Fig 1. Acute loss of endogenous Huntingtin changes cell morphology.

(a) Western blots show Huntingtin signal is reduced in Control 1 fibroblasts treated with siRNA targeting exon1 of Huntingtin (E1-4) compared to cells treated with siRNA against GFP (a control siRNA) or mock treated cells. Full blots are shown in S1a Fig. Graph shows mean ±SD for Huntingtin (Htt) total pixel intensity standardized to GAPDH as percent of mock treated cells from western blots (*p<0.01, n = 4 biological replicates, unpaired t-test compared to GFP. (b) Graph shows total number of Typical and Atypical cells identified using F-actin labeling. Results are shown for mock (no siRNA plus transfection reagent), siRNA targeting GFP or siRNA targeting Huntingtin (E1-4) and in the absence or presence of 100 ng/ml PDGF for 15 min. Total cell number counted for each group was mock, 219 cells; mock + PDGF, 271 cells; GFP siRNA, 269 cells; GFP siRNA + PDGF, 272 cells; Huntingtin siRNA, 203 cells, Huntingtin siRNA+ PDGF, 161cells (3 coverslips per group). Populations were compared using Pearson’s chi square test (p = 1.25 E-22), Mantel-Haenszel analysis and showed a highly significant change in cell shape in response to treatment with Huntingtin (E1-4) when controlled for PDGF treatment (p<2.2e-16; confidence interval is p<0.05) and no change in cell shape in response to PDGF treatment when controlled for treatment with Huntingtin (E1-4) (p = 0.4103). (c) Graph shows breakdown of cell counts for each category shown in Fig 1b and described in d. (d) F-actin labeling in primary fibroblasts. Sample of representative images of cells with “Typical” morphologies (Types 1–3) found in control cultures and “Atypical” morphologies (Types 4–7). “Typical” morphology (Types 1–3) have flat, polarized or non-polarized shape, have cytoplasm is well-spread with stress fibers (Type 1) or without stress fibers (Types 2 and 3), and some with ruffled membranes or lamellapodia (Type 2). “Atypical” morphology (Types 4–7) are moderately spread and have high proportion of ruffling of the plasma membrane (Type 4), are not well spread with some apparent retraction of the cytoplasm toward the nucleus (Type 5, arrow), are rounded with short adherent extensions off the plasma membrane (Type 6, arrow), or are fully rounded with no apparent adhesions (Type 7). Cells stained with Alexa-Phalloidin. Scale bar = 50 μm and applies to all images in d. (e) SiRNA treated Control 1 cells were assessed for morphology Types 1 and 2 in serum starved or stimulated with 100 ng/ml PDGF for 15 minutes. Graph shows mean percent ±SD. *<0.05, paired t-test, n = 3 coverslips. (f) Control 1 (17/19) and two HD human fibroblast lines (HD1 47/44 and HD2 20/50) were assessed in parallel for morphology types described in d. Cells were serum starved or stimulated with 100 ng/ml PDGF for 15 min. Graphs show mean percent ±SD for number of Type 1 (left) and Type 2 (right)’ Typical cells and Type 8 Atypical cells are shown in S2c Fig. A significant loss of Type 1 cells and a gain of Type 2 cells occurs in Control 1 fibroblasts with stimulation reflecting membrane ruffling (Two-way ANOVA and Bonferroni posthoc test, *p<0.05, n = 4 coverslips). Numbers in parentheses on x-axis indicate CAG repeat length for each Huntingtin allele in each cell.