15 research outputs found
<i>Sox10</i> is required by bulb melanocytes postnatally.
<p>(A–B) <i>Sox10<sup>fl/fl</sup></i> (<i>fl/fl; +/+</i>) and <i>Sox10<sup>fl/fl</sup>; Tyr::CreERT2</i> (<i>fl/fl; Cre/+</i>) pups treated with TAM by IP injection to the lactating mother on P0–3 display variegated hypopigmentation on the belly and back and exhibit a white head spot upon the emergence of the morphogenetic coat (P10 shown here, n>5). (C) Number of PAX3<sup>+</sup> melanocytes per hair bulb in skins harvested from these mice at P10 are significantly decreased in <i>Sox10<sup>fl/fl</sup>; Tyr::CreERT2</i> animals compared to similarly-treated <i>Sox10<sup>fl/fl</sup></i> animals (*p = 0.002). (D–E) Adult <i>Sox10<sup>fl/fl</sup>; Tyr::CreERT2</i> mice treated with TAM by IP injection on 0–3dpp exhibit white hairs within the plucked region upon hair regrowth that is not visible in similarly treated <i>Sox10<sup>fl/fl</sup></i> mice (brackets indicate plucked region, lower image is a magnification of plucked region). (F) Number of PAX3<sup>+</sup> melanocytes per hair bulb in skins harvested from similarly-treated mice at 7dpp are significantly decreased in <i>Sox10<sup>fl/fl</sup>; Tyr::CreERT2</i> animals compared to <i>Sox10<sup>fl/fl</sup></i> animals (*p = 0.001). (G–H) Fluorescent and corresponding brightfield images of hair bulbs from mice described in D–E. Arrows and arrowheads indicate PAX3<sup>+</sup>/SOX10<sup>+</sup> and PAX3<sup>+</sup>/SOX10<sup>−</sup> melanocytes, respectively. (I) Distribution of melanocytes double-labeled for PAX3 and SOX10 within pigmented (gray) and non-pigmented (white) hair bulbs in skins from <i>Sox10<sup>fl/fl</sup></i> (n = 3) and <i>Sox10<sup>fl/fl</sup>; Tyr::CreERT2</i> (n = 4) harvested on 7dpp from mice treated with TAM on 0–3dpp (*p<0.006).</p
<i>Sox10</i> is required by LPP melanocytes postnatally.
<p>(A) Number of KIT<sup>+</sup> LPP melanocytes within hairs from <i>Sox10<sup>fl/fl</sup></i> (<i>fl/fl; +/+</i>) and <i>Sox10<sup>fl/fl</sup>; Tyr::CreERT2</i> (<i>fl/fl; Cre/+</i>) mice. P0–3/P10 indicates skins harvested from pups on P10 that were maintained by lactating mothers that were IP injected with TAM on P0–3. 0–3dpp/7dpp indicates skins harvested from adult mice on 7dpp after IP injections of TAM on 0–3dpp. (B) White hairs remain visible in adult <i>Sox10<sup>fl/fl</sup>; Tyr::CreERT2</i> mice that were treated with TAM by IP injection on 0–3dpp, allowed for complete hair regeneration, replucked and allowed for a second round of hair regrowth (brackets indicate plucked/replucked region, lower image is a magnification of plucked region; mouse in 2E and 3B are the same, imaged prior to and after replucking). (C) Number of PAX3<sup>+</sup> bulb melanocytes within hairs from <i>Sox10<sup>fl/fl</sup></i> and <i>Sox10<sup>fl/fl</sup>; Tyr::CreERT2</i> mice treated as described in B but harvested on 7dpp after replucking (0–3dpp/7dpp repluck). (D) Distribution of melanocytes double-labeled for PAX3 and SOX10 within pigmented (gray) and non-pigmented (white) hair bulbs in skins from <i>Sox10<sup>fl/fl</sup></i> (n = 3) and <i>Sox10<sup>fl/fl</sup>; Tyr::CreERT2</i> (n = 3) mice treated as described in B but harvested on 7dpp after replucking (*p<0.002). (E) Persistent hair graying is visible in <i>Sox10<sup>fl/fl</sup>; Tyr::CreERT2</i> mice treated with IP TAM for pulse of five days beginning at five weeks old and imaged at one and two years old.</p
LPP melanocytes are reduced in <i>Tg(DctSox10)</i> homozygotes during hair morphogenesis.
<p>(A) Brightfield images of hairs in <i>Tg(DctSox10)</i> and <i>+/+</i> littermates at P2. (B) Number of DCT<sup>+</sup> melanocytes within the LPP of hairs at P2 (stage 6 hairs) and P7/8. At both time points, LPP melanocytes per hair are reduced in <i>Tg(DctSox10)/Tg(DctSox10)</i> compared to <i>Tg(DctSox10)/+</i> and <i>+/+</i> mice (*p<0.017). (C, D) Quantitative immunohistochemical analysis of stage 6 hairs from P2 skins for DCT and TRP1, or DCT and KIT. The population of DCT<sup>+</sup>/TRP1<sup>+</sup> cells is significantly reduced in <i>Tg(DctSox10)/Tg(DctSox10)</i> in comparison to <i>Tg(DctSox10)/+</i> and +/+ mice (*p<0.008). <i>Tg(DctSox10)</i> also causes a switch in KIT intensity from KIT<sup>hi</sup> in wild type to KIT<sup>low</sup> in <i>Tg(DctSox10)</i> animals (*KIT<sup>lo</sup> and **KIT<sup>hi</sup> comparisons made between +/+ and <i>Tg(DctSox10)/+</i> or <i>+/+</i> and <i>Tg(DctSox10)/Tg(DctSox10)</i>; p<0.005).</p
<i>Tg(DctSox10)</i> results in congenital white spotting and premature hair graying.
<p>(A, B) Ventral and dorsal views demonstrating variable hypopigmentation in <i>Tg(DctSox10)/+</i> and <i>Tg(DctSox10)/Tg(DctSox10)</i> mice during hair morphogenesis and adult hair cycling. (C) Frequency of pigmented (pig+) and non-pigmented (pig−) anagen III/IV (7dpp) hairs that contain (DCT+ LPP cells) or do not contain (no LPP cells) LPP melanocytes within <i>Tg(DctSox10)</i> or <i>+/+</i> mice. The ages of mice analyzed ranged between 9–22 weeks at harvest. Significance determined by chi-square analysis (p<<0.0001) and evaluation of standardized residuals (*, z = −8.84; **, z = 12.24).</p
Alteration of the <i>Tg(DctSox10)</i> phenotype through the reduction of <i>Mitf</i>.
<p>(A–B) Comparison of <i>Tg(DctSox10)/Tg(DctSox10)</i> and <i>Tg(DctSox10)/Tg(DctSox10); Mitf<sup>vga9/+</sup></i> animals at P70. Addition of the <i>Mitf<sup>vga9/+</sup></i> allele reduces the congenital hypopigmentation seen in <i>Tg(DctSox10)/Tg(DctSox10)</i> animals, and is evident in dorsal views (loss of back spotting) and in ventral views (reduction in belly spot size). Premature hair graying of <i>Tg(DctSox10)</i> homozygotes seen at p70 is retained with <i>Mitf<sup>vga9</sup></i> (n = 6). (C) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003644#s1" target="_blank">Introduction</a> of <i>Sox10<sup>lacZ</sup></i> into <i>Tg(DctSox10)/Tg(DctSox10)</i> homozygotes partially rescues both congenital white spotting and premature hair graying (n = 2). (D) At 40 weeks of age, <i>Tg(DctSox10)/+; Mitf<sup>vga9/+</sup></i> double heterozygotes exhibit visibly increased hair graying in comparison to <i>Tg(DctSox10)/+</i>. (E) Hair graying severity was determined in animals 6–10 weeks of age by quantitating the number of non-pigmented anagen III/IV hair bulbs in +/+, <i>Tg(DctSox10)/+, Tg(DctSox10)/+; Mitf<sup>vga9/+</sup></i>, and <i>Mitf<sup>vga9/+</sup></i> skins after plucking and harvesting at 7dpp. <i>Tg(DctSox10)/+; Mitf<sup>vga9/+</sup></i> mice exhibit a significant increase in non-pigmented hair bulbs in comparison to the single heterozygotes or +/+ animals (**p<0.0015). <i>Tg(DctSox10)/+</i> animals also produce more non-pigmented hair bulbs in comparison to +/+ and <i>Mitf<sup>vga9/+</sup></i> animals (*p<0.002). (F) <i>Tg(DctSox10)/+; Mitf<sup>vga9/+</sup></i> animals (from E) display extensive ectopic pigmentation within the LPP of their hair follicles beyond what is normally observed in <i>Tg(DctSox10)/+</i> animals (arrows, n = 4). (G) Number of LPP melanocytes per anagen III/IV hair follicle in <i>Tg(DctSox10)/+, Tg(DctSox10)/+; Mitf<sup>vga9/+</sup>, Mitf<sup>vga9/+</sup></i> animals (from E) that double label for DCT, TRP1, and produce ectopic pigmentation. Hairs from <i>Tg(DctSox10)/+; Mitf<sup>vga9/+</sup></i> animals exhibit significantly more TRP1<sup>+</sup>/PIG<sup>+</sup> LPP melanocytes than in either single heterozygote (*p<0.0125).</p
Overexpression of <i>Sox10</i> results in premature differentiation of LPP melanocytes in anagen hairs.
<p>(A) Number of DCT<sup>+</sup> LPP melanocytes per anagen III/IV hair follicle (independent of the presence or absence of hair pigmentation) is significantly reduced in <i>Tg(DctSox10)/Tg(DctSox10)</i> mice when compared to wild type and <i>Tg(DctSox10)/+</i> mice (*p<0.0003). The ages of mice analyzed ranged between 9–22 weeks at harvest. (B) Eosin-stained skin sections of these hairs demonstrate the presence of ectopic pigmentation in the LPP of <i>Tg(DctSox10)/+</i> and <i>Tg(DctSox10)/Tg(DctSox10)</i> hairs (arrows) that is not see in wild type hairs. In <i>Tg(DctSox10)/+</i> LPP regions, this pigmentation often appeared in cells that were highly dendritic. (C, D) Brightfield and corresponding fluorescent images of anagen III/IV hair follicles double labeled for DCT and TRP1 (C) or KIT (D) in wild type and <i>Tg(DctSox10)/+</i> animals. The intensity of KIT fluorescence expression was variable, and categorized as KIT<sup>lo</sup> (arrows) or KIT<sup>hi</sup> (arrowheads), and did not appear to correlate with the presence or absence of pigmentation. (E,F) Comparison of the number LPP melanocytes per anagen III/IV hair follicle in <i>+/+</i> and <i>Tg(DctSox10)/+</i> animals that express DCT, and TRP1 or KIT, and produce ectopic pigmentation (*p<0.008).</p
RVFV virion-associated host proteins represented in canonical entry pathways.
<p>RVFV virion-associated host proteins represented in canonical entry pathways.</p
Analysis of infectivity of RVFV virions obtained under conditions of HSP inhibition.
<p>Vero cells were pretreated for 2 hours with equivalent volumes of either DMSO (control) or 17-AGG prior to infection with the MP-12 strain of RVFV (MOI 0.1). Following infection, cells were washed with PBS and media containing either DMSO or 17-AAG was added back to the cells. Twenty-four hours later, culture supernatants were collected and viral RNA levels were analyzed by qRT-PCR to measure the amounts of released virus. The supernatant samples were also functionally analyzed by plaque assays as described in the Methods section, and the plaque data was normalized to the amount of released virus based on the q-RT-PCR results. Data are average values from three independent assays.</p
Effects of siRNA silencing of host chaperones on RVFV levels.
<p>(A) Silencer Select siRNA library (Life Technologies) was used to serially assess the importance of identified chaperones in RVFV infection. HeLa cells were transfected with siRNA molecules targeting chaperone proteins, negative control targets (GAPDH, scrambled siRNA) or “no siRNA” controls. Following 48 hr incubation, cells were infected with the MP-12 strain of RVFV and the extent of viral infection assessed by high content imaging, as described in the Methods section. The down-regulation of chaperone proteins indicated in the figure resulted in statistically significant effect on viral infection when compared to the scrambled siRNA, as indicated by the asterisk symbols (P<0.05). Negative controls corresponding to GAPDH, scrambled siRNA and no siRNA control did not impact RVFV infection. The experiment was repeated in three independent trials with samples assayed in duplicate. The displayed data is a representative of the entire sample set. (B) The effects of siRNA treatments on RVFV titers were also measured using qRT-PCR analysis. For each target HSP with a duplicate siRNA set, the siRNA construct with the highest knockdown efficiency was analyzed. HeLa cells were transfected with the siRNA constructs and after 48 hours the transfected cells were infected with the MP-12 strain. The culture supernatants were analyzed 24 hours after infection. Asterisks designate significant changes in viral levels (P<0.05).</p
Effects of host chaperone inhibitors on RVFV levels and timing of HSP effects.
<p>(A) Vero cells were pretreated for 2 hours with equivalent volumes of either DMSO (control), 17-AGG (10 µM), BAPTA (10 µM), or KNK437 (10 µM), prior to infection with the MP-12 strain of RVFV (MOI 0.1). Following infection, cells were washed with PBS and media containing either DMSO or each of the compounds was added back to the cells. Twenty-four hours later, supernatants were collected and viral RNA was measured by qRT-PCR as described in the Methods section. Percent viral RNA is expressed as the percent of the DMSO control. The viability of the cells in the presence of 10 µM inhibitor or in DMSO (vehicle) was assessed by MTT assay, as described in the Methods section. Data are average values from three independent assays. (B) Time of addition studies were performed using 17-AAG and BAPTA-AM inhibitors to analyze the timing of HSP effects during RVFV infection. Vero cells were treated with HSP inhibitors, or with vehicle only (DMSO), at the indicated time points relative to MP-12 infection (MOI 0.1). Following infection, cells were washed with PBS and media containing either DMSO or each of the compounds was added back to the cells. qRT-PCR was performed to measure viral RNA levels in the supernatants collected 24 hours post-infection. Data are average values from three independent assays. (C), and (D) Viral inhibition following HSP inhibitor treatment was measured in real time. One hour prior to infection with rMP12-rLuc (MOI 0.1), HepG2 cells were treated with HSP inhibitors 17-AAG, or BAPTA-AM, or a combination of both inhibitors. Controls included no inhibitor treatment and no infection [Untreated (UT)], no inhibitor treatment but with infection using either the regular MP-12 strain that has no luciferase expression (MP12; UT) or with the luciferase expressing rMP12-rluc strain (Mp12-Luc; UT), and treatment with vehicle only, either without any infection (DMSO) or with infection using the rMP12-rluc strain (Mp12-Luc; DMSO). All samples were analyzed either at 4 hours p.i. (C) or at 8 hours p.i. (D), using the Renilla Luciferase assay. Data are average values from three independent assays. Asterisks designate significant changes in luminescence levels (P<0.05).</p