Journal: eBioMedicine

Article Title: HMGA1 deficiency: a pathogenic link between tau pathology and insulin resistance

doi: 10.1016/j.ebiom.2025.105700

Figure Lengend Snippet: Neuronal differentiation of SH-SY5Y human neuroblastoma cells and HMGA1 and tau e xpression. ( a ) Upper panels: representative optical microscopy images of SH-SY5Y cell morphology are shown at day 1, day 5, and day 10 after starting differentiation with RA. Lower panels: representative immunofluorescence microscopy images of tau protein (green fluorescence), nuclear DAPI (blue fluorescence), and merged signals at the same time points selected for the morphological analysis of SH-SY5Y cells, during differentiation. Scale bar = 20 μm. ( b ) HMGA1 and tau mRNA and protein expression were measured in both undifferentiated (Undiff.) and differentiated (Diff.) SH-SY5Y cells, using Western blot (WB) analysis and qRT-PCR. Values represent the mean ± s.e.m. of three independent experiments. ∗∗∗ p < 0.001 versus undifferentiated cells, in each assay condition. The inset shows the progressive changes in HMGA1 and INSR expression over time (day 1, day 5, and day 10) following the initiation of differentiation with RA, as measured by WB. ( c ) HMGA1 and tau protein levels in the secretome of SH-SY5Y cells before and after neuronal differentiation. Left, representative WB of HMGA1 and tau in cell suspension (PBS) of undifferentiated (Undiff.) and differentiated (Diff.) SH-SY5Y cells. Ponceau S, protein loading control. Right, WB of HMGA1 in 24-h conditioned medium samples from undifferentiated (lanes 1–2) and differentiated (lanes 3–4) SH-SY5Y cells. A representative WB out of six independent experiments is shown. The absolute values of tau protein content in 24-h conditioned medium from undifferentiated (Undiff.) and differentiated (Diff.) SH-SY5Y cells are shown in bar graph (mean ± s.e.m.), together with the levels of p-tau (inset). ∗∗∗ p < 0.001 versus undifferentiated cells. ( d ) Oxidative stress in undifferentiated (Undiff.) and differentiated (Diff.) SH-SY5Y cells. Left, Representative images of the MitoSOX Red Superoxide Indicator assay, showing red fluorescence, which indicates intracellular, mitochondrial-generated superoxide, overlaid with brightfield images. Scale bar = 100 μm. Right, Quantification of reactive oxygen species (ROS) production using the ROS-Glo™ H 2 O 2 Assay. Data are expressed as mean ± s.e.m. of relative light units, normalized to the number of viable cells, from three independent experiments.

Article Snippet: For immunofluorescence assays, monocytes were fixed in glutaraldehyde, permeabilized with 0.1% Triton X-100, and incubated with primary polyclonal rabbit anti-human INSR antibody (N-20, α chain, sc-710; Santa Cruz Biotechnology; RRID: AB_631106 ) for 2 h at room temperature.

Techniques: Microscopy, Immunofluorescence, Fluorescence, Expressing, Western Blot, Quantitative RT-PCR, Suspension, Control, Generated

Journal: eBioMedicine

Article Title: HMGA1 deficiency: a pathogenic link between tau pathology and insulin resistance

doi: 10.1016/j.ebiom.2025.105700

Figure Lengend Snippet: Hmga1, tau and InsR expression in primary cultures of mouse neurons and in Hmga1 -deficient mice . ( a ) Hmga1 and tau protein expression, as well as tau/InsR mRNA levels, were assessed in primary cultured neurons derived from wild-type mouse embryos. pcDNA3-HMGA1a expression plasmid or anti- HMGA1 siRNA (100 pmol) was transfected into cultured neurons and Hmga1 protein and tau/InsR mRNA were measured 48–96 h later. ∗ p < 0.05, ∗∗ p < 0.01 versus control siRNA-transfected cells. Data are shown as the mean ± s.e.m. of three independent experiments. ( b ) Hmga1, InsR and tau mRNA and protein expression (left) in cerebral tissues from wild-type (+/+) and Hmga1 knockout (−/−) mice. mRNA was measured by qRT-PCR and normalized to Rps9 . Data from three independent assays from six animals per genotype are shown, together with representative Western blots (WB) of Hmga1, tau and p-tau (p-Tau1, Ser400/Thr403/Ser404; and p-Tau2, Thr205) (right) along with densitometric analysis of the blots. ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus wild-type mice.

Article Snippet: For immunofluorescence assays, monocytes were fixed in glutaraldehyde, permeabilized with 0.1% Triton X-100, and incubated with primary polyclonal rabbit anti-human INSR antibody (N-20, α chain, sc-710; Santa Cruz Biotechnology; RRID: AB_631106 ) for 2 h at room temperature.

Techniques: Expressing, Cell Culture, Derivative Assay, Plasmid Preparation, Transfection, Control, Knock-Out, Quantitative RT-PCR, Western Blot

Journal: eBioMedicine

Article Title: HMGA1 deficiency: a pathogenic link between tau pathology and insulin resistance

doi: 10.1016/j.ebiom.2025.105700

Figure Lengend Snippet: INSR expression and insulin binding in circulating blood monocytes. ( a ) Immunofluorescence of blood monocytes from AD patients, with and without the rs146052672 variant, fixed and stained with anti-INSR antibody. Representative immunofluorescence microscopy images of cell surface INSR protein (green fluorescence), nuclear DAPI (blue fluorescence), and merged signals. Scale bar = 2 μm. ( b ) Left: biotinylated insulin (B-Insulin) binding to the INSR was measured in intact monocytes from AD patients carrying (n = 6) or not carrying (n = 12) the HMGA1 variant. Fluorescence intensities are shown in bar graph (mean ± s.e.m.) for each HMGA1 genotype. Right, INSR expression was measured in monocytes from wild-type AD patient (n = 12) and AD variant carriers (n = 6), by Western blot (WB) analysis. A representative blot is shown, together with densitometric values (mean ± s.e.m). ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For immunofluorescence assays, monocytes were fixed in glutaraldehyde, permeabilized with 0.1% Triton X-100, and incubated with primary polyclonal rabbit anti-human INSR antibody (N-20, α chain, sc-710; Santa Cruz Biotechnology; RRID: AB_631106 ) for 2 h at room temperature.

Techniques: Expressing, Binding Assay, Immunofluorescence, Variant Assay, Staining, Microscopy, Fluorescence, Western Blot

Journal: NPJ Systems Biology and Applications

Article Title: Mapping the dynamics of insulin-responsive pathways in the blood–brain barrier endothelium using time-series transcriptomics data

doi: 10.1038/s41540-022-00235-8

Figure Lengend Snippet: a , b Scatter plot of cells’ calcein/EthD-1 fluorescence showing the percentage of total counted cells within the live and dead gates in a untreated control and b insulin-treated (300 minutes) cells. c Immunoblots showing IR-β expression in hCMEC/D3 cells treated with insulin for 40 and 300 minutes. d Dynamic changes of insulin receptor gene (INSR) expression in hCMEC/D3 cells treated with insulin up to 300 minutes.

Article Snippet: The proteins were then electroblotted onto a 0.45 μm nitrocellulose membrane, blocked with 5% nonfat dry milk protein (Bio-Rad Laboratories, Hercules, CA), and incubated overnight at 4 °C with primary antibodies against IR-β (1:1000, #3025, Cell Signaling Technology, Denvers, MA) and GAPDH (1:1000, #5174, Cell Signaling Technology, Danvers, MA).

Techniques: Fluorescence, Control, Western Blot, Expressing