Journal: eLife

Article Title: Sensing of nutrients by CPT1C regulates late endosome/lysosome anterograde transport and axon growth

doi: 10.7554/elife.51063

Figure Lengend Snippet: Figure 3. CPT1C-protrudin interaction. (A) CPT1C-protrudin binding was analyzed in HeLa cells by co-immunoprecipitation (co-IP). HeLa cells were co- transfected with mCitrine-protrudin or mCitrine-ER-5 (Citrine-KDEL) and MYC-CPT1C. 36 hr later, citrine-containing proteins were immunoprecipitated with the GFP-Trap assay, and the indicated proteins were detected by immunoblot in whole lysate (input) and immunoprecipitated samples (IP). The co-IP was performed in biological triplicates. A representative image of the experiment is shown. (B–C) Binding evaluation by FRET assay in HEK293 Figure 3 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.51063 15 of 26 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Transfected construct (H. sapiens) EGFP-FYCO1 DOI: 10.1038/nature14359 pDEST-EGFPFYCO1 (AmpR) Transfected construct (H. sapiens) CPT1C-mTurquoise2 other Empty vector: #54843 Vector from Addgene.

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, TRAP Assay, Western Blot

Journal: eLife

Article Title: Sensing of nutrients by CPT1C regulates late endosome/lysosome anterograde transport and axon growth

doi: 10.7554/elife.51063

Figure Lengend Snippet: Figure 5. CPT1C regulates the anterograde transport of LEs depending on malonyl-CoA levels. HeLa cells were co-transfected with mTurquoise-ER, CPT1C-mTurquoise2 or CPT1CM589S-mTurquoise2, and mCherry-FYCO1. After 48 hr, cells were treated with TOFA (1 hr at 20 mg/mL) or vehicle (control, DMSO 1:500) for 1 hr. Trajectories of mCherry-FYCO1 vesicles were analyzed. Representative videos are shown in Figure 5—videos 1–6. (A) Movement pattern of individual mCherry-FYCO1 vesicles in two dimensions. (B) Total distance traveled in anterograde and retrograde directions. (C) Quantification of vesicle velocity. (D) Scheme for the direction quantification. The random movement of the vesicles was considered value 0, and the directional movement value 1. (E) Analysis of vesicle directionality. Results are shown as mean ± SEM of 3 independent experiments performed in Figure 5 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.51063 15 of 26 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Transfected construct (H. sapiens) EGFP-FYCO1 DOI: 10.1038/nature14359 pDEST-EGFPFYCO1 (AmpR) Transfected construct (H. sapiens) CPT1C-mTurquoise2 other Empty vector: #54843 Vector from Addgene.

Techniques: Transfection, Control

Journal: eLife

Article Title: Sensing of nutrients by CPT1C regulates late endosome/lysosome anterograde transport and axon growth

doi: 10.7554/elife.51063

Figure Lengend Snippet: Figure 6. CPT1C is an ER protein localizing to ER-LE/Ly contact sites. HeLa cells were transfected with the plasmids indicated, fixed in 3% FA, stained with the indicated antibodies and analyzed by confocal microscopy. (A) Myc-CPT1C colocalizes with the ER resident protein VAPA. (B) CPT1C-mTurq2 colocalizes with the ER protein protrudin in ER-LE/Ly contact sites, but is not enriched in contact sites, contrary to protrudin (intensity line plot).

Article Snippet: DOI: https://doi.org/10.7554/eLife.51063 15 of 26 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Transfected construct (H. sapiens) EGFP-FYCO1 DOI: 10.1038/nature14359 pDEST-EGFPFYCO1 (AmpR) Transfected construct (H. sapiens) CPT1C-mTurquoise2 other Empty vector: #54843 Vector from Addgene.

Techniques: Transfection, Staining, Confocal Microscopy

Journal: eLife

Article Title: Sensing of nutrients by CPT1C regulates late endosome/lysosome anterograde transport and axon growth

doi: 10.7554/elife.51063

Figure Lengend Snippet: Figure 7. CPT1C regulates the protrudin-mediated transfer of kinesin-1 to LE/Lys in a malonyl-CoA-dependent manner. (A) Plasmids for protrudin silencing and CPT1C overexpression were transfected in HeLa cells at the same time. A scrambled sequence was used as a negative control of protrudin silencing, and an empty vector (Myc) was used as negative control of CPT1C overexpression. 72 hr later, Lamp1 localization was analysed by immunostaining. Results are shown as the percentage of cells with peripheral Lamp1 and given as mean ± SEM of 3 independent experiments Figure 7 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.51063 15 of 26 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Transfected construct (H. sapiens) EGFP-FYCO1 DOI: 10.1038/nature14359 pDEST-EGFPFYCO1 (AmpR) Transfected construct (H. sapiens) CPT1C-mTurquoise2 other Empty vector: #54843 Vector from Addgene.

Techniques: Over Expression, Transfection, Sequencing, Negative Control, Plasmid Preparation, Immunostaining

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A, E) IFM analysis of HeLa cells transfected with control or two different STX1A (1 and 2) siRNA sequences. The cells were stained for LAMP1 and LBPA or CD63 (A). Another set of cells was overexpressed with RFP-LC3 or tfLC3 (rat LC3 fused to mRFP and EGFP, E). RFP-LC3 expressing cells were stained for LAMP1. Individual and merged panels are shown separately. Insets are magnified views of the white boxed areas. Scale bars, 10 µm. (B) Plot represents the Pearson’s correlation coefficient ( r ) between LAMP1 and LBPA, shown in A. (C, D, G) Immunoblotting analysis of control and STX1A knockdown cell lysates. The blots were probed for checking the expression of different lysosome associated proteins in C, cargo proteins in D, and autophagy related proteins in G. γ- tubulin is used as the loading control in all immunoblots. The fold change in band intensities was normalized with internal control and indicated on the blots. (F) Plots represent the number of RFP-LC3 puncta, Pearson’s correlation coefficient ( r value) between LAMP1 and RFP-LC3, and the number of autolysosomes. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns-non-significant, **p≤0.01, and ***p≤0.001.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Transfection, Control, Staining, Expressing, Western Blot, Knockdown

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A) Immunoblotting analysis of STX1A knockdown and control cells for checking the expression of STX1A. * non-specific band highlighted by anti-STX1A antibody. The fold change in band intensities was normalized with internal control and indicated on the blots. (B) Immunoblotting analysis of GFP-STX1A overexpression in HeLa cells. GFP expression is used as a control. The blot was probed with an anti-GFP antibody. γ-tubulin is used as the loading control in all immunoblots. (C) Images represent the GFP-STX1A overexpression in HeLa cells. Undeconvolved (left) and deconvolved (right) images are shown separately. Insets are magnified views of the white boxed areas. (D-F) Live cell imaging microscopy of HeLa cells expressing GFP-STX1A and LAMP1-RFP in D; mCherry-STX1A and GFP-CD63 in E; and GFP-CD63 and LAMP1-RFP in F. Insets are magnified views of the white boxed areas shown in the images at different time points (s, Sec). Scale bars, 10 µm. (G) Subcellular membrane fractionation of HeLa cells. Fractions were probed for STX1A, LAMP1 (lysosome marker), Na + -K + ATPase (plasma membrane marker) and Rab22A (recycling endosome marker). In- Input and numbers represent the membrane (alternative) fractions from top to bottom of the sucrose gradient. (H) Plot represents the number of LysoTracker puncta in non-transfected (-) and GFP-STX1A overexpressing cells. (I) Plot represents the CTCF (A.U.) of DQ-Red intensity in non-transfected (-) and GFP-STX1A overexpressing cells. (J) Plot represents the size of lysosomes (μm 2 ) in non-transfected (-) and GFP-STX1A overexpressing cells. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns-non-significant and *p≤0.1.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Western Blot, Knockdown, Control, Expressing, Over Expression, Live Cell Imaging, Microscopy, Membrane, Fractionation, Marker, Transfection

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A) Schematic representation of STX1A. TM, transmembrane domain. Domain lengths are indicated by amino acid numbers. (A, C) IFM analysis of HeLa cells expressing GFP-STX1A. Cells were stained with antibodies (anti-LAMP1, anti-LAMP2, anti-LBPA, anti-EEA1, anti-p230, anti-GM130) or subjected to uptake (Dextran-AF647, LysoTracker-Red or DQ-Red BSA), stained (WGA-AF594 or phalloidin-AF594) or transfected with Arl8b-tomato or mCherry-UtrCH. Insets are magnified views of the white boxed areas. Scale bars, 10 µm. (B) Plot represents the Pearson’s correlation coefficient ( r ) of GFP-STX1A with various organelle markers as listed. (D) Plot represents the number of lysosomes/cell in the non-transfected (-) or GFP-STX1A expressing cells. (E) Plot represents the fold change in lysosome size in the non-transfected (-) or GFP-STX1A expressing cells. (F) Plots represent the ratio of change in radial profile intensity (represents lysosome dispersion) of lysosomes between 5-15 µm or more than 15 µm with respect to 0-5 µm, respectively. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns, non-significant and **p≤0.01.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Expressing, Staining, Transfection, Dispersion

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A) Schematic representation of GFP-STX1A and GFP-STX1A Δ25-146 . TM, transmembrane domain. Domain lengths are indicated by amino acid numbers. (B) IFM analysis of HeLa cells expressing GFP-STX1A Δ25-146 and stained for LAMP1 (lysosomes) or p230 (Golgi). (C) Plots represent the Pearson’s correlation coefficient ( r ) of GFP-STX1A and GFP-STX1A Δ25-146 with LAMP1 or p230 separately. (D) Plot represents the number of LAMP1 puncta/cell in the non-transfected (-) or GFP-STX1A Δ25-146 expressing cells. (E) Plots represent the size of LAMP1 puncta (μm 2 ) and their fold difference in the non-transfected (-) or GFP-STX1A Δ25-146 expressing cells. (F) Plots represent the ratio of change in radial profile intensity of lysosomes between 5-15 µm distance or more than 15 µm distance with respect to 0-5 µm distance, respectively. (G) IFM analysis of HeLa cells transfected with control siRNA, Rab7 siRNA and Arl8b siRNA. Post 24 h, the cells were again transfected with GFP-STX1A, fixed after 24 h and stained with an anti-LAMP1 antibody. (H) Plot represents the Pearson’s correlation coefficient ( r ) of GFP-STX1A with LAMP1 in siControl, siRab7 and siArl8b cells. (I) HeLa cells were transfected with GFP-STX1A. After 24 h of transfection, cells were treated with either methanol or 3 mM LLOMe for 4 h followed by fixation. The cells were stained for LAMP1 and then analyzed by IFM. Insets in IFM images are magnified views of the white boxed areas. Scale bars, 10 µm. (J) Plot represents the Pearson’s correlation coefficient ( r ) of GFP-STX1A with LAMP1 in methanol or LLOMe treated cells. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns, non-significant, ***p≤0.001 and ****p≤0.0001.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Expressing, Staining, Transfection, Control

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A) IFM analysis HeLa cells co-expressing GFP-STX1A or mCherry-STX1A with GFP-VAMP2, GFP-VAMP3, GFP-VAMP4, VAMP7-L-pHuji or VAMP8-mCherry. (B) IFM analysis of HeLa cells transfected with mCherry-STX1A Δ25-146 and GFP-VAMP2. (C) IFM analysis HeLa cells transduced with lentivirus containing shControl, shVAMP2, shSNAP23, shSNAP25, shSTX7, shSTX8, shVAMP4 or shVAMP7 plasmids. The cells were overexpressed with GFP-STX1A and stained with anti-LAMP1 antibody. In all IFM images, insets are magnified views of the white boxed areas. Scale bars, 10 µm. (D) Subcellular membrane fractionation of HeLa cells. Fractions were probed for STX1A, SNAP23, SNAP25 and VAMP2. The numbers represent the membrane (alternative) fractions from top to bottom of the sucrose gradient.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Expressing, Transfection, Transduction, Staining, Membrane, Fractionation

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A, B, D) IFM analysis of HeLa cells expressing GFP-STX1A WT or GFP-STX1A Δ25-146 . Cells were treated with nocodazole for 1.5 h, fixed and stained with anti-LAMP1 antibody in A. Cells were subjected to DQ-Red BSA processivity assay in B. Cells were subjected to LysoTracker-Red uptake assay in D. (C) Plot represents the CTCF (A.U.) of DQ-Red intensity in non-transfected (-) and GFP-STX1A Δ25-146 expressing cells. (E) Plot represents the number of LysoTracker-Red spots/cell in non-transfected (-) and GFP-STX1A Δ25-146 expressing cells. (F) IFM analysis of HeLa cells co-expressing mCherry-STX1A with GFP-Rab7 WT , GFP-Rab7 Q67L , or GFP-Rab7 T22N . Cells were fixed and stained for LAMP1 (blue). The colocalization efficiency ( r ) between Rab7 and STX1A is indicated on the images. (G) Immunoblotting analysis of Rab7 and Arl8b knockdown cell lysates along with control cell lysate. Cell lysates were probed for the expression of STX1A. γ-tubulin is used as a loading control. (H) IFM analysis of HeLa cells transfected with YFP-GAL3 alone or along with mCherry-STX1A. After 24 h, cells were treated with either methanol or 3 mM LLOMe for 4 h followed by fixation. YFP-GAL3 transfected cells were stained for LAMP1. Insets are magnified views of the white boxed areas. Scale bars, 10 µm. (I) Plot represents the fold change in the number of YFP-GAL3 particles in non-transfected (-) and mCherry-STX1A expressing cells. Cells were treated with LLOMe. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns-non-significant and ***p≤0.001.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Expressing, Staining, Transfection, Western Blot, Knockdown, Control

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A) Immunoblotting analysis of HeLa cell lysates from shControl, shSTX1A-1 and shSTX1A-2 cells. The blot was probed for the expression of STX1A. IFM analysis HeLa cells transduced with lentivirus containing shControl, shSTX1A-1, and shSTX1A-2. Cells were stained with anti-LAMP1 antibody and phalloidin-Alexa Fluor 488, followed by IFM. (B) Immunoblotting analysis of HeLa cell lysates from siControl, siSTX1A-1 and siSTX1A-2 cells. The blot was probed for the expression of STX1A. IFM analysis of HeLa cells transfected with siControl, siSTX1A-1 and siSTX1A-2, fixed and stained with anti-LAMP1 antibody and phalloidin-Alexa Fluor 488. (C) Immunoblotting analysis of siControl and siSTX1A (3’-UTR) HeLa cell lysates expressing GFP-STX1A. Blots were probed with anti-STX1A and anti-GFP antibodies separately. γ-tubulin is used as a loading control in all immunoblotting experiments. (D) IFM analysis of HeLa cells transfected with control or STX1A (3’-UTR) siRNAs. Cells were transfected with GFP-STX1A after 24 h. Insets are magnified views of the white boxed areas. Scale bars, 10 µm. (E) Plots represent the ratio of change in radial profile intensity of lysosomes between 5-15 µm or more than 15 µm distance with respect to 0-5 µm distance, respectively, as a measure of lysosome dispersion in the given conditions. -, non-transfected cells. N=3. ns-non-significant, *p≤0.1, ***p≤0.001 and ****p≤0.0001.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Western Blot, Expressing, Transduction, Staining, Transfection, Control, Dispersion

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A) IFM analysis of HeLa cells transduced with lentivirus containing control shRNA (shControl) or two different shRNAs against STX1A (shSTX1A-1 or shSTX1A-2). Cells were stained with anti-LAMP1 antibody and phalloidin-Alexa Fluor 488 or subjected to the uptake of dextran-Fluorescein (Dextran-F). Individual and merged panels are shown separately. Insets are magnified views of the white boxed areas. Scale bars, 10 µm. (B) Plots represent the ratio of change in radial profile intensity of lysosomes between 5-15 µm or more than 15 µm distance with respect to 0-5 µm distance, respectively, as a measure of lysosome dispersion. (C, D, E) Plots represent the fold change in lysosome number (C), size of the lysosomes (μm 2 ) (D) and fold change in GBA activity (F) in control and STX1A knockdown cells. (F) Immunoblotting of cell lysates probed for cathepsin B processivity. Pro- immature, m- mature, and LC- light chain. * non-specific band highlighted by anti-cathepsin B antibody. γ- tubulin is used as the loading control. The fold change in band intensities was normalized with internal control and indicated on the blots. (G) Plot represents the percentage change in the release of β-hexosaminidase (β-Hex) into the supernatant as a measure of lysosome exocytosis. Similarly, fold change in the release of β-Hex into the supernatant is shown separately. (H) Plots represent the ratio of change in radial profile intensity of lysosomes between 5-15 µm or more than 15 µm distance with respective to 0-5 µm distance, respectively. Cells were either treated with DMSO or ionomycin. (I, J) Plots represent the number of lysosomes/cell (I) and the size of lysosomes (μm 2 ) (J) in the given conditions. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns- nonspecific; *p≤0.1, **p≤0.01; ***p≤0.001 and ****p≤0.0001.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Transduction, Control, shRNA, Staining, Dispersion, Activity Assay, Knockdown, Western Blot

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A) IFM analysis of HeLa cells transduced with lentivirus containing shControl, shSTX1A-1 or shSTX1A-2 plasmids. Cells were subjected to the uptake of LysoTracker Red (left panel) or DQ-Red BSA (right panel), fixed and then imaged. Top plot represents the fold change in the number of LysoTracker-Red puncta/cell in STX1A knockdown compared to control cells. Bottom plot represents the CTCF (A.U.) of DQ-Red intensity in the control and STX1A depleted cells. The average values in mean±s.e.m. are indicated on the graphs. (B) IFM analysis of STX1A knockdown and control cells that were treated with ionomycin, fixed and stained with anti-LAMP1 antibody and phalloidin-Alexa Fluor 488. (C) IFM analysis of STX1A knockdown and control cells that were treated with methanol or LLOMe, fixed and stained with anti-LAMP1 antibody and phalloidin-Alexa Fluor 488. (D) Plots represent the ratio of change in radial profile intensity of lysosomes between 5-15 µm or more than 15 µm distance with respect to 0-5 µm distance, respectively, as a measure of lysosome dispersion in the given conditions (as shown in C). (E) IFM analysis of TFEB-GFP transfected STX1A knockdown and control cells, fixed and staining with anti-LAMP1 antibody. In all IFM images, insets are magnified views of the white boxed areas. Scale bars, 10 µm. (F) Plots represent the ratio of change in radial profile intensity of lysosomes between 5-15 µm or more than 15 µm distance with respect to 0-5 µm distance, respectively, in the given conditions (as shown in E). -, non-transfected cells. N=3. ns-non-significant, *p≤0.1, **p≤0.01, ***p≤0.001 and ****p≤0.0001

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Transduction, Knockdown, Control, Staining, Dispersion, Transfection

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A, E) IFM analysis of HeLa cells transfected with control or two different STX1A (1 and 2) siRNA sequences. The cells were stained for LAMP1 and LBPA or CD63 (A). Another set of cells was overexpressed with RFP-LC3 or tfLC3 (rat LC3 fused to mRFP and EGFP, E). RFP-LC3 expressing cells were stained for LAMP1. Individual and merged panels are shown separately. Insets are magnified views of the white boxed areas. Scale bars, 10 µm. (B) Plot represents the Pearson’s correlation coefficient ( r ) between LAMP1 and LBPA, shown in A. (C, D, G) Immunoblotting analysis of control and STX1A knockdown cell lysates. The blots were probed for checking the expression of different lysosome associated proteins in C, cargo proteins in D, and autophagy related proteins in G. γ- tubulin is used as the loading control in all immunoblots. The fold change in band intensities was normalized with internal control and indicated on the blots. (F) Plots represent the number of RFP-LC3 puncta, Pearson’s correlation coefficient ( r value) between LAMP1 and RFP-LC3, and the number of autolysosomes. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns-non-significant, **p≤0.01, and ***p≤0.001.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Transfection, Control, Staining, Expressing, Western Blot, Knockdown

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A) IFM analysis of HeLa cells transduced with lentivirus containing shControl, shSTX1A, shSNAP23, shSNAP25 or shVAMP2. The cells were stained for LAMP1 and phalloidin-Alexa Fluor 488. Individual and merged panels are shown separately. Insets are magnified views of the white boxed areas. Scale bars, 10 µm. (B, D) Immunoblotting analysis of cell lysates. The efficiency of each gene knockdown is shown in B. The expression of different cargo in the knockdown cells is shown in E. γ-tubulin is used as the loading control in all immunoblots. The fold change in band intensities was normalized with internal control and indicated on the blots. (C) The plots represent the ratio of change in radial profile intensity of lysosomes between 5-15 µm or more than 15 µm distance with respective to 0-5 µm distance, respectively. (E) Plot represents the percentage change in the release of β-Hex into the supernatant (as a measure of lysosome exocytosis) of different SNAREs knockdown cells. Similarly, fold change in the release of β-Hex into the supernatant is shown separately. The average values in mean±s.e.m. are indicated on the graph. N=3 or 5. ns-non-significant, *p≤0.1, **p≤0.01 and ***p≤0.001.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Transduction, Staining, Western Blot, Knockdown, Expressing, Control

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A) qRT-PCR analysis of different SNAREs in the cell lysates of control and STX4 knockdown cells. The fold change in band intensities was normalized with GAPDH and indicated on the gels. (B) Immunoblotting analysis of control and STX4 knockdown cell lysates. The blots were probed for checking the expression of different SNAREs and lysosome associated and degradative proteins. γ-tubulin is used as the loading control in all immunoblots. The fold change in band intensities was normalized with internal control and indicated on the blots. (C) IFM analysis of HeLa cells transfected with control or STX4 siRNAs. Cells were fixed and stained with anti-LAMP1 antibody and phalloidin-Alexa Fluor 488. Individual and merged panels are shown separately. Insets are magnified views of the white boxed areas. Scale bars, 10 µm. (D) Plots represent the ratio of change in radial profile intensity of lysosomes between 5-15 µm or more than 15 µm distance with respect to 0-5 µm distance, respectively, as a measure of lysosome dispersion in the control and STX4 depleted cells. (E) Plot represents the fold change in lysosome number between control and STX4 depleted cells. (D) Plot represents the size of lysosomes (μm 2 ) in control and STX4 depleted cells. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns-non-significant and **p≤0.01.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Quantitative RT-PCR, Control, Knockdown, Western Blot, Expressing, Transfection, Staining, Dispersion

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A) TIRF imaging of HeLa cells transduced with lentivirus containing shControl or shSTX1A mix (sh1+sh2). Cells were transfected with LAMP1-GFP and analyzed by live cell imaging. Insets are magnified views of the white boxed areas. Scale bars, 10 µm. Top: Plots represent the relative number of LAMP1- GFP vesicles at the cell surface and their kinetics in control and STX1A depleted cells. Bottom: Plots illustrate the resident time and the kinetics of LAMP1-GFP vesicles at the cell surface in control and STX1A knockdown cells. (B) GFP trap pulldown of GFP (G) or GFP-STX1A (G-S1A) expressing HeLa cell lysates. Pulldown samples were probed for input (top blot) and possible interacting SNAREs (bottom blot), including the negative controls such as VAMP7, LAMP1 and EGFR. IP – immunoprecipitated sample. (C) Model representing the lysosome exocytosis in HeLa cells. During this process, lysosome localized STX1A (Qa, blue) form a complex with SNAP23 or SNAP25 (Qbc, purple) and facilitate the fusion by interacting with plasma membrane (PM) SNARE VAMP2 (R, orange).

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Imaging, Transduction, Transfection, Live Cell Imaging, Control, Knockdown, Expressing, Immunoprecipitation, Membrane

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A, E) IFM analysis of HeLa cells transfected with control or two different STX1A (1 and 2) siRNA sequences. The cells were stained for LAMP1 and LBPA or CD63 (A). Another set of cells was overexpressed with RFP-LC3 or tfLC3 (rat LC3 fused to mRFP and EGFP, E). RFP-LC3 expressing cells were stained for LAMP1. Individual and merged panels are shown separately. Insets are magnified views of the white boxed areas. Scale bars, 10 µm. (B) Plot represents the Pearson’s correlation coefficient ( r ) between LAMP1 and LBPA, shown in A. (C, D, G) Immunoblotting analysis of control and STX1A knockdown cell lysates. The blots were probed for checking the expression of different lysosome associated proteins in C, cargo proteins in D, and autophagy related proteins in G. γ- tubulin is used as the loading control in all immunoblots. The fold change in band intensities was normalized with internal control and indicated on the blots. (F) Plots represent the number of RFP-LC3 puncta, Pearson’s correlation coefficient ( r value) between LAMP1 and RFP-LC3, and the number of autolysosomes. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns-non-significant, **p≤0.01, and ***p≤0.001.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Transfection, Control, Staining, Expressing, Western Blot, Knockdown