Journal: JCI insight

Article Title: A Syx-RhoA-Dia1 signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in glioblastoma.

doi: 10.1172/jci.insight.157491

Figure Lengend Snippet: Figure 4. Syx regulates the expression of cyclins and cyclin-dependent kinase inhibitors. (A–D) Immunoblotting (A and C) and qPCR (B and D) analyses of the expression of p21 (CDKN1A), p27 (CDKN1B), Cyclin D1, Cyclin E2 (CCNE2), Cyclin A2 (CCNA2), and Cyclin B1 (CCNB1) in U251 (A and B) and LN229 (C and D) cells expressing indicated shRNAs, grown at subconfluency. For Western blots (A and C), different biological samples are separated by dashed lines. Sam- ples from each blot set were run in parallel, except Cyclin A2 and the corresponding GAPDH from the same gel blot. The same samples for the Cyclin D1 blot with equal loading amounts as other Cyclins blots were run at different times (C, indicated by larger white space). See Figure 2B for the GAPDH loading control for p21 and p27 blots in C. Bar graphs (B and D) represent mean ± SEM of 3–4 biological replicates of relative mRNA expression of indicated genes normalized by GAPDH or β-actin. One-way ANOVA with Dunnett’s multiple-comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies used for immunoblotting and immunofluorescence included: Syx (Abnova, H00057449-M01, clone 5A9), GAPDH (Cell Signaling Technology, 2118), α-tubulin (Sigma, T5168), pS10-HH3 (Cell Signaling Technology, 9701), HH3 (Cell Signaling Technology, 4499), Cdc20 (Cell Signaling Technology, 4823), Survivin (Cell Signaling Technology, 2808), PARP (Cell Signaling Technology, 9542), cCas3 (Cell Signaling Technology, 9661), pS139-H2AX (γH2AX, Cell Signaling Technology, 2577), H2AX (Cell Signaling Technology, 7631), p27 (Santa Cruz Biotechnology, sc528), p21 (Cell Signaling Technology, 2946), Cyclin D1 (Abcam, ab134175; or Cell Signaling Technology, 2978), Cyclin E2 (Cell Signaling Technology, 4132), Cyclin A2 (Abcam, ab38), Cyclin B1 (Cell Signaling Technology, 4138), Dia1 (BD, 610848), YAP1/TAZ (Santa Cruz Biotechnology, sc-101199), YAP1 (Cell Signaling Technology, 14074), TAZ (BD, 560235), pS127YAP/pS89TAZ (Cell Signaling Technology, 4911), pS397YAP1 (corresponding to pS381 of YAP2, Cell Signaling Technology, 13619), BiP/GRP78 (Cell Signaling Technology, 3177), IRE1α (Cell Signaling Technology, 3294), CHOP (Cell Signaling Technology, 2895), pT183/Y185-JNK (Cell Signaling Technology, 9251S), JNK (Santa Cruz Biotechnology, sc-474), LC3-I/II (Acris, AM20212PU-N), and p62/SQSTM1 (Cell Signaling Technology, 5114S).

Techniques: Expressing, Western Blot, Control, Comparison

Journal: JCI insight

Article Title: A Syx-RhoA-Dia1 signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in glioblastoma.

doi: 10.1172/jci.insight.157491

Figure Lengend Snippet: Figure 5. Dia1 is a downstream effector of Syx for the regulation of cell growth and gene expression. (A–C) Immunoblot analysis of Dia1 and GAPDH in lysates of U251 (A), LN229 (B), or GBM12 (C) cells transduced with Dia1 shRNAs (top). Cell viability over the indicated time for each cell population was measured by the MTT assay (bottom). Shown are representative graphs (mean ± SD) of 3 biological repeats with 3 technical replicates each. (D and E) Immunoblot (D) and qPCR (E) analyses of phosphorylated histone 3 at Ser-10 (pHH3), total histone 3 (HH3), Cdc20, Survivin (BIRC5), p21 (CDKN1A), p27 (CDKN1B), Cyclin E2 (CCNE2), Cyclin A2 (CCNA2), and Cyclin B1 (CCNB1) in U251 cells expressing indicated shRNAs. Different biological samples are sepa- rated by dashed lines. Dia1 and Survivin blots were run at different times (indicated by larger white space). All other blots were run in parallel. Graph (E) represents the mean ± SEM of 3–5 biological replicates of relative mRNA expression of indicated genes normalized by GAPDH. (F) Representative images of immunofluorescence staining (left) show γH2AX (red) foci in the nucleus (DAPI, blue) of U251 cells expressing indicated shRNAs. Scale bar: 5 μm. Bar graph (right) depicts the average ± SEM number of γH2AX foci per cell in U251 cells transduced with indicated shRNAs (n > 60 cells per group). One-way ANOVA with Dunnett’s multiple-comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies used for immunoblotting and immunofluorescence included: Syx (Abnova, H00057449-M01, clone 5A9), GAPDH (Cell Signaling Technology, 2118), α-tubulin (Sigma, T5168), pS10-HH3 (Cell Signaling Technology, 9701), HH3 (Cell Signaling Technology, 4499), Cdc20 (Cell Signaling Technology, 4823), Survivin (Cell Signaling Technology, 2808), PARP (Cell Signaling Technology, 9542), cCas3 (Cell Signaling Technology, 9661), pS139-H2AX (γH2AX, Cell Signaling Technology, 2577), H2AX (Cell Signaling Technology, 7631), p27 (Santa Cruz Biotechnology, sc528), p21 (Cell Signaling Technology, 2946), Cyclin D1 (Abcam, ab134175; or Cell Signaling Technology, 2978), Cyclin E2 (Cell Signaling Technology, 4132), Cyclin A2 (Abcam, ab38), Cyclin B1 (Cell Signaling Technology, 4138), Dia1 (BD, 610848), YAP1/TAZ (Santa Cruz Biotechnology, sc-101199), YAP1 (Cell Signaling Technology, 14074), TAZ (BD, 560235), pS127YAP/pS89TAZ (Cell Signaling Technology, 4911), pS397YAP1 (corresponding to pS381 of YAP2, Cell Signaling Technology, 13619), BiP/GRP78 (Cell Signaling Technology, 3177), IRE1α (Cell Signaling Technology, 3294), CHOP (Cell Signaling Technology, 2895), pT183/Y185-JNK (Cell Signaling Technology, 9251S), JNK (Santa Cruz Biotechnology, sc-474), LC3-I/II (Acris, AM20212PU-N), and p62/SQSTM1 (Cell Signaling Technology, 5114S).

Techniques: Gene Expression, Western Blot, Transduction, MTT Assay, Expressing, Immunofluorescence, Staining, Comparison

Journal: Physiological reports

Article Title: Transmembrane protein 14A protects glomerular filtration barrier integrity.

doi: 10.14814/phy2.15847

Figure Lengend Snippet: FIGURE 1 Glomerular TMEM14A expression was diminished before onset of proteinuria. (a) Urinary albumin excretion is significantly higher in Dahl rats compared to SHR from 6 weeks of age. Y-axis shows urinary albumin excretion in mg/24 h, and the X-axis shows animal age in weeks. (b) Glomerular TMEM14A mRNA expression in Dahl rats is significantly lower than in SHR controls at all time points. The Y-axis shows mRNA expression as relative expression compared to Hprt1, and the X-axis shows animal age in weeks. No correlation was observed between TMEM14A expression and urinary albumin excretion in both SHR (r = − 0.80, CI −0.98 to 0.27) and Dahl (r = −0.60, CI −0.97 to 0.60) using Pearson's correlation coefficient. (c) Glomerular staining for Tmem14a protein is lower in Dahl rats than in SHR after 2 weeks. This difference is significant at Weeks 4 and 8 of age. The columns represent mean semiquantitative score. Dahl rats develop proteinuria at 6 weeks of age. Thus, both mRNA and protein expression levels drop before onset of proteinuria. ANOVA with Tukey's post hoc analysis was used.(d–m) Representative images of glomeruli of spontaneously proteinuric Dahl (d–h) and SHR (i–m) rats at respectively 2 (d and i), 4 (e and j), 6 (f and k), 8 (g and l), and 10 (h and m) weeks of age. (a–c) Horizontal lines indicate mean with SD. Scale bar = 50 μm *p < 0.001.

Article Snippet: A commercially available polyclonal goat anti-TMEM14A antibody (Santa Cruz Biotech, sc-248,899, Dallas, TX) was used for immunohistochemical staining of TMEM14A on human and rat material.

Techniques: Expressing, Staining

Journal: Physiological reports

Article Title: Transmembrane protein 14A protects glomerular filtration barrier integrity.

doi: 10.14814/phy2.15847

Figure Lengend Snippet: FIGURE 2 Knocking down TMEM14A mRNA translation causes proteinuria. Knocking down mRNA translation of the zebrafish homologue of TMEM14A through morpholino injection results in proteinuria. (a–f) Representative immunofluorescence images of transversal sections of zebrafish proximal tubule cells after injection of a mixture of red labeled 3 kDa dextran tracer (a, c, e) and green labeled 70 kDa dextran tracer (b, d, f) in controls (a,b), TMEM14A knockdowns (C and d), and PAN injected positive controls (e,f). Dextran tracers that passed the GFB are reabsorbed by proximal tubule epithelial cells in endosomes. Thus, reabsorbed dextran tracer appears as fluorescent droplets. The number of proximal tubule reabsorption droplets was counted in a blinded manner in sections as those shown here. The arrowheads in B point out examples of counted droplets. The circled areas show high fluorescence due to dextran present in the peritubular capillaries. These areas are not counted as reabsorption droplets. The sharpness of the images in panels A through F has been enhanced by overlaying them with a digital high pass filter. (g and h) Uptake of the red 3 kDa marker (g) was used to assess tubular reabsorption function, which was intact in both TMEM14A knockdown animals and controls. In the knockdown model, significantly more 70 kDa droplets (h) have passed the GFB and were subsequently reabsorbed. Puromycin aminonucleoside (PAN) injected zebrafish were used as positive controls. Students t-test was used.(g and h) Horizontal lines indicate mean with SD. Scale bar = 20 μm.* = p < 0.05.

Article Snippet: A commercially available polyclonal goat anti-TMEM14A antibody (Santa Cruz Biotech, sc-248,899, Dallas, TX) was used for immunohistochemical staining of TMEM14A on human and rat material.

Techniques: Injection, Immunofluorescence, Labeling, Fluorescence, Marker, Knockdown

Journal: Physiological reports

Article Title: Transmembrane protein 14A protects glomerular filtration barrier integrity.

doi: 10.14814/phy2.15847

Figure Lengend Snippet: FIGURE 3 TMEM14A was primarily expressed by podocytes. In vitro experiments of relative TMEM14A mRNA expression show expression relative to GAPDH expression when comparing mRNA extracts from whole kidney, purified glomeruli, human umbilical vein endothelial cells (Huvec), human embryonic kidney (HEK), and finally podocytes. Expression in podocytes, HEK and Huvec was significantly higher than in whole kidney (p < 0.001 for all groups) and then purified glomeruli (p < 0.001 for podocytes and HEK, p < 0.05 for Huvec). Podocyte expression was highest of all cells and significantly so compared to Huvec (p < 0.05). Mean and SD shown. ANOVA with Tukey's post hoc analysis was used.

Article Snippet: A commercially available polyclonal goat anti-TMEM14A antibody (Santa Cruz Biotech, sc-248,899, Dallas, TX) was used for immunohistochemical staining of TMEM14A on human and rat material.

Techniques: In Vitro, Expressing, Purification

Journal: Physiological reports

Article Title: Transmembrane protein 14A protects glomerular filtration barrier integrity.

doi: 10.14814/phy2.15847

Figure Lengend Snippet: FIGURE 4 Glomerular TMEM14A expression was increased in human proteinuric renal diseases. (a) Glomerular TMEM14A protein expression was examined in human kidney biopsies from patients with diabetic nephropathy (DN), lupus nephritis (LN), IgA nephropathy (IgAN), minimal change disease (MCD), and healthy controls. Compared to controls, TMEM14A protein expression is significantly more extensive in IgAN, LN, and MCD, but not in DN. (b–f) Representative images of glomeruli stained for TMEM14A in healthy controls (b), diabetic nephropathy (c), lupus nephritis (d), IgA nephropathy (e), and minimal change disease (f). Slides were stained with goat anti-TMEM14A antibody and immunoreactivity was assessed by diaminobenzidine. This results in a brown color which then indicates TMEM14A localization. Counterstaining with hematoxylin results in blue-purple coloring of cell nuclei. Boxes in a show the range of values between the lower and upper quartile, the whiskers show 5th–95th percentile, triangles show values lying outside the 5th–95th percentile, the line in the box shows the median, and “+” indicates the mean. The scale bar in B applies to B through f and indicates 50 μm. * = p < 0.001. ANOVA with Tukey's post hoc analysis was used.

Article Snippet: A commercially available polyclonal goat anti-TMEM14A antibody (Santa Cruz Biotech, sc-248,899, Dallas, TX) was used for immunohistochemical staining of TMEM14A on human and rat material.

Techniques: Expressing, Staining

Journal: Cancer medicine

Article Title: The expression and role of SUZ12 in lung adenocarcinoma.

doi: 10.1002/cam4.70190

Figure Lengend Snippet: FIGURE 7 The effect of SUZ12 on CDKs and cyclins expression was tested by qRT-PCR and western blotting. sh-SUZ12 decreased CDK3 mRNA (A), CDK2/3 (C), and cyclin D1 (F) protein expression, while increased cyclin E1 protein expression (F), without significantly effected the cyclins mRNA (E). oe-SUZ12 increased CDK3/6 mRNA (B) and CDK3 protein expression (D). *p < 0.05, **p < 0.001.

Article Snippet: The list of primary antibodies: SUZ12 (1 μg/mL, Abcam Cambridge, cat no: ab12073), CDK2 (1:1000; Proteintech, USA, cat. no. 10122- 1- AP), CDK3 (1:2000; Proteintech, USA, cat. no. 55103- 1- AP), CDK6 (1:2000; Proteintech, USA, cat. no. 14052- 1- AP), cyclin D1 (1:5000; Proteintech, USA, cat. no. 26939- 1- AP), cyclin E1 (1:1000; Proteintech, USA, cat. no. 11554- 1- AP), p18 (1:1000, BOSTER China, cat. no. M03299- 1), p19 (1:1000, BOSTER China, cat. no. MA1075), p53 (1:5000; Proteintech, USA, cat. no. 60283- 2- Ig), p- p53 (1:2000; Proteintech, USA, cat. no. 28961- 1- AP), p57 (1:1000, BOSTER China, cat. no. BM4129), Rb (1:1000, BOSTER China, cat. no. BM4500), pRb (1:1000, BOSTER China, cat. no. BM4338), Bcl- 2 (1:1000; Proteintech, USA, cat. no. 26593- 1- AP), Bax (1:2000; Proteintech, USA, cat. no. 50599- 2- lg), Ecadherin (1:5000; Proteintech, USA, cat. no. 20874- 1- AP), N- cadherin (1:3000; Proteintech, USA, cat. no. 22018- 1- AP), vimentin (1:4000; Proteintech, USA, cat. no. 10366- 1- AP), MMP1 (1:1000, BOSTER China, cat. no. A00733- 1), MMP2 (1:500, BOSTER China, cat. no. BM4075), MMP9 (1:1000, BOSTER China, cat. no. PB0709), MMP14 (1:1000, BOSTER China, cat. no. BM4119), TIMP1 (1:1000, Bioss China, cat. no. bs0415R), TIMP2 (1:1000, Bioss China, cat. no. bs- 10395R), TIMP3 (1:1000; Proteintech, USA, cat. no. 10858- 1- AP), ITGB1 (1:1000, BOSTER China, cat. no. BM4308), ITGB3 (1:1000, BOSTER China, cat. no. BA1670), ITGB5 (1:1000, BOSTER China, cat. no. A04201- 1), nm23 (1:1000, BOSTER China, cat. no. BA3787), PD- L1 (1:3000; Proteintech, USA, cat. no. 66248- 1- Ig), and βactin (1:5000; Proteintech, USA, cat. no. 66009- 1- Ig).

Techniques: Expressing, Quantitative RT-PCR, Western Blot