Neural cultures derived from KLC1-suppressed hESC have reduced neural microtubule-associated markers.

<p>(A) Cultures were harvested at seven weeks <i>in vitro</i> and equal protein from mouse PA6 feeders cultured with control, <i>shKLC1-1</i> or no hESC (PA6 feeder cells lane) were analyzed by Western blotting for actin, GAPDH and SOD1. Note that unlike Actin, mouse and human GAPDH and SOD1 have different electrophoretic mobilities (arrows). (B–C) Control or <i>shKLC1-1</i> hESC were cultured for seven weeks with PA6 feeder cells and then harvested. Equal protein from control and <i>shKLC1-1</i> cultures was analyzed by Western blotting. (B) Representative immunoblots of Actin, NSE, GFAP, α-Tubulin, β-III-Tubulin, MAP2, pNF-H, pNF-M, Tau and pTau. (C) Quantification of protein levels relative to control and normalized to Actin. Based on n = 6 wells each *p<0.05, **p<0.01, ***p<0.001 by 2 tailed t-test compared to control.</p

Human neural cultures with reduced KLC1 exhibit altered APP metabolism.

<p>(A) APP proteolytic processing by either β-and γ-secretases or α- and γ-secretases produces sAPPβ and Aβ (shaded dark grey) or sAPPα and p3 fragments, respectively. (B–C) PA6 feeder neural differentiation cultures were harvested after seven weeks and equal protein from control and <i>shKLC1-1</i> cultures were analyzed using Western blots. (B) Representative immunoblots for full length APP in control and <i>shKLC1-1</i> neural differentiation lysates. Results for both amino (APP-N; LN27) and carboxy terminal (APP-C) antibodies are shown. The APP carboxyl terminal cleavage fragments were not reliably detected. (C) Quantification of full length APP levels relative to NSE. (D) Levels of extracellular human Aβ peptides 38, 40 or 42 amino acids in length detected in media conditioned by control or <i>shKLC1-1</i> hESC co-cultured with PA6 feeder cells for seven weeks. Human Aβ was not detected from PA6 feeder only cultures. (E) Levels of Triton X-100 soluble intracellular human Aβ-40 in control or <i>shKLC1-1</i> PA6 feeder differentiation cultures aged seven weeks. Intracellular Aβ peptides 38 or 42 amino acids long were not detectable. (F) Levels of human extracellular sAPPα and sAPPβ were detected in media conditioned by control or <i>shKLC1-1</i> PA6 feeder cocultures aged <i>in vitro</i> for seven weeks. Based on n = 6 each line; *p<0.05, **p<0.01 by 2-tailed t-test.</p

Undifferentiated KLC1-suppressed hESC exhibit normal morphology, pluripotency marker expression and karyotypes.

<p>(A) Representative images of control, <i>shKLC1-1</i> and <i>shKLC1-2</i> undifferentiated hESC cultures showing bordered colony morphology typical of pluripotent cells (arrows). Scale bar 200 micrometers. (B) Immunofluorescence staining of KLC1 in undifferentiated hESC control, <i>shKLC1-1</i> and <i>shKLC1-2</i> colonies. Merged images show overlay of KLC1 (red) and DAPI-stained nuclei (blue). Scale bar 50 micrometers. (C) Equal protein from undifferentiated control, <i>shKLC1-1</i> and <i>shKLC-2</i> culture lysates were analyzed by Western blot for KLC1 and Actin. Bar graph shows Actin normalized KLC1 levels relative to control. n = 3; **p<0.01, ***p<0.001 by 2-tailed t-test compared to control. (D–E) Immunofluorescence imagesof undifferentiated control, <i>shKLC1-1</i> and <i>shKLC1-2</i> cultures for pluripotency markers Oct-4 (D) and TRA-1-81 (E). Merged images show overlay of Oct-4 (D; red) or TRA-1-81 (E; red) and DAPI-stained nuclei (blue). Scale bar 50 micrometers. (F) Bivariate plots show distribution of cells in control, <i>shKLC1-1</i> and <i>shKLC1-2</i> undifferentiated cultures Oct-4+TRA-1-81+ (in blue). Data is representative of three experiments.</p

Neural induction cultures made from KLC1-suppressed hESC have reduced cell densities and proportions of NPs.

<p>(A–D) Control, hESC were subjected to neural induction conditions for eighteen days using PA6 feeder or EB methods as indicated. (A) Bright field image of control EB neural induction culture (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029755#pone-0029755-g002" target="_blank">Figure 2A</a> for image of PA6 differentiation). Arrows point to rosettes. Insets show close-ups of indicated rosettes. Scale bars: 200 µm for main images, 50 µm for insets. (B) Percent of cells in control EB and control PA6 feeder differentiation cultures with CD184^hiCD24^hiCD44^loCD271^lo NP cell surface marker signature (C) Quantification of cell density in EB control, <i>shKLC1-1</i> and <i>shKLC1-2</i> hESC EB neural induction cultures. EB cultures were dissociated enzymatically and counted using a hemocytometer. (D). Percent of cells within EB control, <i>shKLC1-1</i> and <i>shKLC1-2</i> hESC differentiation cultures exhibiting CD184^hiCD24^hiCD44^loCD271^lo NP cell surface marker signature after. For (B–C), control n = 9, <i>shKLC1-1</i> n = 6, <i>shKLC1-2</i> n = 3. For (D), n = 3 each line. **p<0.01, ***p<0.001 by 2-tailed t-test compared to control.</p

KLC1 and Kinesin-1C subunits are reduced in neural cultures derived from KLC1-suppressed hESC.

<p>(A) Control, <i>shKLC1-1</i> and <i>shKLC1-2</i> hESC were differentiated for seven weeks using the PA6 feeder method. Representative bright field images of control, <i>shKLC1-1</i> and <i>shKLC1-2</i> PA6 feeder cocultures collected at nine, eighteen, twenty-two and forty-eight days after plating. Arrows point to rosettes. Insets show close-ups of indicated rosettes. Arrowheads denote axon-like projections emanating from hESC derived cell clusters. Scale bars: 200 µm for main images, 50 µm for insets. (B) PA6 neural differentiation cultures were harvested after seven weeks <i>in vitro</i> and equal protein from control, <i>shKLC1-1</i> and <i>shKLC1-2</i> cultures analyzed by Western blotting for KLC1, Kinesin-1C and Actin. Bar graphs show relative quantification of KLC1 and Kinesin-1C levels relative to Actin. Based on n = 7 control and <i>shKLC1-1</i>; n = 3 <i>shKLC1-2</i>, ***p<0.001 by two-tailed Student's t-test compared to control.</p