Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: Antibodies used for different experiments in this report

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques:

Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: Primers used for RT-PCR

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques: Sequencing

Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: siRNA duplexes used for RNAi experiments

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques: Negative Control

Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: a Using mRNAs extracted from testes (T) of adult rats, Sertoli cells (SC) from 20-day-old rats (day 4 cultures), germ cells (GC) freshly isolated from adult rat testes, small intestines (Int), and liver (Liv) from adult rats for RT-PCR with the corresponding primer pairs specific for Dvl1, Dvl2, Dvl3 (Table ), all three Dishevelled mRNAs were found to be expressed by Sertoli and germ cells in the testis. Representative data from a single experiment, and n = 3 independent experiments yielded similar results. b Immunoblot analysis illustrating the specificity of the anti-Dvl3 antibody (Table ), which detected a prominent band of 90 kDa using lysates (40 µg protein per lane) of either Sertoli cells (SC) or testes (T), consistent with the Mr of Dvl3 as earlier reported . c Dvl3 (green fluorescence) distribution in Sertoli cells cultured in vitro for 3 days was shown herein, which localized prominently to the Sertoli cell–cell interface, with some Dvl3 staining in cytosol and cell nuclei, consistent with its localization in cell cortical zone, cytosol, and cell nuclei as shown in other mammalian cells as earlier reports , . No Dvl3 staining was noted when the primary antibody was substituted by normal rabbit IgG (negative control), illustrating the specificity of staining for Dvl3 shown in micrographs in the left panel. Scale bar, 40 µm, which applies to other micrographs. d Localization of Dvl3 in cross-sections of testes, illustrating possible stage-specific expression of Dvl3 in the seminiferous epithelium of adult rat testes. No Dvl3 staining was found in control sections in which the primary antibody was substituted with the corresponding normal rabbit IgG, illustrating staining specificity. Scale bar, 400 µm, which applies to other micrograph in the same panel. e Schematic drawing in the upper panel, illustrating stage-specific localization of Dvl3 at the apical ES during the epithelial cycle, which was prepared based on the observations noted in the images in the lower panel. Fluorescent images regarding the distribution of Dvl3 (green fluorescence) across the seminiferous epithelium in representative stages of the epithelial cycle, in particular the enlarged boxed rectangles in red, illustrating changes in Dvl3 distribution at the apical ES. In brief, Dvl3 prominently expressed around the entire spermatid heads at the apical ES in stage I–IV and V tubules. In stage VI–VII, Dvl3 began to move away from the spermatid head, more concentrated to the tip of spermatid heads, and Dvl3 no longer wrapped around the spermatid head in stage VIII tubules, apparently being phagocytosed (or endocytosed) by Sertoli cells, appearing as droplets inside the Sertoli cell. Scale bar, 80 µm; 40 µm in inset, which apply to corresponding micrographs and insets

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Fluorescence, Cell Culture, In Vitro, Staining, Negative Control, Expressing, Control

Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: a Dvl3 (green fluorescence) was found to colocalize with F-actin (red fluorescence) at the site of the BTB/basal ES (annotated by yellow arrowheads), apical ES, and also track-like structures across the epithelium. Scale bar, 400 µm and 40 µm, which apply to corresponding micrographs in the same panel. b Dvl3 (green fluorescence) also colocalized with α-tubulin (red fluorescence; α- together with β-tubulin create the α-/β-tubulin dimers, which are the building blocks of MTs), and the MT-conferred tracks were notably detected in the epithelium, which laid perpendicular to the basement membrane at the base of the seminiferous epithelium. Scale bar, 400 µm and 40 µm, which apply to corresponding micrographs in the same panel

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques: Fluorescence, Membrane

Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: a Using the adjudin animal model that is known to mimic the sequence of events leading to the release of sperm at spermiation to study the biology of spermiation , it was shown that adjudin treatment (50 mg/kg b.w., by oral gavage) rapidly downregulated the expression of Dvl3 within 6 h. Uncropped immunoblots (IBs) were shown in Figure . b Distribution of Dvl3 (green fluorescence) was rapidly perturbed following adjudin treatment, and this disruptive changes in Dvl3 distribution closely mimicked adjudin-induced disruption of the actin (red fluorescence)- and MT (red fluorescence)-based cytoskeletal organization. Following adjudin treatment, the track-like structures conferred by F-actin surrounding the developing spermatids in these stage VI–VII tubules were grossly disrupted; and virtually no elongating/elongated spermatids were noted in the seminiferous epithelium in all the tubules examined by 96 h when compared with control testes. Scale bar, 40 µm, which applies to corresponding micrographs in the same panel

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques: Animal Model, Sequencing, Expressing, Western Blot, Fluorescence, Disruption, Control

Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: a Regimen used in this study to monitor the effects of Dvl3 knockdown by RNAi to assess: (i) changes in the steady-state level of target gene expression by RT-PCR or protein level by immunoblotting (IB) and also protein expression by immunofluorescence analysis (IF); or (ii) changes in Sertoli cell TJ-permeability barrier function by quantifying transepithelial resistance (TER) across the Sertoli cell epithelium on the bicameral units. Transfection of Sertoli cells with siRNA duplexes (including negative non-targeting control siRNA duplexes) was performed on day 3 for 12 h prior to their removal by washing (thrice) with F12/DMEM medium, based on pilot experiments. b Representative findings of an RT-PCR experiment, illustrating specificity of Dishevelled protein knockdown, in which knockdown of Dvl1, Dvl2, or Dvl3 using corresponding specific siRNA duplexes (Table ) only downregulated the expression of the intended Dvl gene expression. c Representative IB results illustrating a knockdown of Dvl3 by almost ~ 80% (see bar graph on the right panel also) failed to perturb the expression of multiple BTB-associated proteins except a considerably down-regulation of Eps8 (an actin barbed end capping and bundling protein) and up-regulation of MARK4 (a Ser/Thr protein kinase known to stabilize MT ) (see also right panel for composite data of n = 3 independent experiments), thereby perturbing the corresponding actin- and MT-based cytoskeletal function. Uncropped IBs were shown in Figure . Each bar in the histogram shown in the right panel is a mean ± SD of n = 3 independent IB experiments. * P < 0.01, compared to the corresponding control by Student’s t test. d Fluorescent staining of Dvl3 (green fluorescence) at the Sertoli cell–cell interface (and some in cell cytosol and in cell nuclei), illustrating a ~ 80% knockdown of Dvl3 following transfection of Sertoli cells with Dvl3-specific siRNA duplexes vs. non-targeting negative control siRNA duplexes (see also bar graph on the right panel for the composite data). Successful transfection was indicated by siGLO (red fluorescence) transfection reagent. Scale bar, 40 µm, which applies to all other micrographs. Each bar in the bar graph on the right panel is a mean ± SD of n = 3 independent experiments. * P < 0.01, by Student’s t test. e A knockdown of Dvl3 (vs. Dvl1 and Dvl2 when compared with control) was considerably more effective to perturb the Sertoli cell TJ-permeability barrier function than Dvl1 or Dvl2 alone, and it was just as effective as Dvl1/2/3 triple knockdown. Each data point is a mean ± SE of n = 4 quadruple bicameral units of a representative experiment. * P < 0.05, when compared to the corresponding control by Student’s t -test. This experiment was repeated thrice with n = 3 independent experiments and yielded similar results

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques: Knockdown, Targeted Gene Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Permeability, Transfection, Control, Gene Expression, Staining, Fluorescence, Negative Control

Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: A knockdown of Dvl3 by RNAi (Dvl3 RNAi) using specific siRNA duplexes vs. non-targeting negative control siRNA duplexes (Ctrl RNAi) was found to considerably perturb the distribution of TJ proteins (green fluorescence) CAR (an integral membrane protein) and ZO-1 (a TJ adaptor protein), and basal ES proteins (green fluorescence) N-cadherin (an integral membrane protein) and β-catenin (a basal ES adaptor protein). These proteins were localized at the Sertoli cell cortical zone in control cells (see white brackets). However, following Dvl3 knockdown by ~ 80% by RNAi, these proteins were rapidly endocytosed, no longer tightly localized at the Sertoli cell–cell interface (see yellow brackets) to confer the TJ-permeability barrier. These changes thus led to a loss of TJ-barrier function as noted in Fig. . See Figure for negative control IF images, illustrating the staining shown herein was specific to the corresponding marker protein. Scale bar, 40 µm, which applies to all other micrographs. Bar graphs on the right panel is the composite data in which each bar is a mean ± SD of n = 3 independent experiments using different batches of Sertoli cells, which yielded similar results. About 100 Sertoli cells from an experiment were randomly scored for analysis. *, P < 0.01 by Student’s t test when compared with the corresponding control group. Sertoli cell nuclei were stained with DAPI (blue fluorescence), and siGLO (red fluorescence) transfection indicator illustrates successful transfection

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques: Knockdown, Negative Control, Fluorescence, Membrane, Control, Permeability, Staining, Marker, Transfection

Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: a In control cells transfected with non-targeting negative control siRNA duplexes (Ctrl RNAi), F-actin stretched across the Sertoli cell cytosol as linear bundles. However, following Dvl3 knockdown, actin filaments were extensively truncated and mis-organized, appearing as considerably branched filaments, which are clearly noted in the enlarged images boxed in yellow. These changes appeared to be the result of changes in spatial expression of Arp3 and Eps8 as these actin regulatory proteins no longer tightly localized at the Sertoli cell–cell interface to confer the Sertoli cell TJ-barrier function but endocytosed to Sertoli cell cytosol. For instance, Arp3 tightly associated at the Sertoli cell–cell interface in controls (see white brackets) were diffusely localized in Dvl3 silenced Sertoli cells (see yellow brackets). Sertoli cell nuclei were visualized by DAPI (blue fluorescence). See Figure for negative control IF images, illustrating the staining shown herein was specific to the corresponding marker protein. Scale bar, 40 µm, which applies to corresponding micrographs in this panel; inset, 20 µm, which applies to other insets. b Findings of a representative biochemical assay, illustrating changes in actin polymerization kinetics following a knockdown of Dvl3, which impeded the ability of Sertoli cells to polymerize actin into linear filaments. This experiment was repeated with n = 3 using different batches of Sertoli cells, which yielded similar observations. In brief, polymerization of fluorescent pyrene-labeled actin was monitored by enhanced fluorescence emission at 395–440 nm over time (top panel). Changes in polymerization during the first 10 min, illustrating the rate of actin polymerization (increase in fluorescence intensity over time) during the initial linear phase in the first 10 min was plotted (lower left panel) and estimated by linear regression (lower right panel) to show the relative polymerization rate in Dvl3 RNAi vs. non-targeting negative control group. Each bar is a mean ± SD of n = 3 experiments. *, P < 0.01 compared with control RNAi by Student’s t test. c An actin-spin-down assay to monitor the relative levels of F- vs. G-actin, illustrating the ability of the Sertoli cell lysates to maintain polymerized actin filaments in these cell cultures. Dvl3 RNAi considerably perturbed the level of F-actin in cell lysates. These findings were results of a representative experiment of n = 3 experiments, and the composite data are shown in the lower panel. Uncropped IBs were shown in Figure . Histogram is the composite data wherein each bar is a mean ± SD of n = 3 experiments. *, P < 0.01 when compared with corresponding control by Student’s t -test

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques: Control, Transfection, Negative Control, Knockdown, Expressing, Fluorescence, Staining, Marker, Labeling, Spin Down Assay

Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: a In control cells transfected with non-targeting negative control siRNA duplexes (Ctrl RNAi), MTs (visualized by α-tubulin staining, green fluorescence, which together with β-tubulin create the α-/ß-tubulin dimers, which are the building blocks of MTs) stretched across the Sertoli cell cytosol. However, following Dvl3 knockdown, MTs were retracted from cell peripheries and stayed closer to the Sertoli cell nuclei. Sertoli cell nuclei were visualized by DAPI (blue fluorescence). The lower panel illustrates changes in the organization of detyrosinated α-tubulin, the more stabilized forms of α-tubulin , as in control cells, detyrosinated α-tubulin appeared as linear while mildly branched MTs; however, after Dvl3 knockdown, α-tubulin appeared as extensively branched/truncated network (see magnified image in yellow boxed area). See Figure for negative control IF images, illustrating the staining shown herein was specific to the corresponding marker protein. Scale bar, 40 µm, which applies to corresponding micrographs in this panel; 15 µm in boxed inset, which apply to corresponding insets. b Results of a representative biochemical assay that monitored the ability of Sertoli cell lysates to polymerize tubulins into MTs, which were recovered in the pellet vs. free tubules in S/N (supernatant). It was noted that a knockdown of Dvl3 by RNAi perturbed the ability of the Sertoli cells to polymerize MTs considerably. Uncropped IBs were shown in Figure . The histogram shown on the right panel is the composite data of n = 3 independent experiments using different batches of Sertoli cells following knockdown of Dvl3 with each bar a mean ± SD of three experiments, and each experiment had triplicate cultures. *, P < 0.05 when compared with the corresponding control by Student’s t test

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques: Control, Transfection, Negative Control, Staining, Fluorescence, Knockdown, Marker

Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: a Regimen used to treat adult rats with n = 6 rats per treatment or control group. Testes received the Dvl1, Dvl2, and Dvl3-specific siRNA duplexes (triple knockdown) vs. non-targeting negative control siRNA duplexes on day 0, 2, and 4 (thrice) and rats were killed on day 6 for either IB, histological analysis, or immunofluorescence (IF) analysis. This regimen was established based on results of pilot experiments as a knockdown of Dvl3 alone failed to induce considerable phenotype in the testis, thus a triple knockdown by RNAi was used instead. b Representative IB data from n = 3 experiments, illustrating successful knockdown of Dvl1, Dvl2, and Dvl3, without affecting the expression of other PCP proteins such as Fzd3 and Vangl2, nor other BTB-associated except for a considerable downregulation of actin barbed end capping and bundling protein Eps8 (known to maintain actin filament bundles at the ES), and upregulation of MARK2 regulatory protein (known to promote MT catastrophe, i.e., destabilizing MTs), consistent with findings in vitro (see Fig. ). Uncropped IBs were shown in Figure . Histograms in the lower panel are the composite IB data such as those shown in the upper panel from n = 3 independent experiments. Each bar is a mean ± SD of three experiments. *, P < 0.01 when compared with the corresponding control by Student’s t test. c Histological analysis was performed using paraffin sections (~ 5 µm thick) of testes (fixed in modified Davidson fixative) and stained with hematoxylin and eosin. Control testes transfected with non-targeting negative control siRNA duplexes had normal spermatogenesis in the seminiferous epithelium (left panel). However, considerable defects in spermatogenesis were detected in the epithelium from testes following Dvl1/2/3 knockdown by RNAi as noted in the second and third columns; selected areas in these two columns were boxed in either red or blue and magnified in the corresponding insets to illustrate defects. These include: (i) step 19 spermatids (white arrowheads) found in stage I–IV tubules, co-existing with steps 15–17 spermatids, and also some multinucleated round spermatids (green arrowheads); (ii) step 19 spermatids remained trapped deep inside the epithelium in stage VII or VIII tubules (blue arrowheads) when they should have been transported near the tubule lumen to prepare for their release at spermiation, also defects in spermatid polarity were noted with these spermatids no longer pointed toward the basement membrane but deviated by 90°–180° from the intended orientation (yellow arrowheads); (iii) step 19 spermatids were detected in the epithelium, co-existing with step 9,10, 11, or 12 spermatids (white arrowheads) and mult-nucleated round spermatids (green arrowheads) in stages IX–X and XI–XII tubules, and some step 19 spermatids had defects in polarity (yellow arrowhead); and (iv) considerable thinning of the epithelium was also detected, possibly due to unwanted release of elongated spermatids as a result of defects in cytoskeletal organization. Scale bar, 80 µm in first panel, which applies to all other images in first, second and third panels; 40 µm in red and blue insets, which apply to all corresponding insets. d Percentage (%) of defective tubules from at least 100 randomly scored tubules from cross-sections of a rat testis with n = 4 rats. *, P < 0.01 compared with the control group by Student’s t test

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques: Control, Knockdown, Negative Control, Immunofluorescence, Expressing, In Vitro, Modification, Staining, Transfection, Membrane

Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: a Distribution of Dvl3 (green fluorescence) and its co-localization with F-actin (red fluorescence) in the seminiferous epithelium in control testes noted herein were similar to findings shown in Figs and , wherein Dvl3 appeared as track-like structures co-localized with F-actin in stage V–VI tubules, associated with elongating spermatids. However, the expression of Dvl3 across the seminiferous epithelium was considerably diminished following Dvl1/2/3 knockdown by RNAi in the testis. The disruptive changes in the organization of F-actin across the seminiferous epithelium in tubules (such as stage V–VI tubules) were remarkable in magnified images shown in the lower panel following a knockdown of Dvl1/2/3 as illustrated by Dvl3 (green fluorescence) immunofluorescence staining. For instance, F-actin no longer highly expressed at the apical ES and orderly aligned across the seminiferous epithelium to support elongating/elongated spermatids. Scale bar, 350 µm and 80 µm, in the top and bottom panel, respectively, which apply to other images in the corresponding panel. b In control testes, Arp3 (red fluorescence, an branched actin polymerization protein, causing linear actin filaments to assume a branched configuration) or Eps8 (red fluorescence, an actin barbed end capping and bundling protein, causing linear actin filaments to assemble into bundles as noted in the ES) and F-actin (green fluorescence) were predominantly expressed and localized (and also co-localized) at the concave (ventral) side of elongated spermatid heads (see enlarged images in red boxed areas), and also at the basal ES/BTB (annotated by yellow arrowheads) as noted in the corresponding upper and lower panels in stage VII tubules. However, following knockdown of Dvl1/2/3 by RNAi, Arp3, and Eps8 no longer expressed prominently at the apical ES, but considerably downregulated and no longer restrictively expressed at the concave side of spermatid heads (see corresponding enlarged images in red boxed areas). It appeared that Dvl1/2/3 knockdown did not considerably affect spatial expression of Arp3 or Eps8, nor distribution of F-actin, at the basal ES/BTB. Scale bar, 80 µm, which applies to corresponding micrographs in the same panel. See Figure for negative control IF images, illustrating the staining shown herein was specific to the corresponding marker protein

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques: Fluorescence, Control, Expressing, Knockdown, Immunofluorescence, Staining, Negative Control, Marker

Journal: Cell Death & Disease

Article Title: Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

doi: 10.1038/s41419-019-1394-7

Figure Lengend Snippet: a Distribution of Dvl3 (green fluorescence) and its colocalization with α-tubulin (red fluorescence, which together with β-tubulin that create the α-/β-tubulin dimers are the building blocks of MTs) in the seminiferous epithelium in control testes noted herein were similar to findings shown in Fig. , wherein Dvl3 appeared as track-like structures colocalized with MTs in stage V–VI tubules, associated with elongating spermatids. However, the expression of Dvl3 across the seminiferous epithelium was considerably diminished following Dvl1/2/3 knockdown by RNAi in the testis. The disruptive changes in the organization of MTs across the seminiferous epithelium in tubules (such as stage V–VI tubules) were remarkable in magnified images shown in the lower panel following a knockdown of Dvl1/2/3 as illustrated by Dvl3 immunofluorescence staining. For instance, the track-like structures conferred by MTs that laid perpendicular to the basement membrane were extensively truncated, no longer extended across the entire seminiferous epithelium. Scale bar, 350 µm and 80 µm, in the top and bottom panel, respectively, which apply to other images in the corresponding panel. b In control testes, EB1 (red fluorescence, a + TIP protein that is known to stabilize MTs ) or MARK4 (red fluorescence, a Ser/Thr protein kinase known to phosphorylate MAPs (e.g., MAP1a), causing their dissociation from MTs, thereby leading to MT catastrophe , ) and α-tubulin (green fluorescence) were predominantly expressed and localized (and also colocalized) at the concave (ventral) side of elongated spermatid heads, and also the track-like structures across the epithelium as noted in the corresponding upper and lower panels in stage VII tubules. However, following knockdown of Dvl1/2/3 by RNAi, EB1, and MARK4 no longer expressed prominently at the apical ES (and fewer elongated spermatids were detected in the seminiferous epithelium) and across the seminiferous epithelium, but considerably down-regulated. Scale bar, 80 µm, which applies to corresponding micrographs in the same panel. See Figure for negative control IF images, illustrating the staining shown herein was specific to the corresponding marker protein

Article Snippet: Dvl3 (AB_2230636) , Rabbit , Cell Signaling Technology , 3218 , 1:1000 , .

Techniques: Fluorescence, Control, Expressing, Knockdown, Immunofluorescence, Staining, Membrane, Negative Control, Marker