Additional file 1 of Progranulin haploinsufficiency mediates cytoplasmic TDP-43 aggregation with lysosomal abnormalities in human microglia
Creators: name: Sung, Wonjae
name: Noh, Min-Young
name: Nahm, Minyeop
name: Kim, Yong Sung
name: Ki, Chang-Seok
name: Kim, Young-Eun
name: Kim, Hee-Jin
name: Kim, Seung Hyun
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Immunology
subject: FOS: Clinical medicine
subject: Mental Health
subject: Virology

description: Additional file 1: Materials and Methods S1. For immunostaining, the following primary antibodies were used in this study: Galectin-3 (Gal-3, 1:100, Proteintech Cat# 14979–1-AP, RRID: AB_2136768) and cleaved-caspase3 (1:100, Cell Signaling, Cat# #9661S, RRID: AB_2910623). For determining the number of galectin-3 puncta per cell in the microscopy images were quantified using the software ImageJ. For colocalization analysis of LAMP1 and BOPIDY, Fiji software of ImageJ was used for colocalization analysis. The regions of interest (ROIs) were created with the circular enlargement of local fluorescence intensity maxima or lasso tool. The overlap and direct apposition of segmented ROIs were collectively defined as an association. The overlap was defined as colocalization. To quantify apoptosis, cleaved-caspase-3 positive cells were counted using the counter plugin and divided by the number of DAPI + nuclei in each field. Figure S1. Family members who underwent DNA analysis for segregation data are indicated with V/W for a heterozygous variant and W/W for a homozygous reference allele. Solid black denotes patients affected with FTD. A black-shaded pattern indicates patient presenting with only dementia symptoms without evidence of FTD. A single midline indicates an individual carrying a variant without clinical symptoms at the time of family pedigree generation. C Proband and his older son carried the same variant affecting the initiation codon (c.1A &gt; G), while other family members did not present with variants. Figure S2. A, C Images of the patient carrying an impaired GRN initiation codon presenting with asymmetric cortical atrophy of the left cerebral hemisphere in MRI. FDG–PET images demonstrate metabolic impairment in the left parieto-temporal cortices, medial frontal cortex, and right parietal cortex. B, D, and E. Brain MRI of the patient with a premature stop codon in GRN revealed mild atrophy of left frontal and temporal lobes. The left anterolateral temporal cortex showed a prominent decrease in metabolic activity. The left fronto-temporo-parietal cortex showed diffuse metabolic impairment. However, amyloid deposits were absent in both lateral temporal, frontal, posterior cingulate, and parietal cortices. Figure S3. A Representative fluorescence images of phosphorylated TDP-43 at Ser409/410 (pTDP-43, green) in FTD–GRN patients-derived fibroblast and control-derived fibroblast. Nuclei were stained with DAPI. Scale bar, 10 µm. B Western blot of TDP-43, pTDP-43, ubiquitin, and p62 in soluble and insoluble fractions from FTD–GRN patients-derived fibroblast and control-derived fibroblast. GAPDH was used as a loading control in soluble fractions. C Quantification of normalized TDP-43, pTDP-43, ubiquitination, p62, and PGRN expression in soluble and insoluble fractions (n = 3). Values are presented as mean ± SEM. Comparisons were made against control-derived fibroblasts (**p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001; one-way ANOVA with post hoc Tukey’s test). Figure S4. A Representative fluorescence images of IBA1 (green) and cleaved-caspase 3 (red) to identify dead cells in FTD–GRN patients-derived iMGs and control-derived iMGs. Nuclei were stained with DAPI. Scale bar, 10 µm. B Average cell death rates of GRN mutations were measured as the percentage of cleaved-caspase 3-positive cells in IBA1-positive cells. Over 30 cells were quantified per experiment from three biologically independent experiments. Values are presented as mean ± SEM. Comparisons were made against control-derived iMGs (****p &lt; 0.0001; one-way ANOVA with post hoc Tukey’s test). Figure S5. A Representative fluorescence images of Galectin-3 (Gal-3, red) in FTD–GRN patients-derived iMGs and control-derived iMGs. Nuclei were stained with DAPI. Scale bar, 10 µm. B Quantification of the percentage of galectin-3 puncta in (A). Galectin-3 puncta is the marker of vesicle rupture. Over 30 cells were quantified per experiment from three biologically independent experiments. Values are presented as mean ± SEM. Comparisons were made against control-derived iMGs (****p &lt; 0.0001; one-way ANOVA with post hoc Tukey’s test. Figure S6. A Representative fluorescence images of BODIPY (lipid droplets, green) and LAMP1 (lysosome, red) in FTD–GRN patients-derived iMGs and control-derived iMGs. The right panel shows higher magnification views of white box regions. Nuclei were stained with DAPI. Scale bar, 10 µm. B Quantification of colocalization between LAMP1 and BODIPY in (A). Over 30 cells were quantified per experiment from three biologically independent experiments. Values are presented as mean ± SEM. Comparisons were made against control-derived iMGs (****p &lt; 0.0001; one-way ANOVA with post hoc Tukey’s test). Figure S7. full uncropped Gels and Blots images. Table S1. Clinical characteristics of patients with FTD–GRN and controls.