The supplementary materials for the article "A mechanism of lysosomal calcium entry" by Matthew Zajac et al. provide comprehensive supporting data and detailed information to enhance the understanding of the study. The materials include: 1. **Supplementary Text**: - ** Localization of WT LCI**: Discusses the localization of wild-type human LCI in HeLa cells and its potential role on lysosomal membranes. - **Lysosomal Calcium Measurements**: Explains the methods used to measure lysosomal calcium levels in worms and cells, including the use of *CalipHluor2.0* and *CalipHluor*^mLy^ probes. - **Homologous Regions of Human LCI**: Analyzes the homology between human LCI and other proteins, such as vcx1, to understand the mechanism of Ca²⁺ transport. - **Yeast Color Change**: Describes the use of yeast mutants to demonstrate the rescue effect of human LCI on vacuolar dysfunction. 2. **Figures S1 to S18**: - **Figure S1**: Shows the rescue of *cup-5*+/+ worm phenotypes by human LCI. - **Figure S2**: Demonstrates the rescue of *lci-1*+/+ worm phenotypes by cup-5. - **Figure S3**: Displays the localization of human LCI disease mutants in COS-7 cells. - **Figure S4**: Confirms the lysosomal localization of human LCI in COS-7 cells. - **Figure S5**: Illustrates the rescue of organism and cellular phenotypes in *lci-1*+/+ worms by human LCI variants. - **Figure S6**: Shows the rescue of lysosomal Ca²⁺ dysregulation in *lci-1*+/+ worms by human LCI. - **Figure S7**: Provides a homology-based model of human LCI based on vcx1. - **Figure S8**: Demonstrates the rescue of K665 strain phenotypes by human LCI. - **Figure S9**: Shows the rescue of organism and cellular phenotypes in *lci-1*+/+ worms by human LCI loss-of-function mutants. - **Figure S10**: Displays the expression and localization of human LCI mutants in HeLa cells. - **Figure S11**: Measures lysosomal Ca²⁺ in *lci-1*+/+ worms expressing human LCI mutants. - **Figure S12**: Justifies the technology used for single-lysosome Ca²⁺ measurements. - **Figure S13**: Reduces lysosomal Ca²⁺ in TMEM165 KO HeLa cells. - **Figure S14**: Shows 2-IM maps of lysosomal Ca²⁺ and pH in TMEM165 KO HeThe supplementary materials for the article "A mechanism of lysosomal calcium entry" by Matthew Zajac et al. provide comprehensive supporting data and detailed information to enhance the understanding of the study. The materials include: 1. **Supplementary Text**: - ** Localization of WT LCI**: Discusses the localization of wild-type human LCI in HeLa cells and its potential role on lysosomal membranes. - **Lysosomal Calcium Measurements**: Explains the methods used to measure lysosomal calcium levels in worms and cells, including the use of *CalipHluor2.0* and *CalipHluor*^mLy^ probes. - **Homologous Regions of Human LCI**: Analyzes the homology between human LCI and other proteins, such as vcx1, to understand the mechanism of Ca²⁺ transport. - **Yeast Color Change**: Describes the use of yeast mutants to demonstrate the rescue effect of human LCI on vacuolar dysfunction. 2. **Figures S1 to S18**: - **Figure S1**: Shows the rescue of *cup-5*+/+ worm phenotypes by human LCI. - **Figure S2**: Demonstrates the rescue of *lci-1*+/+ worm phenotypes by cup-5. - **Figure S3**: Displays the localization of human LCI disease mutants in COS-7 cells. - **Figure S4**: Confirms the lysosomal localization of human LCI in COS-7 cells. - **Figure S5**: Illustrates the rescue of organism and cellular phenotypes in *lci-1*+/+ worms by human LCI variants. - **Figure S6**: Shows the rescue of lysosomal Ca²⁺ dysregulation in *lci-1*+/+ worms by human LCI. - **Figure S7**: Provides a homology-based model of human LCI based on vcx1. - **Figure S8**: Demonstrates the rescue of K665 strain phenotypes by human LCI. - **Figure S9**: Shows the rescue of organism and cellular phenotypes in *lci-1*+/+ worms by human LCI loss-of-function mutants. - **Figure S10**: Displays the expression and localization of human LCI mutants in HeLa cells. - **Figure S11**: Measures lysosomal Ca²⁺ in *lci-1*+/+ worms expressing human LCI mutants. - **Figure S12**: Justifies the technology used for single-lysosome Ca²⁺ measurements. - **Figure S13**: Reduces lysosomal Ca²⁺ in TMEM165 KO HeLa cells. - **Figure S14**: Shows 2-IM maps of lysosomal Ca²⁺ and pH in TMEM165 KO He