A randomized controlled trial to evaluate the role of postoperative strict prone positioning for one day versus three days in anatomical closure of idiopathic full thickness macular hole: a preliminary study

<p>A randomized controlled trial to evaluate the role of postoperative strict prone positioning for one day versus three days in anatomical closure of idiopathic full thickness macular hole: a preliminary study. Rajpal Vohra, Rajive Kumar, Koushik Tripathy, Pradeep Venkatesh, S. N. Dwivedi, Rajvardhan Azad<br>The 2nd Asia-Pacific Glaucoma Congress held in conjunction with the 10th International Symposium of Ophthalmology – Hong Kong (APGC-ISOHK 2014 Hong Kong)<br>2014-09 | conference-paper</p> <p> </p

Demographic and clinicopathological characteristics of bladder TCC patients.

<p>Demographic and clinicopathological characteristics of bladder TCC patients.</p

Effect of SPAG9 expression on migration and invasion ability of UM-UC-3 cells.

<p>Knockdown of SPAG9 expression inhibited (<b>A</b>) Migration, (<b>B</b>) Invasion and (<b>C</b>) Wound healing assay are depicted in only cells scrambled siRNA and SPAG9 siRNA transfected cells.</p

Humoral response against SPAG9 in bladder TCC patients.

<p>(<b>A</b>) Circulating anti-SPAG9 antibodies in sera from bladder TCC patients. Line X denotes cutoff value taken at 490 nm (mean+2 SD) from healthy donors (n = 50) for positivity above or negativity below the line. Marker- molecular size marker, CBR- Coomassie brilliant blue stained recombinant SPAG9 protein, Rat antibody- antibodies raised in rat against recombinant SPAG9 protein: positive control (<b>B</b>) Western blotting confirmed the circulating anti-SPAG9 antibodies in five representative patient’s serum. No reactivity detected in healthy donors. Neutralization experiments resulted in complete loss of immunoreactivity [patient’s sera preincubated with recombinant SPAG9 protein (15 µg/ml)].</p

<i>SPAG9</i> Silencing and its effect on cellular proliferation and cell cycle.

<p>(<b>A</b>) Western blot depicts ablation of SPAG9 protein in UM-UC-3 cells transfected with SPAG9 siRNA. β-Actin was used as a control. (<b>B</b>) Knockdown of <i>SPAG9</i> by siRNA inhibited cell growth in UM-UC-3 cells <i>in vitro</i> determined by MTT assay. (<b>C</b>) Silencing of <i>SPAG9</i> expression arrested cell cycle progression in G₀–G₁ phase in SPAG9 siRNA transfected cells as compared with only cells or scrambled siRNA transfected cells. (<b>D</b>) Histogram analysis confirmed that in SPAG9 siRNA transfected cells accumulated in G₀–G₁ phase. (<b>E</b>) Western blot analysis of cell cycle molecules in SPAG9 ablated UM-UC-3 cells depicted up-regulation of CDK inhibitors including p21 and p16. The up-regulation of p21 and p16 was preceded by down-regulation of cyclin E, cyclin D, cyclin B, CDK4 and CDK1. β-Actin was used as an internal loading control. [M1- G₀–G₁ phase_; M2- S phase and M3- G₂-M phase].</p

Cell type <i>SPAG9</i> gene expression in bladder TCC patient’s tissues.

<p>Representative H&E stained tissue specimens showing cytostructure of Ta, Tis, T1, T2 and T3 stages (left panel). <i>In situ</i> RNA hybridization showing SPAG9 expression in matched serial tissue section from same specimens of Ta, Tis, T1, T2 and T3 stages showed a strong chocolate brown color with digoxigenin-labeled <i>SPAG9</i> antisense riboprobes (middle panel). However, <i>SPAG9</i> sense riboprobe failed to show the signal (right panel). Original magnification, ×400; objective, 40×.</p

Validation of SPAG9 protein expression in bladder TCC patient’s tissue.

<p>(<b>A</b>) Representative H&E stained tissue specimens of Ta, Tis, T1, T2 and T3 stages (I panel). Immunohistochemical analysis of SPAG9 protein expression in matched serial tissue section from the same specimens of Ta, Tis, T1, T2 and T3 stages probed with anti-SPAG9 antibodies showed SPAG9 cytoplasmic localization (II panel). Control IgG showed no reactivity (III panel). Panel IV and V representing different tissue specimens sections of Ta, Tis, T1, T2 and T3 stages probed with anti-SPAG9 antibodies showed SPAG9 protein expression (<b>B</b>) Matched ANCT specimens probed with anti-SPAG9 antibodies showed no reactivity. (<b>C</b>) Representative photomicrographs showing immunoreactivity of anti-SPAG9 antibody before and after neutralization. Neutralization of anti-SPAG9 antibody raised in rats revealed loss of immunoreactivity in IHC which confirms the specificity of anti-SPAG9 antibody. Original magnification, ×400; objective, 40×.</p

SPAG9 expression in bladder cancer cell lines.

<p>(<b>A</b>) <i>SPAG9</i> mRNA expression assessed by RT-PCR revealed the presence of <i>SPAG9</i> transcript in all bladder cancer cells. However, NHU cells did not reveal <i>SPAG9</i> transcript. <i>β-Actin</i> was used as an internal control. (<b>B</b>) Western blot analysis of SPAG9 protein expression. (<b>C</b>) Indirect immunofluorescence revealed SPAG9 cytoplasmic localization in all bladder cancer cells. Nuclear staining was done by DAPI. (<b>D</b>) UM-UC-3 cancer cells were probed for SPAG9 and various markers for cell organelles. Co-localization of SPAG9 was examined by indirect immunofluorescence assay which revealed SPAG9 co-localization with ER and Golgi marker. Nuclear envelop and mitochondria did not show co-localization with SPAG9. Immunofluorescence staining was detected by a laser-scanning confocal microscope. The images are merged for co-localization of SPAG9 (green) and marker co-staining (red). Original magnification, ×630; objective, 63×). (<b>E</b>) Flow cytometric analysis demonstrated surface localization of SPAG9 protein in fixed cancer cells (green histogram) compared with cells probed with control IgG (black histogram) or stained with secondary antibody only (red histogram). Results from 1 of 3 representative experiments are shown.</p

Additional file 6: Figure S5. of Heat shock protein 70-2 (HSP70-2) overexpression in breast cancer

Quantitative PCR analysis of various genes involved in different signaling cascades in breast cancer tumor xenograft. (PPTX 93 kb