Journal: HardwareX

Article Title: OpenPore: A low-cost, portable, battery-powered exponential decay pulse generator for electroporation

doi: 10.1016/j.ohx.2025.e00730

Figure Lengend Snippet: Microfluidic electroporation validation using U2 OS cells and YO-PRO™-1. (A) Photograph of assembled custom microfluidic electroporation chamber. The chamber comprises a pair of aluminum electrodes 20 mm in length and spaced 1 mm apart. The coverslip glass measures 50 mm by 25 mm and can be fitted to standard microscope stages and environmental chambers. The microfluidic channel holds approximately 2.5 µL of cell resuspended cell culture. The channel was mounted on cover slip glass, allowing for higher resolution microscopy. (B) & (C) Composite fluorescence micrographs of adherent U-2 OS cells within the microfluidic electroporation chamber in the presence of YO-PRO™-1 before and after electroporation, respectively. Scale = 50 µm.

Article Snippet: U-2 OS osteosarcoma human cells (ATCC) were used and resuspended at approximately 6 × 10 6 cells/mL in DMEM – High Glucose (Gibco).

Techniques: Electroporation, Biomarker Discovery, Microscopy, Cell Culture, Fluorescence

Journal: PLOS One

Article Title: Quantitative and temporal analysis of autophagy: Differential Response to amino acid and glucose starvation

doi: 10.1371/journal.pone.0340957

Figure Lengend Snippet: (A) MTORC1 and AMPK activity is influenced by amino acids and glucose, respectively, and regulate ULK1 activity, which induces autophagy. (B-E) U2OS cells were treated with full media (FM), FM lacking glucose (-Glc), or FM lacking amino acids (-aa) for the indicated times and cell lysates analyzed by immunoblot for ACC, total and phosphorylated at S79 (B, D) , and S6K1 phosphorylated at T389 and actin as a loading control (C, E) . Immunoblots were quantified and signal normalized to total ACC (for D) or actin loading control (for E) plotted. Symbols represent mean of 3 independent experiments and bars are s.e.m. (F-I) Monoclonal U2OS cell lines expressing DFCP1 (F) , WIPI2B (G) , WIPI1 (H) , or ATG5 ( I ) were treated with FM (blue), -Glc (green), or -aa media (red) for 6 hours and subjected to live-cell fluorescent imaging. GFP-puncta (objects) for each reporter were quantified from single cells. Trajectories include mean objects per cell (symbols); bars represent 95% CI. (J-K) GFP-LC3B objects were quantified from cells treated with FM, -aa, or -Glc in the presence of BafA1 (to prevent lysosome degradation) or a vehicle control in 2 hour increments (J) . The number of GFP-LC3 puncta synthesized (solid symbols) and degraded (open symbols) from time 0 min was calculated and plotted. The dashed lines demarcate where individual datasets were collected and data stitched together. Trajectories include mean puncta per cell (symbols); bars represent standard deviation.

Article Snippet: The human osteosarcoma cell line U2OS (HTB-96) was purchased from American Type Culture Collection, and cells maintained in RPMI-1640 medium (Gibco, 11-875-119) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Activity Assay, Western Blot, Control, Expressing, Imaging, Synthesized, Standard Deviation

Journal: PLOS One

Article Title: Quantitative and temporal analysis of autophagy: Differential Response to amino acid and glucose starvation

doi: 10.1371/journal.pone.0340957

Figure Lengend Snippet: (A-B) U2OS cells were treated for 6 hours with FM (blue), indicated as 100% aa (the concentration found in RPMI-1640), or 10% (green), 5% (orange), or 0% (red) of that amino acid concentration. Cells were lysed and ULK1 phosphorylated at S758 quantified (relative to actin loading control and normalized to time 0 controls) (A) . Bars represent means of 3 biological replicates. The data in (A) was fit to a sigmoidal dose-response curve (dashed line) to generate an EC 50 of 6% aa (B) . (C-D) Cells were treated with the medias described in A and imaged live from hours 4-6 in the presence of BafA1 (as in ). GFP-LC3 puncta were quantified from cells and sum intensity plotted (this is the sum of the intensity of all GFP-positive pixels, an output used to avoid potential issues with aggregated vesicles). Trajectories include mean objects per cell (symbols); bars represent s.e.m.; black lines represent simple linear regression (C) . The GFP-LC3 synthesis rates from the linear regression lines in (C) across amino acid concentrations were fit to a sigmoidal dose-response curve (dashed line) to generate an EC 50 of 7% aa (D) . (E) The rate of GFP-LC3 synthesis (derived from linear regression analysis, shown in (C) and the relative level of pULK1-S758 (from A) plotted to show a negative, linear association (dashed line, r 2 = 0.815).

Article Snippet: The human osteosarcoma cell line U2OS (HTB-96) was purchased from American Type Culture Collection, and cells maintained in RPMI-1640 medium (Gibco, 11-875-119) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Concentration Assay, Control, Derivative Assay

Journal: PLOS One

Article Title: Quantitative and temporal analysis of autophagy: Differential Response to amino acid and glucose starvation

doi: 10.1371/journal.pone.0340957

Figure Lengend Snippet: (A) GFP-WIPI1 (green, left Y-axis) and GFP-WIPI2B (purple, right Y-axis) object counts over the 6 hour -aa treatment were overlaid. The gray region indicates the immediate starvation period (0-1 hours), and the yellow highlights the period of delayed autophagy under sustained starvation (3-6 hours). (B) EGFP-2xFYVE puncta (PI(3)P-positive cell membranes) were quantified from cells under FM (blue) or -aa (red) treatment. Note a lack of substantial puncta increase in the immediate (0-1 hour) period (gray shading). (C) The fraction of cells containing at least 1 GFP-WIPI2B puncta is plotted with time of -aa starvation. The dashed line represents 50% of the cell population. (D) Representative EGFP-2xFYVE puncta in U2OS cells treated with a VPS34 inhibitor (1 μM compound 31, lower panels) or vehicle control (upper panels). Blue = Hoechst nuclear stain; green = EGFP-2xFYVE; captured with a 60x oil objective. Scale bars in left panels are 20 μm and scale bars in right panels (insets) are 5 μm. (E) GFP-LC3 synthesis with BafA1 in the presence of compound 31 (1 μM) or vehicle control. BafA1 was added for 1 hour during either the first hour of -aa starvation (“0-1 hr” bars) or after 4 hours of -aa starvation (“4-5 hr” bars). Data shown represent GFP-LC3 puncta synthesis relative to vehicle control. Symbols represent mean and bars are s.e.m. **** = adjusted p < 0.0001, one-way ANOVA.

Article Snippet: The human osteosarcoma cell line U2OS (HTB-96) was purchased from American Type Culture Collection, and cells maintained in RPMI-1640 medium (Gibco, 11-875-119) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Control, Staining

Journal: PLOS One

Article Title: Quantitative and temporal analysis of autophagy: Differential Response to amino acid and glucose starvation

doi: 10.1371/journal.pone.0340957

Figure Lengend Snippet: (A) Cells were cultured with or without amino acids for 6 hours prior to a restimulation phase of 60 min with FM (containing amino acids). (B-C) Representative images of GFP-DFCP1 (B) or GFP-WIPI2B (C) puncta in U2OS cells that were starved of amino acids for 6 hours and subject to aa-restimulation for 0 min (left), 10 min (middle) or 20 min (right). Insets show 2x magnification of indicated region to highlight disappearance of puncta. (D-G) DFCP1 (D) , WIPI2B (E) , WIPI1 (F) , and LC3B (G) quantified from cells during the restimulation period following -aa (red) or FM (blue) treatments. Symbols are mean GFP-positive puncta per cell and bars are s.e.m. Solid lines are non-linear regression models (one phase exponential decay). Gray shaded area emphasizes restoration to FM levels within 20 min of aa restimulation.

Article Snippet: The human osteosarcoma cell line U2OS (HTB-96) was purchased from American Type Culture Collection, and cells maintained in RPMI-1640 medium (Gibco, 11-875-119) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Cell Culture