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Page 51 Ryan et al. J Transl Genet Genom. 2025;9:48-61 https://dx.doi.org/10.20517/jtgg.2024.87

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chromatography, and data processing parameters, has been previously published .

Temporal visualization of cellular components using CellLight staining
To visualize lysosomes and cell components in cultured podocytes in real time, the cells were transfected
with CellLight reagents, which express fluorescently tagged proteins targeted to specific organelles. On Day
8 of differentiation, cells were plated at 2,500 cells per well in black-walled 96-well plates coated with Geltrex
(Gibco) and incubated for 24 h under optimal conditions (37 °C, 5%CO , and humidity). On Day 9, the cells
2
were transfected with CellLight Lysosomes-green fluorescent protein (GFP; LAMP-1, Invitrogen) at
25 particles per cell, which emits green fluorescence (Excitation: 488 nm, Emission: 510 nm), and CellLight
Plasma Membrane-cyan fluorescent protein (CFP, Invitrogen), which targets the plasma membrane via the
myristoylation/palmitoylation sequence from Lck tyrosine kinase, transfected at 25 particles per cell
(Excitation: 435 nm, Emission: 485 nm).

After a 16-h incubation period to allow for the expression of the fluorescently tagged proteins, on Day 10,
the iPSC-podocytes were stained with 1 µM Hoechst 33342 to visualize the nuclei. Live-cell imaging was
performed using the Perkin Elmer Opera Phenix High Content Screening Microscope in a 37 °C, 5%CO ,
2
humid environment to ensure cell viability during imaging. Cells were re-transfected on Day 19 with
CellLight Lysosomes-GFP at 10 particles per cell, CellLight Plasma Membrane-CFP at 10 particles per cell,
and CellLight Late Endosomes-red fluorescent protein (RFP, Invitrogen), which targets Rab-7a and emits
red fluorescence (Excitation: 555 nm, Emission: 584 nm), at 25 particles per cell. This enabled continued
monitoring of lysosome/organelle dynamics over the time of podocyte differentiation. In addition,
podocytes were incubated with 50 nM LysoTracker Red (Invitrogen), diluted in pre-warmed podocyte
differentiation medium (without additional supplements) for 30 min to allow for lysosome-specific staining,
or immunofluorescence staining with a mouse monoclonal LAMP-2 antibody (Abcam, clone H4B4).
Podocytes were imaged 20 days post differentiation using the Perkin Elmer Phenix High Content Screening
Microscope under optimal incubation conditions.

Analysis of LAMP-1+ lysosomal accumulation
The built-in Harmony software of the Perkin Elmer Opera Phenix High Content Screening Microscope
enabled real-time quantitative analysis of the fluorescence intensity of CellLight and LysoTracker-stained
lysosomes. Key metrics such as the number of LAMP-1+ vesicles and cellular area were recorded over time
of culture in FD podocytes and controls. Baseline manual adjustments were made to the image analysis
parameters, such as threshold settings for fluorescence intensity, to exclude background noise and focus on
lysosome-specific signals within the cellular boundaries. Artifacts or any non-specific fluorescence were
excluded through stringent gating parameters to ensure high specificity and consistency of the analysis
across comparisons. In addition to quantifying lysosomes, their spatial distribution within the cell cytosol
was examined. This provided a more comprehensive understanding of lysosomal dynamics in iPSC-
podocytes during their differentiation into mature podocytes over the 20 days of differentiation.

Statistical analysis
The data collected from the image analysis were exported from the Harmony software v5.1.2167.302 and
processed for further statistical evaluation, with outputs per cell including lysosome number, area, and
intensity. Data were analyzed using GraphPad Prism v10.0.02, with results presented as mean ± SEM. The
non-parametric Kruskal-Wallis test was used for comparing groups, with Dunn’s post-hoc test applied for
multiple comparisons. Statistical significance was defined as P < 0.05.