Fig 2 - uploaded by John Pearson
Content may be subject to copyright.
In vivo zymography reveals increased collagenase activity in timp mutant ovaries. Control and timp mutant ovaries were incubated in culture medium with human Collagen IV-FITC for two hours then fixed and stained to show filamentous actin (F-actin). Green fluorescence corresponds to Col IV-FITC molecules cleaved by zymogen activity in the ovaries. (A) Fluorescence along each ovariole was measured at multiple positions within the following regions: the TF (terminal filament, 12 measurements/ovariole), germarial regions 1-2a (15 measurements/ovariole), and 2b-3 (9 measurements/ovariole), interfollicular stalks (12 measurements/ovariole) and successive egg chamber s (ECh, 12 measurements/ovariole). 5 control and 3 experimental ovarioles were measured. (B) Control ovariole. Note the Col IV-FITC staining decorating the
Source publication
Contexts in source publication
Context 1
... order to confirm that the proteins identified in the iTRAQ study represented changes due to the loss of timp activity, we performed a series of tests. First, we used an LTQ-Orbitrap ion trap mass spectrometer to compare a fraction of the proteome of 1-week old w 1118 (wild- type) ovaries with that of ovo D1 /+;; timp 28 /TM3 of the same age, as determined by the iTRAQ study. The LTQ-Orbitrap analysis identified 610 proteins with a MASCOT score above 50 and a peptide hit ! 2.380 of these (62.3%) were also found in the ovo D1 /+;; timp 28 /TM3 iTRAQ analysis (S1B- S1D Fig and S1 Table). A Gene Ontology analysis utilizing the GeneCodis tool to search for biological annotations significantly associated to both sets of identified genes ren- dered similar results, with over 90% of the clustered protein hits in the iTRAQ and LTQ approaches falling in the same Biological Process categories (S1C and S1D Fig). These results corroborated that the iTRAQ experiment identified a representative proteome of the normal tissue. Second, we performed two-dimensional DIfference Gel Electrophoresis (2D-DIGE) to validate proteins differentially expressed either in control ovaries (w 1118 ;; timp 28 /TM3) or in experimental ones (w 1118 ;; timp 28 /Df). Protein samples isolated from both types of ovaries were labeled with different fluorescent dyes and separated in two-dimensional electrophoresis, which allowed the isolation and identification by mass-spectrometry of three proteins. Two of these were up-regulated in control samples (Chorion protein S16 and Vitelline membrane 26Aa, with standardized logarithm abundance ratios of 3.02 and 3.67, respectively) and the third one was more abundant in timp mutant ovaries (Regucalcin, average ratio of -1.56). In agreement with these findings, the iTRAQ experiment identified another of the chorion pro- teins in the Drosophila genome (S15) as over-represented in control ovaries, while Regucalcin was found over-expressed in mutant ovaries (S2A- S2C Fig and S2 Table). Third, to rule out the possibility that the genetic combination used in the iTRAQ study (a deletion for the timp gene in trans to a larger deficiency for the locus) could identify as false-positives flanking genes removed by the deficiency or the deletion, we checked individually all the genes removed by both deletion and deficiency (Syn and timp) or by the deficiency alone (CG12814, CG12817, CG12420, CG34107, CG42795, CG3999, CG43143, CG6293, CG12818, CG12592, CG18545, CG31406, Best1, Sirt6, pont, Bruce, sle and jumu). None of the proteins encoded by these genes were identified in the iTRAQ ...
Context 2
... test if timp is involved in the regulation of Collagen IV turnover, most likely by preventing excessive MMP-mediated degradation, we performed an in vivo zymography assay on live ova- ries. We incubated 1-week old control and timp mutant ovaries with human Collagen IV-FITC, fixed them, co-stained them with Rhodamine-Phalloidin and measured the intensity of the FITC signal accumulated in the basement membrane of the treated ovaries. FITC fluorescence levels are proportional to Collagen IV-FITC cleavage and the proteolytic activity of the target tissue. Treatment of control and mutant ovaries in parallel rendered significantly different fluo- rescent intensity values, with the ECM of timp ovaries consistently showing stronger signal lev- els (Fig 2). Importantly, pre-incubating the samples 30' with 1mM 1, 10-Phenanthroline-a general MMP inhibitor (see for instance [25]-blocked Collagen IV-FITC degradation (S3 Fig). Thus, assuming that Collagen IV-FITC incorporates at equal levels in both control and In vivo zymography reveals increased collagenase activity in timp mutant ovaries. Control and timp mutant ovaries were incubated in culture medium with human Collagen IV-FITC for two hours then fixed and stained to show filamentous actin (F-actin). Green fluorescence corresponds to Col IV-FITC molecules cleaved by zymogen activity in the ovaries. (A) Fluorescence along each ovariole was measured at multiple positions within the following regions: the TF (terminal filament, 12 measurements/ovariole), germarial regions 1-2a (15 measurements/ovariole), and 2b-3 (9 measurements/ovariole), interfollicular stalks (12 measurements/ovariole) and successive egg chamber s (ECh, 12 measurements/ovariole). 5 control and 3 experimental ovarioles were measured. (B) Control ovariole. Note the Col IV-FITC staining decorating the experimental BMs, we conclude that Drosophila timp modulates Collagen IV degradation by regulating MMP activity in the ovarian ...
Similar publications
Relationship between co-locations and calls over time.
(PDF)
For each $n\geq 3$ we establish the existence of a nodal solution $u$ to the Yamabe problem on the round sphere $(\mathbb{S}^n,g)$ which satisfies $$\int_{\mathbb{S}^n}|u|^{2^*}dV_g < 2m_n\mathrm{vol}(\mathbb{S}^n),$$ where $m_3=9,$ $m_4= 7,$ $m_5=m_6=6$, and $m_n= 5\ \text{if }n\geq 7.
