Fig 1: DIAPH3 regulates ciliogenesis and cilia length.A–C, impairment of ciliation and cilia length by DIAPH2 depletion cannot be rescued by other isoforms. hTERT-RPE1 cells were transfected with siCtr or siDIAPH2#1 for 48 h, and cells were rescued using wild-type constructs of DIAPH1-myc, DIAPH2-myc, and DIAPH3-myc. A, western blots of samples blotted for human DIAPH2, myc, and GAPDH. Quantifications of (B) ciliation and (C) cilia length are shown. Error bars in B represent ± SD of three independent experiments; n = 50 or more each. Graph of cilia length measurement (C) is displayed in box-and-whisker plot, where the upper and lower quartiles are indicated as the ends of the box, the median is marked by a horizontal line inside the box, mean is marked by a cross sign inside the box, and whiskers are two lines outside the box that extend to the highest and lowest observations, while dots outside this range represent outliers. Two-tailed t-test analysis was done to compare control siRNA to all samples or siDIAPH1 to rescue samples, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001. D–F, reduction of ciliation and cilia length by DIAPH3 depletion cannot be rescued by other isoforms. hTERT-RPE1 cells were transfected with siCtr or siDIAPH3#1, and cells were rescued using wild-type constructs of DIAPH1-myc, DIAPH2-myc, or DIAPH3-myc. D, western blots of rescued cells blotted for human DIAPH3, myc, and GAPDH. E and F, quantification of ciliation and cilia length in hTERT-RPE1 cells treated with siRNA for 48 h, respectively. Error bars in E represent ±SD of three independent experiments; n = 50 or more each. Two-tailed t-test analysis was done to compare control siRNA to all samples or siDIAPH1 to rescue samples, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001. Graph of cilia length measurement is displayed in a box-and-whisker plot as in C. G and H, DIAPH2 and DIAPH3 localize at the base of cilia. hTERT-RPE1 cells were grown to confluence and serum starved for 24 h. Cells were stained with Actub (axonemal marker) and centrin (CNT) for cilia basal marker. Cells were costained for (G) DIAPH2 and CNT or DIAPH2 and Actub. H, cells were costained for DIAPH3 and CNT or DIAPH3 and Actub. Scale bars in G and H are 10 μm in the left panels and 1 μm in the right-side panels.
Fig 2: Cytoplasmic Rb colocalizes with mDia1 in atrophied skeletal muscle.(A–C′) Normal and atrophied tibialis anterior muscles were surgically excised from cancer patients. Cross sections (A) and longitudinal sections (B–C′) were stained. (A) Hematoxylin and eosin-stained cryosections. Scale bar, 400 μm. (B–C′) Localization of Rb and mDia1 in normal and atrophied tibialis anterior muscles. Confocal images of cryosections stained with anti-mDia1 and anti-α-actinin antibodies (B) or anti-Rb and anti-mDia1 antibodies (C, magnified in C′). Scale bar, 10 μm.DOI: http://dx.doi.org/10.7554/eLife.01228.013
Fig 3: mDia1 is critical for sarcomeric organization.(A and B) HSMMs were infected with adenoviruses expressing control non-target shRNA or shRNA against mDia1 at a MOI of 5 pfu/nucleus for 4 days. (A) Depletion of mDia1 protein was verified by immunoblotting. (B) Confocal images for F-actin and α-actinin. Scale bar, 10 μm.DOI: http://dx.doi.org/10.7554/eLife.01228.015
Fig 4: Functional DEGs analysis. A The diagram of expression data for DEGs with known biological functions and their participation in signaling pathways from KEGG database. The analysis of signaling pathways demonstrated four major categories, such as: environmental information processing, metabolism, human diseases, organismal systems (central pie chart), and 13 subcategories (lateral pie chart). The most enriched biological pathway in lumbar SC from STZ-treated mice was the PI3K-Akt signaling pathway (mmu04151). The cloud indicates genes involved in PI3K-Akt signaling pathway. B Functional analyses of DEGs by REVIGO gene ontology (GO) enrichment analysis [50]. The most representative GO terms for biological process (BP) were utilized for REVIGO functional annotation clustering that relies on semantic similarity measures. Bubble color corresponds the P-value obtained from GO enrichment analysis, whereas the bubble size corresponds to the GO term prevalence in the UniProt-GOA database for Mus musculus. Arabic numeral indicates the number of DEGs in SC. See also Supplementary Table 2 for details on the GO-BP terms. C Gene interaction network constructed with a use of GeneMANIA for selected set of genes, i.e. HOXB13, HAO1, NET1, RHOJ, TXNIP, CTSE, AGER, DIAPH1, ACTB (limited to Mus musculus). The selected genes are in a striped circle. The colors of the line suggest the type of interaction (see legend). In silico analysis indicated that ACTB gene has the most direct interactions (double green circle). Green circles indicate genes that have a direct interaction with ACTB, i.e. NET1, TMSB4X, SUSD6, EEF1A1, DIAPH1, RAC1, ACTG1, RHOJ, PTPRO genes. See also Supplementary Table 6 for details on the interaction network. D Venn diagram [29] representing DEGs in SC of T1D, SN of T1D and T2D [27, 28]. The analysis showed that the expression of a one, single gene, i.e. CTSE was altered for all tissue transcriptome profiles
Fig 5: TNF-α-induced sarcomeric disorganization is prevented by constitutively active mDia1.(A–F) HSMMs were infected with adenoviruses expressing GFP or GFP-mDia1 ΔGBD/ΔDAD at a MOI of 1 pfu/nucleus and then treated with TNF-α for 2 days. (A) The expression was analyzed by immunoblotting. (B) Confocal images for α-actinin. Scale bar, 5 μm. (C) TNF-α-induced contractile dysfunction is diminished by constitutively active mDia1. EPS was applied to HSMMs. The percentage of beating cells from a total of 100 HSMMs is shown. Results are presented as mean ± SD from three independent experiments. *p<0.002; **p<0.02, determined by the Student’s t-test. (D and E) Constitutively active mDia1 recovers TNF-α-induced sarcomeric disorganization. Confocal images for α-actinin (D) and merged images of F-actin (green) and α-actinin (red) (E). Scale bar, 10 μm in D and 5 μm in E. (F) Autocorrelation analyses of the α-actinin distribution. Scale bar, 5 μm.DOI: http://dx.doi.org/10.7554/eLife.01228.014
Supplier Page from Abcam for Anti-DIAPH1 antibody