Fig 2: Comparison of VM- versus FB-Patterned Graft CompositionPhenotypic characterization of hESC-derived VM-patterned (A–F) and FB-patterned cells (G–L) 6 months after grafting to the nigra.(A, B, G, and H) VM- (A) and FB-patterned cells (G) generated grafts of comparable size, as visualized by human NCAM staining. VM- (B) but not FB-patterned grafts (H) generated TH+ neurons in vivo.(C and I) In VM-patterned grafts (C) but not FB-patterned grafts (I), TH+ neurons also co-expressed the midbrain DA neuron marker FOXA2 (arrowheads).(D–F and J–L) FB- (J) but not VM-patterned graft cells (HuNu+) (D) expressed NKX2.1 and FOXG1 (E and K), and included DARPP-32+ neurons (F and L). See also Figure S1 for in vitro characterization of cell preparations.All graft neurons expressed a rabies tracing construct and, thus, nuclear GFP. Scale bars represent 1 mm (A, B, G, and H) and 20 μm (C–F and I–L). Images in (A), (B), (G), and (H) are digitally stitched from multiple high-magnification images. DARPP-32, dopamine- and cAMP-regulated phosphoprotein; FB, forebrain; FOXA2, forkhead box A2; FOXG1, forkhead box protein G1; hESCs, human embryonic stem cells; HuNu, human nucleus; NKX2.1, NK2 homeobox 1; TH, tyrosine hydroxylase; Tx, transplant; VM, ventral midbrain.

Fig 3: Pre-clinical models of resistance to anti-HER2 agents. A. Growth of tumors derived from subcutaneous implantation of CTR, T, and TP BT474 cells. B. RNA-Seq heatmap of the 1000 genes differentially expressed in TP as compared to T resistant cells (FC>1), showed in CTR, T, P, and TP cell populations (SkBr3 and BT474). C. Schematic representation of hyman PPP1R1B gene and exon organization of DARPP32 and its truncated isoform t-DARPP. D. Distribution of HER2 positivity among PPP1R1B amplified and wild type breast cancer cases from Metabric series. E. mRNA levels of t-DARPP and DARPP-32 in resistant BT474 and SkBr3 cells, represented as Fold Change as compared to parental cell population. F. Western Blot analysis revealing t-DARPP expression in resistant and parental BT474 and SkBr3 cell populations. The results are presented as mean +/- standard deviation of three independent experiments (*p < 0.05; **p < 0.001; ***p < 0.0001).

Fig 4: Colocalization analysis of MET‐GFP with layer 6 projection neuron markers, in mPFC and V1 across developmental ages. (a) Representative images at P2 (left panel) and P15 (right panel) of MET‐GFP (green) and DARPP‐32 (blue) overlayed expressions in layer 6 mPFC. Arrows denote examples of colocalization between MET‐GFP and DARPP‐32. (b) Quantification of the percentage of layer 6 mPFC MET‐GFP+ neurons that co‐express DARPP‐32 at P2, P9, and P15. n = 6 for P2 and P9, n = 7 for P15. “****” indicates p < .0001, analyzed by ordinary one‐way ANOVA followed by Tukey's multiple comparisons test. (c) Quantification of the percentage of layer 6 mPFC DARPP‐32+ neurons that co‐express MET‐GFP at P2, P9, and P15. n = 6 for P2 and P9, n = 7 for P15. “***” indicates p < .001, “****” indicates p < .0001, analyzed by ordinary one‐way ANOVA followed by Tukey's multiple comparisons test. (d) Representative images at P7 (top panel) and P15 (bottom panel) of MET‐GFP (green) and PCP4 (blue) overlayed expressions in layer 6 V1. (e) Representative images at P7 (top panel) and P21 (bottom panel) of MET‐GFP (green), CTIP2 (magenta), and SATB2 (yellow) overlayed expressions in layer 6 V1. White arrows denote examples of colocalization between MET‐GFP, CTIP2, and SATB2. Magenta arrows denote examples of MET‐GFP+; CTIP2+(SATB2−) neurons. (f) Quantification of the percentage of layer 6 V1 MET‐GFP+ neurons that are SATB2+(CTIP2−) at P7, P12, P15, and P21. n = 5 for each age. “*” indicates p ≤ .05, “***” indicates p < .001, analyzed by ordinary one‐way ANOVA followed by Tukey's multiple comparisons test. (g) Quantification of the percentage of layer 6 V1 MET‐GFP+ neurons that co‐express both CTIP2 and SATB2 at P7, P12, P15, and P21. n = 5 for each age. “*” indicates p ≤ .05, “***” indicates p < .001, analyzed by ordinary one‐way ANOVA followed by Tukey's multiple comparisons test. (h) Quantification of the percentage of layer 6 V1 SATB2+ neurons that express CTIP2 at P7, P12, P15, and P21. n = 5 for each age. “*” indicates p ≤ .05, analyzed by ordinary one‐way ANOVA followed by Tukey's multiple comparisons test. All scale bars = 50 µm. The brightness and contrast of each channel were increased globally in images for visualization purposes.

Fig 5: Striatal infusion of the high dose of cholesterol rescues muHTT aggregation in the striatum of HD mice A–GImmunolabeling of muHTT aggregates (red) in R6/2 ACSF and R6/2 chol‐high mice (N = 3/group). Zoom of representative confocal images of immunostaining against muHTT aggregates (red) showing muHTT aggregates positive for EM48 antibody in the infused and contralateral striatum (A) and relative quantification of number (B) and size (C) of aggregates. Hoechst (Ho, blue) was used to counterstain nuclei. 18 images/animal were analyzed from 9 sections throughout the entire striatum. Representative confocal images of immunostaining against muHTT (red) and DARPP32 (D) or GFAP (F) (green) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification (E, G). Hoechst (Ho, blue) was used to counterstain nuclei. All values are expressed as % above the mean of aggregates in the contralateral striatum of R6/2 ACSF or of R6/2 chol‐high. The data in (B, C, E, and G) are shown as scatterplots with means ± standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 10 μm (A) and 5 μm (D, F). Statistics: one‐way ANOVA followed by Newman–Keuls multiple comparison tests (*P < 0.05; **P < 0.01; ****P < 0.0001).HTEM images of EM48 pre‐embedding immunogold labeling showing muHTT aggregates in the striatal neuron cell bodies of R6/2 ACSF mice and R6/2 chol‐high mice (upper panels). In the lower panels, muHTT aggregates are clearly visible and the magnifications show the area indicated by the black square in the upper images. Arrows indicate large muHTT aggregates with a fibrous structure in the nucleus, and arrowheads indicate single 10‐nm gold particles in MSN. Nucleus (N), nucleolus (n), mitochondrion (m), rough endoplasmic reticulum (rer), Golgi apparatus (g), and white arrows indicate the nuclear envelope (N = 3 animals/group). Scale bars: 700 nm and 300 nm.I–MQuantification of aggregated and total muHTT in the infused striatum of HD mice after 4‐week cholesterol infusion by TR‐FRET analysis using different antibody pairs. Schematic representation of employed TR‐FRET assay (I). Preliminary assessment of the sustainability of the assay in wt and R6/2 striata (N = 5/group) using 4C9‐4C9 and 2B7‐MW1 antibodies in combination to detect, respectively, muHTT aggregates (J), and total muHTT (K). Quantification of muHTT aggregates (L) and soluble and other muHTT species (M) in the infused striata of R6/2 ACSF and R6/2 chol‐high mice. Data in (J–M) are shown as scatterplots with means ± standard error. Each dot corresponds to the value obtained from one striatum. Statistics: Student's t‐test (*P < 0.05).