Journal: Journal of Bacteriology

Article Title: Cytoplasmic Copper Detoxification in Salmonella Can Contribute to SodC Metalation but Is Dispensable during Systemic Infection

doi: 10.1128/JB.00437-17

Figure Lengend Snippet: Bacterial strains and plasmids

Article Snippet: The temperature-sensitive plasmid pCP20 carrying FLP recombinase ( 59 ) was used to remove antibiotic resistance cassettes containing FLP recombination target (FRT) sites when necessary. table ft1 table-wrap mode="anchored" t5 TABLE 4 caption a7 Strain or plasmid Genotype a Deletion endpoints (bp) b Source or reference c Strain 14028 Wild type ATCC JS192 Δ sodCI-1 ::Aph 9 JS1176 Δ sodCII :: sodCII 1–133 sodCI 136–157 ( sodCIIres ) Δ sodCI-108 ::Cm 14 JS2085 Δ dsbC105 ::Kn 3222825–3223540 JS2086 Δ golT1 ::Kn 399253–401541 JS2087 Δ copA1 ::Cm 558639–561140 JS2088 Δ dsbG106 ::Cm 669962–670696 JS2089 Δ cueP2 ::Cm 3850516–3851055 JS2090 Δ golT1 ::Kn Δ copA1 ::Cm JS2091 Δ golT1 Δ copA1 JS2092 Δ golT1 Δ copA1 Δ cueP2 ::Cm JS2093 Δ sodA101 Δ sodB102 Δ sodCI-105 Δ sodCII-103 JS2094 Δ sodA101 Δ sodB102 Δ sodCI-105 Δ sodCII-103 Δ cueP2 Δ golT1 Δ copA1 ::Kn JS2095 Δ golT1 Δ copA1 Δ sodCI-105 ::Cm JS2096 Δ cueP2 ::Cm Δ sodCI-1 ::Aph JS2097 Δ golT1 Δ copA1 Δ cueP2 ::Cm Δ sodCI-1 ::Aph JS2098 Δ dsbC105 ΔdsbG106 ::Cm JS2099 Δ dsbC105 ΔdsbG106 ::Cm Δ sodCI-1 ::Aph JS2100 Δ sodCI-105 Δ sodCII-103 JS2101 Δ cueP2 ::Kn Δ sodCII :: sodCII 1–133 sodCI 136–157 ( sodCIIres ) Δ sodCI-108 ::Cm JS2102 Δ sodCI-105 Δ sodCII-103 Δ cueP2 ::Cm Plasmids pMR101 pWSK29:: sodCI + pMR102 pWSK29:: sodCII + Open in a separate window a All Salmonella strains are isogenic derivatives of S. enterica serovar Typhimurium strain 14028.

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: TBC1D15 potentiates lysosomal regeneration from damaged membranes

doi: 10.1101/2022.12.14.520480

Figure Lengend Snippet: a) HeLa cells were treated with control, TFEB or TFE3 siRNA for 48 h. Lysosomal damage was induced by 1mM LLOMe for 2 h. The LLOMe containing media was then replaced with fresh media to allow recovery. Cells were fixed at different time points after LLOMe washout. LysoTracker Red was loaded 30 min before the cells were fixed for confocal imaging. LysoTracker Red intensity was measured at different time points before and after LLOMe washout. Data are means ± SEM of 60 cells taken from three independent experiments. b) Schematic of the RFP GFP dual-tagged Gal3 (tfGal3) reporter construct used for the lysosomal regeneration flux assay. c) HeLa cells with stable expression of tfGal3 were treated with control or TFEB siRNA for 48 h. Lysosomal damage was induced by treating cells with LLOMe (1mM, 2 h). Cells were cultured in LLOMe-free medium for different durations similar to panel (a). The number of red Galectin3 puncta was counted at each time point before and after LLOMe washout. TFEB knockdown efficiency is noted by western blotting. d) Graph showing numbers of red Galectin3 puncta per cell obtained from the experiments in panel (c). Data are means ± SEM of 50 cells taken from three independent experiments. e) Schematic of the LAMP1-APEX proximity labelling assay. f) Volcano plot to compare the biotinylated landscape of APEX2-tagged LAMP1 with and without treatment with 1mM LLOMe for 2 h. Green and yellow circles indicate biotinylated proteins that are significantly enriched and significantly depleted in LLOMe-treated cells, respectively. g) Major categories and candidate proteins enriched by LLOMe treatment that are relevant to this study following pathway analysis in Perseus.

Article Snippet: The siRNAs for TBC1D15 (sc-95660), TFEB (sc-38509), TFE3 (sc-38507), ATG3 (sc-72582), ATG12 (sc-72578), ATG13 (sc-97013), ATG16L (sc-72580), Rubicon (sc-78326), DNM2 (sc-35236), LIMP2 (sc-41546) and TMEM192 (sc-89327) were purchased from Santa Cruz Biotechnology.

Techniques: Control, Imaging, Construct, Flux Assay, Expressing, Cell Culture, Knockdown, Western Blot

Journal: bioRxiv

Article Title: TBC1D15 potentiates lysosomal regeneration from damaged membranes

doi: 10.1101/2022.12.14.520480

Figure Lengend Snippet: Role of TFEB, TFE3, and mTOR in lysosomal regeneration following LLOMe-mediated damage. a) HeLa cells were treated with siRNA to deplete endogenous TFEB/TFE3. Lysosomal damage was induced with 1mM LLOMe treatment for 2 h followed by washout for the indicated durations. Cells were loaded with LysoTracker Red for 30 min, fixed, and stained with a TFEB/TFE3 specific antibody. b) HeLa cells were treated with siRNA to deplete endogenous TFEB and LAMP1-RFP was transfected to mark lysosomes. These cells were then subjected to LLOMe treatment (1mM, 2 h) followed by washout for indicated timepoints. DQ-BSA was loaded for the last 30 minutes of different treatment conditions prior to imaging the samples by confocal microscopy. TFEB knockdown efficiency was checked by western blotting c) DQ-BSA signal intensities were compared between control cells and cells depleted for endogenous TFEB as a percentage of the signal observed in control siRNA treated cells without LLOMe. Data are means ± SEM from at least 30 cells taken from three independent experiments. d) mTOR activity was assessed by monitoring the phosphorylation status of p70-S6K by western blotting for indicated treatments. LLOMe:1mM, 2 h, EBSS: 4 h, Torin1: 500nM, 6 h.

Article Snippet: The siRNAs for TBC1D15 (sc-95660), TFEB (sc-38509), TFE3 (sc-38507), ATG3 (sc-72582), ATG12 (sc-72578), ATG13 (sc-97013), ATG16L (sc-72580), Rubicon (sc-78326), DNM2 (sc-35236), LIMP2 (sc-41546) and TMEM192 (sc-89327) were purchased from Santa Cruz Biotechnology.

Techniques: Staining, Transfection, Imaging, Confocal Microscopy, Knockdown, Western Blot, Control, Activity Assay, Phospho-proteomics