1. Ganglioside GM3 Mediates Glucose-Induced Suppression of IGF-1 Receptor–Rac1 Activation to Inhibit Keratinocyte Motility 
Duncan Hieu M. Dam, Xiao-Qi Wang, Sarah Sheu, Mahima Vijay, Desmond Shipp, Luke Miller, Amy S. Paller 
Journal of Investigative Dermatology 
Volume 137, Issue 2, Pages (February 2017)
DOI: /j.jid
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2. Figure 1
Manipulation of GM3 levels. (a) GM3 quantification by ELISA from human diabetic (n = 7, DM) and age- and sex-matched normal (n = 5, Ctrl) plantar epidermis. (b) Left: pathways for GM3 synthesis and metabolism. Right: GM3 manipulation effects. (c) After 72 hours, treatment with two different GM3S siRNA SNAs (GM3S si), bispecific M2D3 AS SNA, supplemental glucose, or controls, GM3S protein was detected by immunoblotting with anti-GM3S antibody. (d) After 72 hours treatment with GM3S siRNA or M2D3 AS SNA, GM3S, GD3S, and GM2/GD2S mRNA expression was detected by quantitative real-time reverse transcriptase–PCR. (e) NHEKs were treated with SNAs, supplemental glucose, glucosylceramide synthase inhibitors (GCSIs, GZ 161, and C9), or controls. GM3 expression was detected by confocal microscopy with anti-GM3 antibody. Scale bar = 20 μm. ∗∗∗P < AS, antisense DNA; Cer, ceramides; Ctrl, control; DM, diabetic; GlcCer, glucosylceramide; LacCer, lactosylceramide; NT, no treatment; Scr, scrambled; Se, sense; si, small interfering RNA; SNA, spherical nucleic acid; Veh, vehicle.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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3. Figure 2
GM3 levels regulate NHEK migration. Representative scratch assays after GM3 modulation in the presence of (a, c) standard medium and (b, d) medium supplemented with 12 mmol/L glucose (total = 18 mmol/L). (a, b) Dotted lines highlight the wound edge. Graphs showing wound closure in (c) standard medium and (d) high glucose; the central key identifies lines in both graphs. (e) Boyden chemotaxis assay. Treated NHEKs were grown in standard complete (left) or high glucose (right) medium or were starved overnight (basal medium) and stimulated with IGF1 (100 ng/ml) for 18 hours of migration. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < Scale bar in a and b = 500 μm. AS, antisense DNA; h, hours; NHEK, normal human epidermal keratinocyte; NT, no treatment; Scr, scrambled; SE, sense; si, small interfering RNA; Veh, vehicle.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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4. Figure 3
GM3 depletion increases NHEK velocity and directional migration. Single-cell movement was monitored in 4-minute intervals for 100–140 minutes using live cell imaging and computerized analysis. (a) Vector maps (rose diagrams) of IGF-1 stimulated cell migration from start (0) to end as distance in μm. shown are 15 representative cells of the total 60–100 NHEKs analyzed per condition. (b) Velocity, (c) net displacement, and (d) persistence coefficient (also called processivity; net displacement/total distance with 1 representing a linear path) of modulated NHEKs in standard complete (left) and high glucose (right) medium. Mean ± standard deviation. ∗∗∗P < AS, antisense DNA; Glu, glucose; IGF-1, insulin-like growth factor-1; min, minutes; NHEK, normal human epidermal keratinocyte; NT, no treatment; Scr, scrambled; Se, sense; si, small interfering RNA; Veh, vehicle.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

5. Figure 4
GM3 expression regulates Rac1 versus RhoA activation. (a–d) NHEKs with and without GM3 modulation were starved overnight and treated for 2 minutes with or without IGF-1. Phosphorylation and expression of (a) IGF1R and (b) cofilin were examined by Western blotting. Activation of (c) Rac1 or (d) RhoA was determined by G-LISA. (e) IGF1R, Rac1, and Rho-associated protein kinase were inhibited with small molecule inhibitors (Inh) or shRNA (see Supplementary Figure S5). Cell migration was accessed by scratch assay. Scale bar (upper left image) = 100 μm. ∗∗P < 0.01, ∗∗∗P <  AS, antisense DNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G-LISA, small G-protein activation ELISA; Glu, glucose; h, hours; IGF-1, insulin-like growth factor-1; IGF1R, insulin-like growth factor-1 receptor; Inh, inhibitor; IR, insulin receptor; NHEK, normal human epidermal keratinocyte; NT, no treatment; Scr, scrambled; SE, sense; shRNA, short hairpin RNA; si, small interfering RNA; SNA, spherical nucleic acid; Veh, vehicle.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
Quantification of percentage of wound closure of GM3-modulated NHEKs in the presence of inhibitors. Each condition in the key is shown in descending order of the percentage wound closure at 18 hours, expressed as mean ± standard deviation. ** P < 0.01; ∗∗∗P < Ctrl, control; IGF1R, insulin-like growth factor-1 receptor; inh, inhibitor; IR, insulin receptor; NHEK, normal human epidermal keratinocyte; NT, untreated control; Scr, scrambled control; ShRNA, short hairpin RNA; si, small interfering RNA; Veh, phosphate buffered saline vehicle control.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

7. Figure 6
Schematic of proposed impact of GM3 modulation on IGF1R and Rac1 signaling. GM3 depletion promotes IGF1R activation, triggering a cascade of phosphorylations that leads to Rac1 activation, cofilin dephosphorylation to polymerize actin, and directional lamellipodial protrusion and increases in both cell velocity and displacement. In contrast, increases in GM3 content suppress IGF1R phosphorylation, leading to RhoA activation, cofilin phosphorylation, and inhibition of cytoskeletal polarization and cell motility. AS, antisense DNA; GCSI, glucosylceramide synthase inhibitor; IGF-1, insulin-like growth factor-1; IGF1R, insulin-like growth factor-1 receptor; NHEK, normal human epidermal keratinocyte; si, small interfering RNA; SNA, spherical nucleic acid; TNF, tumor necrosis factor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions