Erastin + 30 min recovery), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 + 30 min recovery with Erastin), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and Erastin + 15 min recovery), ns P > 0.9 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and Erastin + 30 min recovery), **P = 0.00103 (2 h YM201636 + 30 min recovery with Erastin vs. 2 h YM201636 and Erastin + 15 min recovery), ****P = < 0.9 ×10 −14 (2 h YM201636 + 30 min recovery with Erastin vs. 2 h YM201636 and Erastin + 30 min recovery). (I) Live-cell imaging of cells expressing LAMP1-GFP and treated with YM201636 together with the VDAC1 inhibitor VBIT-4. Cells were subsequently washed and recovered in media containing only VBIT-4 to test the effect of VBIT-4 alone, as described in (B). ROIs show the LAMP1-positive compartments in representative parts of cells. (J) Quantification of the average area of LAMP1-positive structures for (I). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n = 3 biological replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ns P > 0.9 ×10 −14 ( 2 h YM201636 + 30 min recovery vs. 2 h YM201636 and VBIT-4 + 30 min recovery), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 + 30 min recovery with VBIT-4), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and VBIT-4 + 15 min recovery), ns P =0.2364 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and VBIT-4 + 30 min recovery), **P = 0.29 ×10 −4 (2 h YM201636 + 30 min recovery with VBIT-4 vs. 2 h YM201636 and VBIT-4 + 15 min recovery), ****P = < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery with VBIT-4 vs. 2 h YM201636 and VBIT-4 + 30 min recovery).

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Schematic representation of calcium transport during ELR. (B) Schematic representation of the assay used to inhibit the lysosomal calcium exporter (TRPML1) or the mitochondrial calcium importer (VDAC) to assess its effect on ELR. (C) Live-cell imaging of cells co-transfected with LAMP1-GFP (green) and the mitochondrial calcium sensor mt-RCAMP1h (magenta). ELR was initiated with 2 h YM201636 treatment and washout. Cells were imaged for 30 min at 15-min intervals. D) Mean fluorescence intensity of mt-RCAMP1h (magenta) was quantified and plotted for 0 min, 15 min and 30 min recovery timepoints. Each dot represents the gray value of an individual cell. The plot shows the mean fluorescence intensity of 30 cells from n = 3 biological replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ****P = 0.5257 × 10 −4 (YM201636 vs. 15 min recovery), ****P = 0.03522 × 10 −6 (YM201636 vs. 30 min recovery), ns P > 0.9473 (15 min recovery vs. 30 min recovery). E) Live-cell imaging of cells expressing LAMP1-GFP and treated with YM201636 together with the TRPML1 inhibitor ML-SI3. Cells were subsequently washed and recovered in media containing only ML-SI3 to test the effect of ML-SI3 alone, as described in (B). ROIs show the LAMP1-positive compartments in representative parts of cells. (F) Quantification of the average area of LAMP1-positive structures for (E). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n = 3 biological replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ns P > 0.9 ×10 −14 ( 2 h YM201636 + 30 min recovery vs. 2 h YM201636 and ML-SI3 + 30 min recovery), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 + 30 min recovery with ML-SI3), ****P = 0.691 × 10 −12 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and ML-SI3 + 15 min recovery), ns P > 0.9 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and ML-SI3 + 30 min recovery), ****P = 0.4885 ×10 −4 (2 h YM201636 + 30 min recovery with ML-SI3 vs. 2 h YM201636 and ML-SI3 + 15 min recovery), ****P = < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery with ML-SI3 vs. 2 h YM201636 and ML-SI3 + 30 min recovery). (G) Live-cell imaging of cells expressing LAMP1-GFP and treated with YM201636 together with the VDAC2/3 inhibitor erastin. Cells were subsequently washed and recovered in media containing only erastin to test the effect of erastin alone, as described in (B). ROIs show the LAMP1-positive compartments in representative parts of cells. (H) Quantification of the average area of LAMP1-positive structures for (G). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n = 3 biological replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ns P > 0.9 ×10 −14 ( 2 h YM201636 + 30 min recovery vs. 2 h YM201636 and Erastin + 30 min recovery), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 + 30 min recovery with Erastin), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and Erastin + 15 min recovery), ns P > 0.9 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and Erastin + 30 min recovery), **P = 0.00103 (2 h YM201636 + 30 min recovery with Erastin vs. 2 h YM201636 and Erastin + 15 min recovery), ****P = < 0.9 ×10 −14 (2 h YM201636 + 30 min recovery with Erastin vs. 2 h YM201636 and Erastin + 30 min recovery). (I) Live-cell imaging of cells expressing LAMP1-GFP and treated with YM201636 together with the VDAC1 inhibitor VBIT-4. Cells were subsequently washed and recovered in media containing only VBIT-4 to test the effect of VBIT-4 alone, as described in (B). ROIs show the LAMP1-positive compartments in representative parts of cells. (J) Quantification of the average area of LAMP1-positive structures for (I). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n = 3 biological replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ns P > 0.9 ×10 −14 ( 2 h YM201636 + 30 min recovery vs. 2 h YM201636 and VBIT-4 + 30 min recovery), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 + 30 min recovery with VBIT-4), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and VBIT-4 + 15 min recovery), ns P =0.2364 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and VBIT-4 + 30 min recovery), **P = 0.29 ×10 −4 (2 h YM201636 + 30 min recovery with VBIT-4 vs. 2 h YM201636 and VBIT-4 + 15 min recovery), ****P = < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery with VBIT-4 vs. 2 h YM201636 and VBIT-4 + 30 min recovery).

Techniques: Live Cell Imaging, Transfection, Fluorescence, Expressing

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