ml si3  (TargetMol)

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).

Article Snippet: Erastin (Cat. HY-15763/ MCE), VBIT-4 (Cat. HY-129122/MCE), ML-SI3 (Cat. HY-139426/ MCE), CCCP (Cat. C2759/ Sigma), Nigericin (Cas.

Techniques: Live Cell Imaging, Transfection, Fluorescence, Expressing