Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

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

Article Snippet: N-cadherin (AB_647794) , Rabbit , Santa Cruz Biotechnology , sc-7939 , 1:200; -.

Techniques:

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: A knockdown of Dync1h1 by RNAi or an inactivation of dynein 1 by specific inhibitor ciliobrevin D in Sertoli cells cultured in vitro perturbs the tight junction (TJ)-permeability barrier function via changes in the distribution of TJ and basal ectoplasmic specialization (ES) proteins at the Sertoli cell-cell interface. A: treatment regimens by RNAi [Dync1h1-specific siRNA duplexes vs. non-targeting negative control siRNA duplexes as a control (Ctrl)] or ciliobrevin D (vs. DMSO serving as a vehicle Ctrl) to knockdown Dync1h1 or inactivate dynein 1 to obtain Sertoli cells for RT-PCR, qPCR, immunoblotting (IB), and immunofluorescence analysis (IF), and the number of independent experiments performed for each corresponding study. B: lysates from Sertoli cell cultures were used for IB following a knockdown of Dync1h1 or treatment with ciliobrevin D vs. the corresponding control cultures with the corresponding specific antibodies listed in Table 1. Collectively, these data (i.e., representative findings of n = 3 independent experiments) have shown that the steady-state protein level of Dync1h1 was considerably downregulated following Dync1h1 knockdown (but not treatment with the dynein 1 inhibitor nor any other functional groups of blood-testis barrier (BTB)-associated proteins examined herein), illustrating the knockdown of Dync1h1 is specific to dynein 1 expression without any apparent off-target effects. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, and vimentin served as protein loading controls. C: steady-state mRNA or protein level of Dync1h1 was downregulated by ~70% when examined by qPCR (left) or IB (right), but only in the RNAi group, and dynein 1 inhibitor ciliobrevin D had no effects on Dync1h1 mRNA or protein expression. Each bar in the histogram is a mean ± SD of n = 3 independent experiments. **P < 0.01 when compared with the corresponding control by Student’s t-test. D: knockdown of Sertoli cell Dync1h1 or treatment of Sertoli cells with ciliobrevin D perturbed the Sertoli cell TJ-permeability barrier function. Each data point is a mean ± SD of quadruplicate bicameral units from a representative experiment. A total of n = 3 independent experiments were performed, which yielded similar results. *P < 0.05; **P < 0.01 when compared with the corresponding control by Student’s t-test. E: IF analysis was used to confirm considerable decline in Dync1h1 expression following RNAi but not treatment of Sertoli cells with ciliobrevin D (see top). Histogram (middle) summarized the relative fluorescence intensity of Dync1h1 in Dync1h1 RNAi group vs. the corresponding control cells, illustrating a ~70% knockdown. Sertoli cell Dync1h1 knockdown also perturbed the distribution of TJ proteins CAR and ZO-1 and basal ES proteins N-cadherin and β-catenin. In both control groups, TJ and basal ES proteins were tightly localized to the Sertoli cell-cell interface (see white brackets in control groups). After Dync1h1 knockdown or dynein 1 inactivation, these proteins were diffusively localized at the cell-cell interface (see yellow brackets in treatment groups). It is likely that these BTB-associated proteins were internalized and moved from the cell cortical zone into the cell cytosol. Co-transfection of siRNA duplexes with siGLO Red Transfection Indicator (red fluorescence; Dharmacon/GE/Thermo Fisher) used to illustrate successful transfection. Histograms (bottom) provide semiquantitative analysis regarding changes in the relative distribution of fluorescence at the cell-cell interface. Each bar is a mean ± SD of n = 3 independent experiments. **P < 0.01 when compared with corresponding control by Student’s t-test. Scale bar, 20 µm, which applies to all other micrographs. TER, transepithelial electrical resistance.

Article Snippet: N-cadherin (AB_647794) , Rabbit , Santa Cruz Biotechnology , sc-7939 , 1:200; -.

Techniques: Knockdown, Cell Culture, In Vitro, Permeability, Negative Control, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Functional Assay, Expressing, Fluorescence, Cotransfection, Transfection

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: A knockdown of Dync1h1 or an inactivation of dynein by inhibitor ciliobrevin D in the testis in vivo perturbs distribution of basal ectoplasmic specialization (ES)- and tight junction (TJ)-associated proteins at the blood-testis barrier (BTB). A: in control testes, basal ES proteins N-cadherin and β-catenin and TJ proteins occludin and ZO-1 were tightly associated with the BTB (see white bracket) located near the basement membrane (annotated by a dashed white line) as noted in selected stage V tubules. However, following Dync1h1 knockdown or dynein 1 inactivation, these proteins were diffusely localized at the BTB by extending considerably away from BTB site (see yellow brackets), well beyond the basement membrane (annotated by a dashed white line). Co-transfection of rhodamine-siGLO indicator (red fluorescence) illustrated successful transfection. Histograms (bottom) summarized results of fluorescence analysis (top) regarding changes in the relative distribution of basal ES or TJ proteins in treatment vs. corresponding control groups. Each bar in the histogram is a mean ± SD of n = 4 rats, and 50 randomly selected cross sections of tubules in each testis were scored. **P < 0.01 by Student’s t-test. Scale bar, 40 µm. B: results of an in vivo BTB integrity assay in which the BTB found in control testes from the two control groups were similar to normal rat testes (-ve control group) and were capable of blocking the diffusion of membrane impermeable EZ-LinkSulfo-NHS-LC-Biotin across the immunological barrier. However, as noted in the positive control group (+ve) in which rats were treated with CdCl2 (3 mg/kg body weight, ip) for 5 days, known to disrupt the BTB function (94), biotin freely diffused across the immunological barrier to reach into the tubule lumen, which was visualized by Alexa Fluor 488-streptavidin (green fluorescence) with cell nuclei stained by DAPI. It was noted that the BTB became “leaky” in both Dync1h1 knockdown and dynein 1 inactivated testes. The histogram (bottom) summarized results of the BTB integrity assay in which each bar represents a mean ± SD of n = 3 rats that illustrated the ratio of the distance traveled by the biotin vs. the radius of a seminiferous tubule. For oblique sectioned tubules, the radius of the tubule was obtained by averaging the longest and shortest distance from the tubule lumen. **P < 0.01, by Student’s t-test. Scale bar, 350 µm and 80 µm (magnified micrograph), which apply to corresponding micrographs.

Article Snippet: N-cadherin (AB_647794) , Rabbit , Santa Cruz Biotechnology , sc-7939 , 1:200; -.

Techniques: Knockdown, In Vivo, Control, Membrane, Cotransfection, Fluorescence, Transfection, Integrity Assay, Blocking Assay, Diffusion-based Assay, Positive Control, Staining