Journal: EMBO Reports

Article Title: Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

doi: 10.15252/embr.202050145

Figure Lengend Snippet: A NIH3T3 fibroblasts co‐expressing DsRed‐CA7 and EGFP‐CA2 ( n = 4 independent replicates). B–E Co‐localization of EGFP and the two CA isoforms with filamentous actin studied in fibroblasts expressing (B) EGFP, (C) EGFP‐CA2, or (D, E) EGFP‐CA7 and stained with phalloidin‐594 to visualize F‐actin ( n = 4, 10, and 8 independent replicates, respectively). A magnification of the area marked with the yellow rectangle in (C) and (D) shows the localization of EGFP‐CA2 and EGFP‐CA7 compared to phalloidin‐594. (E) EGFP‐CA7 caused a prominent overexpression phenotype with thick and curvy cytosolic actin bundles (arrow) and plasmalemmal protrusions (arrowhead). F–H The normalized fluorescence emission intensity profiles for F‐actin (red line) and (F) EGFP, (G) EGFP‐CA2, or (H) EGFP‐CA7 (black line). Data information: For the plots, pixel intensities were measured through the cross‐section of the cell indicated by the yellow line in panels (B–D). Scale bar in (A–E) 20 µm.

Article Snippet: Constructs containing full‐length CA2 and CA7 (human isoform 1) coding sequences were obtained from ImaGenes (human CA2 and human CA7 including start and stop codon; OCAAo5051H1054 and OCAAo5051E0588, respectively) and GeneCopoeia (human CA2 without stop codon and mouse CA7 including start and stop).

Techniques: Expressing, Staining, Over Expression, Fluorescence

Journal: EMBO Reports

Article Title: Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

doi: 10.15252/embr.202050145

Figure Lengend Snippet: A–C NIH3T3 fibroblasts expressing EGFP‐CA7‐mutant1 (A), EGFP‐CA7‐mutant2 (B), and EGFP‐CA7‐mutant3 (C). F‐actin is visualized with Phalloidin‐594. In the right‐most panels of (A–C) are the normalized fluorescence intensity profiles of the mutated CA7 EGFP signal (black) and actin (red), and the yellow line in left‐most panels indicates the cross‐section from which the pixel intensities were measured. Scale bars 20 µm. D Analysis of the mutated CA7 and F‐actin co‐localization in cultured fibroblasts. Scatterplots of fluorescent intensities per pixel (EGFP vs. Phalloidin‐594) along a cross‐section through a representative cell. Pearson’s correlation coefficient ( r ) for the analyzed cells is given in each panel. E Pearson’s correlation coefficient values calculated for the depicted constructs and compared to CA7. F‐actin had a strong positive correlation coefficient with EGFP‐CA7 ( r = 0.91 ± 0.02, n = 26 cells). Neither EGFP alone ( r = 0.01 ± 0.03, n = 56) nor EGFP‐CA2 ( r = 0.09 ± 0.03, n = 53) co‐localized with F‐actin ( P < 0.0001 for both constructs, when compared to CA7). From the five mutated CA7 constructs, EGFP‐CA7‐mutant1 ( r = 0.51 ± 0.04, P < 0.0001, n = 25) and EGFP‐CA7‐mutant3 ( r = 0.72 ± 0.03, P = 0.05, n = 21) co‐localized less with F‐actin actin when compared to CA7. The co‐localization of the other four mutated CA7 constructs, EGFP‐CA7‐mutant2 ( r = 0.92 ± 0.02, P = 0.99, n = 18), EGFP‐CA7‐R223E ( r = 0.86 ± 0.02, P = 0.99, n = 24), and EGFP‐CA7‐H96/98C ( r = 0.79 ± 0.04, P = 0.14, n = 18) did not differ significantly from that of CA7. Data are shown as mean ± SEM. Data did not pass Shapiro–Wilk test for normality, statistical comparison against CA7 was done with Kruskal–Wallis test corrected for multiple comparisons. Source data are available online for this figure.

Article Snippet: Constructs containing full‐length CA2 and CA7 (human isoform 1) coding sequences were obtained from ImaGenes (human CA2 and human CA7 including start and stop codon; OCAAo5051H1054 and OCAAo5051E0588, respectively) and GeneCopoeia (human CA2 without stop codon and mouse CA7 including start and stop).

Techniques: Expressing, Fluorescence, Cell Culture, Construct, Comparison

Journal: EMBO Reports

Article Title: Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

doi: 10.15252/embr.202050145

Figure Lengend Snippet: Actin co‐sedimentation assay shows that CA7 binds to F‐actin. The binding is enhanced at more acidic pH (6.5 vs. 7.4). n = 3 independent replicates at each actin concentration, two‐way ANOVA, P < 0.001. Fluorescence time‐lapse images of F‐actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labeled non‐muscle actin was polymerized in the absence (PBS control, upper panel) or presence of mCA7 (lower panel). Numbers in images indicate the time after the onset of the experiment (0, 5, and 23 min). Intensity‐based fire‐coloring (Fiji) was used to visualize intensity changes. Scale bar 10 µm. Quantification of the mean increase in filament length ( n = 10 filaments at each time point) and the mean relative fluorescence intensity values of cross‐sections for individual filaments /bundles ( n = 30–31) in the absence and presence of mCA7 (1.12 µM). The data were analyzed using a general mixed model with time as a within‐unit factor and the presence of CA7 as a between‐unit factor. n = 3 independent repetitions, experiment repeats were included as a covariate and were non‐significant. Kymographs showing different time points in the line of interest (line width 1 µm) from the experiments analyzed in (C). Kymographs were generated with Fiji Multi Kymograph function. Total time is 159 frames = 26.5 min. Scale bar 5 μm (full height is 20 μm). F‐actin bundles can be detected as clear lines in kymographs. Fluorescence time‐lapse images of F‐actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labeled non‐muscle actin was polymerized in the presence of mCA7 (0.11 µM). Numbers above images indicate the time after the onset of the experiment (22–23 min 20 s). Intensity‐based fire‐coloring (Fiji) was used to visualize intensity changes. White arrows highlight the bundling filaments. Scale bar 5 μm. These data are from a representative video ( n = 8 independent repeats). Data information: Data are presented as mean ± SEM in (A) and mean ± SD in (C). Source data are available online for this figure.

Article Snippet: Constructs containing full‐length CA2 and CA7 (human isoform 1) coding sequences were obtained from ImaGenes (human CA2 and human CA7 including start and stop codon; OCAAo5051H1054 and OCAAo5051E0588, respectively) and GeneCopoeia (human CA2 without stop codon and mouse CA7 including start and stop).

Techniques: Sedimentation, Binding Assay, Concentration Assay, Fluorescence, In Vitro, Labeling, Control, Generated

Journal: EMBO Reports

Article Title: Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

doi: 10.15252/embr.202050145

Figure Lengend Snippet: A, B Actin co‐sedimentation assay was carried out at five different concentrations of β/γ‐actin and with 1 µM (A) CA7 or (B) CA2 at two different pH (7.4 or 6.5). After centrifugation, the supernatant (S) and pellet (P) fractions were separated and resolved by SDS‐PAGE. Staining the gels with Coomassie Blue showed that CA7 co‐sedimented in the pellets with actin, whereas CA2 was found only in the supernatant fraction. CA7; three repetitions and CA2; one repetition at each of the four actin concentrations/pH. C Analysis of the CA2 gels confirmed that the isoform does not interact with actin at either pH tested. D In the presence of mCA7, existing filaments assemble thus increasing elongation velocity in a stepwise manner. Fluorescence time‐lapse images of F‐actin bundling in the in vitro bundling assay. A mixture of unlabeled and Rhodamine labeled non‐muscle actin was polymerized in the absence (PBS control, upper row) or in the presence of mCA7 (1.12 µM, lower row). Numbers above images indicate the time after the onset of the experiment (images every 10 s) (First frame 200 s = 3 min 20 s, last frame 500 = 8 min 20 s). The filament/bundle ends that were followed over the experiment are indicated with white arrows. Intensity‐based fire‐coloring (Fiji) was used. Scale bar 5 μm. E Quantification of the filament/bundle length for shown frames. Measured lengths were plotted to Excel x = time in seconds, y = length in μm.

Article Snippet: Constructs containing full‐length CA2 and CA7 (human isoform 1) coding sequences were obtained from ImaGenes (human CA2 and human CA7 including start and stop codon; OCAAo5051H1054 and OCAAo5051E0588, respectively) and GeneCopoeia (human CA2 without stop codon and mouse CA7 including start and stop).

Techniques: Sedimentation, Centrifugation, SDS Page, Staining, Fluorescence, In Vitro, Labeling, Control

Journal: EMBO Reports

Article Title: Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

doi: 10.15252/embr.202050145

Figure Lengend Snippet: A, B NIH3T3 cells transfected with DsRed (A) or DsRed‐CA7 (B) were incubated in growth medium with 5 µM Latrunculin B for 0, 2, 5, 10, or 30 min, or with an equal amount of DMSO for 60 min. Analyses of experiments show that in cells transfected with DsRed‐CA7 F‐actin structures collapse more slowly (0 min: 83% “normal”; 2 min: 80%, P = 0.44; 5 min: 74%, P = 0.39; 10 min: 27%, P = 0.08; 30 min: 16%, P = 0.02; DMSO: 82%, P = 0.99; tested against 0 min with two‐way ANOVA, Dunnett’s multiple comparison test) than in the DsRed‐transfected ones (0 min: 89% “normal”; 2 min: 48%, P = 0.14; 5 min: 31%, P = 0.04; 10 min: 2%, P = 0.001; 30 min: 0.7%, P = 0.001; DMSO: 88%, P = 0.8; tested against 0 min with two‐way ANOVA, Dunnett’s multiple comparison test). For the analysis, cells were categorized to three groups as “normal”, “some shape/F‐actin left”, and “round”. The upper panel shows example images of the cells in all three categories for (A) DsRed‐ or (B) DsRed‐CA7 transfected cells (actin visualized with Phalloidin‐488). Data information: One hundred cells per each time point from each experiment ( n = 3) were counted and categorized. Scale bar 50 µm. Data are presented as mean ± SEM. Source data are available online for this figure.

Article Snippet: Constructs containing full‐length CA2 and CA7 (human isoform 1) coding sequences were obtained from ImaGenes (human CA2 and human CA7 including start and stop codon; OCAAo5051H1054 and OCAAo5051E0588, respectively) and GeneCopoeia (human CA2 without stop codon and mouse CA7 including start and stop).

Techniques: Transfection, Incubation, Comparison

Journal: EMBO Reports

Article Title: Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

doi: 10.15252/embr.202050145

Figure Lengend Snippet: A, B Three‐dimensional representation of (A) CA2 and (B) CA7 structure. The amino acids 101–105 (SLDGQ in CA2 and KKHDV in CA7), 113, 115, and 237–242 (GEPEEL in CA2 and DDERIH in CA7) form a ridge at the protein surface (highlighted in yellow). R223 is located close to the ridge. C Superimposing the CA7 α‐helix‐6 (CA7 in blue, areas where mutations1–3 are located is in yellow, and the putative actin‐interacting CA7 helix in red) on the Twf‐C/G‐actin structure (gray) shows a sterically compatible structure. D Sequence alignment of human CA7 and CA2 protein sequences generated using the Clustal O (1.2.1) multiple sequence alignment. Residues forming a ridge at the CA7 protein surface in the CA7 3D structure are highlighted in the CA7 sequence (bold/underlined). R223 is marked in turquoise and H96 and H98 (mutated to gain a catalytically loss‐of‐function mutant) are highlighted in green. The row below sequence alignment indicates structural features of CA7 based on the UniProt database (B = beta strand, H = helix and, T = turn). E Schematic representation of four mutants with a full (mutant1) or partial (mutant2 and mutant3) replacement of the amino acids encoding the ridge in CA7 by the corresponding CA2 sequence. In the reversed mutant (CA2‐revCA7), the amino acids replaced in mutant1 were introduced to CA2. Data information: Panels (A–C) were prepared using PyMOL (PyMOL The PyMOL Molecular Graphics System, Version 1.4.1 Schrödinger, LLC.).

Article Snippet: Constructs containing full‐length CA2 and CA7 (human isoform 1) coding sequences were obtained from ImaGenes (human CA2 and human CA7 including start and stop codon; OCAAo5051H1054 and OCAAo5051E0588, respectively) and GeneCopoeia (human CA2 without stop codon and mouse CA7 including start and stop).

Techniques: Sequencing, Generated, Mutagenesis

Journal: EMBO Reports

Article Title: Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

doi: 10.15252/embr.202050145

Figure Lengend Snippet: Sequence alignment of the catalytically active (CA1, CA2, CA3, CA5A, CA5B, CA7, and CA13) and catalytically inactive (CA8 and CA10) human cytosolic CA protein sequences generated using the Clustal O (1.2.1) multiple sequence alignment. The amino acids that were characterized as part of a putative actin‐binding site of CA7 (highlighted in red) are not conserved in the other cytosolic CA isoforms (highlighted in gray). An asterisk below the aligned sequences indicates fully conserved residues, a colon indicates residues with strongly similar properties (scoring > 0.5 in the Gonnet PAM 250 matrix) and a period indicates residues with weakly similar properties (scoring ≤ 0.5 in the Gonnet PAM 250 matrix).

Article Snippet: Constructs containing full‐length CA2 and CA7 (human isoform 1) coding sequences were obtained from ImaGenes (human CA2 and human CA7 including start and stop codon; OCAAo5051H1054 and OCAAo5051E0588, respectively) and GeneCopoeia (human CA2 without stop codon and mouse CA7 including start and stop).

Techniques: Sequencing, Generated, Binding Assay

Journal: EMBO Reports

Article Title: Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

doi: 10.15252/embr.202050145

Figure Lengend Snippet: A–G (A) Transfection with EGFP‐CA7‐mutant2 modified cellular F‐actin structures in a similar manner than CA7. (B) Introduction of KKHDV and DDERIH motifs to CA2 (EGFP‐CA2‐revCA7) did not affect the diffuse cytosolic localization of the isoform 2. (C) EGFP‐CA7‐R223E and (D) the catalytically loss‐of‐function mutant EGFP‐CA7‐H96/98C co‐localized with F‐actin. F‐actin is visualized with Phalloidin‐594 in (A–D). The normalized fluorescence emission intensity profiles of (E) EGFP‐CA2‐revCA7 (F) EGFP‐CA7‐R223E, and (G) EGFP‐CA7‐H96/98C (black lines) and F‐actin (red line). The yellow line in (B–D) indicates the cross‐section of the cell from which the pixel intensities were measured. Analysis of co‐localization is shown in lower panels of (E–G). Representative single‐cell pixel intensities of EGFP and phalloidin‐594 channels were plotted against each other and the Pearson’s correlation coefficient value ( r ) was calculated. n = 3–4 independent transfections/construct. Scale bars in (A–D) is 20 µm. H A representative Western blot showing the expression levels of EGFP‐CA fusion proteins in 10 µg of lysate collected 24 h after transfection. The EGFP‐tagged CA fusion proteins are visible at approximately 60 kDa, and the β‐actin loading control is visible at 42 kDa. I Quantification of the fusion proteins expression levels in NIH3T3 cells. Expression level of EGFP‐CA7 was set at 1 for each Western blot. n = 5 independent transfections for all fusion proteins except for CA7‐mutant2, for which n = 4 (data passed Shapiro–Wilk test for normality). Transfections’ efficacy of different mutant fusion proteins was compared to CA7 WT using one‐way ANOVA with Dunnett’s multiple comparisons test. Data are given as mean + SEM. Source data are available online for this figure.

Article Snippet: Constructs containing full‐length CA2 and CA7 (human isoform 1) coding sequences were obtained from ImaGenes (human CA2 and human CA7 including start and stop codon; OCAAo5051H1054 and OCAAo5051E0588, respectively) and GeneCopoeia (human CA2 without stop codon and mouse CA7 including start and stop).

Techniques: Transfection, Modification, Mutagenesis, Fluorescence, Construct, Western Blot, Expressing, Control

Journal: EMBO Reports

Article Title: Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

doi: 10.15252/embr.202050145

Figure Lengend Snippet: A–H (A) Control experiment with neurons co‐expressing mCherry‐actin and EGFP. (B) Neuron transfected with mCherry‐actin and EGFP‐CA2. Compared to the spine‐targeted mCherry‐actin, CA2 localizes more diffusely along dendritic shafts and spines. Both (C) EGFP‐CA7 and (D) EGFP‐CA7‐mutant2 show a highly overlapping localization with mCherry‐actin and disruption of dendritic spine morphology. Spines were replaced by thick, filopodia‐like dendritic protrusions, which lack spine heads. The loss‐of‐function constructs (E) EGFP‐CA7‐mutant3 and (F) EGFP‐CA7‐mutant1 are more homogenously present in both dendrites and spines. (G) EGFP‐CA7‐R223E and (H) the catalytically inactive EGFP‐CA7‐H96/98C showed overlapping localization with mCherry‐actin. Scale bar 5 µm (A–C, G, H), 10 µm (D–F). I EGFP‐CA7 expression disrupted normal spine morphology in cultured neurons. Control, only mCherry‐actin: spines with head 0.34 ± 0.04, thin spines/filopodia 0.21 ± 0.02, abnormal spines 0.00 ± 0.00, total 0.54 ± 0.05 spines/µm; n = 10 cells, 509 spines, 973 µm analyzed dendrite. EGFP‐CA2: spines with head 0.35 ± 0.04, thin spines/filopodia 0.17 ± 0.02, abnormal spines 0.00 ± 0.00, total 0.53 ± 0.06 spines/µm; n = 10 cells, 535 spines, 992 µm analyzed dendrite; EGFP‐CA7: spines with head 0.05 ± 0.02, thin spines /filopodia 0.10 ± 0.02, abnormal spines 0.34 ± 0.03, total 0.49 ± 0.05 spines/µm; n = 10 cells, 493 spines, 10,256 µm dendrite. Analyzed cells were pooled from two independent experiments. The bar diagrams show the mean. Source data are available online for this figure.

Article Snippet: Constructs containing full‐length CA2 and CA7 (human isoform 1) coding sequences were obtained from ImaGenes (human CA2 and human CA7 including start and stop codon; OCAAo5051H1054 and OCAAo5051E0588, respectively) and GeneCopoeia (human CA2 without stop codon and mouse CA7 including start and stop).

Techniques: Control, Expressing, Transfection, Disruption, Construct, Cell Culture

Journal: EMBO Reports

Article Title: Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

doi: 10.15252/embr.202050145

Figure Lengend Snippet: A Isoform‐specific subcellular localization shown in cultured hippocampal neurons (DIV14) co‐expressing DsRed‐CA7 ( left ) and EGFP‐CA2 ( middle ). B, C Representative confocal images of precocious in vivo expression of (B) EGFP‐CA2 and (C) EGFP‐CA7 in P40 mouse cortical layer 2/3 pyramidal neurons. Neurons were transfected at E14.5 with EGFP‐CA2 or EGFP‐CA7 using in utero electroporation and images were taken from fixed slices. Right panels in (B) and (C) show higher magnification of the primary apical dendrite marked with a box. D The strong expression of EGFP‐CA7 disrupted the normal spine morphology and induced the formation of thick, filopodia‐like protrusions. Data information: n = 5 independent repeats for cultured neurons and two animals/construct in vivo . Scale bar in (A) 5 µm; (B and C): 5 µm, insets in (B, C) and panel (D): 25 µm.

Article Snippet: Constructs containing full‐length CA2 and CA7 (human isoform 1) coding sequences were obtained from ImaGenes (human CA2 and human CA7 including start and stop codon; OCAAo5051H1054 and OCAAo5051E0588, respectively) and GeneCopoeia (human CA2 without stop codon and mouse CA7 including start and stop).

Techniques: Cell Culture, Expressing, In Vivo, Transfection, In Utero, Electroporation, Construct

Journal: EMBO Reports

Article Title: Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

doi: 10.15252/embr.202050145

Figure Lengend Snippet: Comparison of mEPSCs in cortical layer 2/3 pyramidal neurons from P30 to P40 WT and CA7 KO mice. Sample traces of mEPSC recordings from WT and CA7 KO neurons, low‐pass filtered at 1 kHz (left). The data are summarized in the bar diagrams (right). mEPSC frequency ( P = 0.63) and amplitude ( P = 0.90) were not significantly different between WT and CA7 KO and neurons ( n = 7 and 5 neurons, respectively, Student’s independent samples t ‐test). Representative confocal images of apical dendrites from Lucifer Yellow‐injected cortical layer 2/3 pyramidal neurons from WT and CA7 KO mice. The dendritic spine density and spine head size were examined in fixed slice preparations from P34 to P37 mice. Scale bar 2 µm. Summary of the spine density analysis done from the Lucifer Yellow‐injected neurons. Spine density was a higher in CA7 KO neurons both in apical and basal dendrites ( n = 28 neurons for both) compared to WT ( n = 29 neurons for apical and n = 30 for basal dendrite analysis) ( P = 0.000002 for apical dendrites, analyzed with Mann–Whitney test, and P = 6.8 × 10 −8 for basal dendrites, Student’s t ‐test with Welch correction.) A total of 8279 spines were analyzed from four CA7 KO mice and 8730 spines from two WT control mice. The spine head width distribution differed significantly between the genotypes ( n = 467 spines from WT and n = 421 spines from CA7 KO animals, 15 neurons analyzed from both genotypes, Wilcoxon rank‐sum test with continuity correction, W = 134,540, P < 0.001). Data information: Data are given as mean ± SEM in (A) and as mean + SEM in (C). Source data are available online for this figure.

Article Snippet: Constructs containing full‐length CA2 and CA7 (human isoform 1) coding sequences were obtained from ImaGenes (human CA2 and human CA7 including start and stop codon; OCAAo5051H1054 and OCAAo5051E0588, respectively) and GeneCopoeia (human CA2 without stop codon and mouse CA7 including start and stop).

Techniques: Comparison, Injection, MANN-WHITNEY, Control

Journal: Cancers

Article Title: Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis

doi: 10.3390/cancers12123528

Figure Lengend Snippet: Structure of IRX1 and its methylation analysis in lung cancer. ( a ) The IRX1 gene encodes a polypeptide of 480 aa and the protein contains a homeobox at position 145–184, a HARE-HTH (HB1, ASXL, restriction endonuclease helix-turn-helix) domain at position 188–247 and the Iroquois (IRO) box at position 313–326 (NCBI tool for conserved domain search) ( b ) IRX1 is located on chromosome 5p15.33 in an 8.6 kb CpG island (dark green box) flanked by the two indicated CTCF binding sites (5′- and 3′-CTCFBS) . ( c ) Overview of the analyzed 675 bp sequence located in the proximal IRX1 promoter region. The transcriptional start site (TSS; arrow) and individual CpG sites (black lines) are depicted by Python vs. CoBRA . For methylation analysis, two Taq I sites of CoBRA, nine CpGs sites (1, 2, 3, 4, 5, 6, 7, 8, and 9) of bisulfite pyrosequencing, and three CpG sites (cg05534710, cg06689918, and cg09232937) of 450K array are marked. Positions of guide#1 and guide#2 for promoter deletion are marked as blue boxes upstream of the IRX1 TSS. ( d ) A 272 bp fragment of the IRX1 promoter was analyzed by CoBRA in five NSCLC (A549, A427, HCC15, H322, and H358), 11 SCLC (HTB171, HTB175, SCLC21H, SCLC22H, SCLC24H, H82, HTB187, H209, H510, H1092, and H1184), HeLa and in vitro methylated DNA (ivm). PCR products were mock (−) or Taq I (+) digested and analyzed on 2% agarose gels together with a 100 bp ladder. Unmethylated (u), partially methylated (pm), and methylated (m) samples are indicated.

Article Snippet: The full length IRX1 coding sequence (BC166635) was obtained from BioCat (Heidelberg, Germany) and cloned into pEYFP (Clontech, Saint-Germain-en-Laye, France).

Techniques: Methylation, Binding Assay, Sequencing, Combined Bisulfite Restriction Analysis Assay, In Vitro

Journal: Cancers

Article Title: Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis

doi: 10.3390/cancers12123528

Figure Lengend Snippet: Methylation and expression of IRX1 in cancer cell lines. ( a ) Methylation level of the IRX1 promoter at the three CpG sites (cg05534710, cg06689918, and cg09232937) were analyzed by Infinium Human Methylation 450 BeadChip (ilmnhm450K array) through the R2 Genomics Analysis and Visualization Platform . Methylation of 244 normal lung samples (GSE52401) were compared to methylation of 1028 cancer cell lines (GSE68379). IRX1 methylation levels are plotted in beta-value (1 = 100% methylation) and p -value was calculated by one-way ANOVA. ( b ) Correlation analysis of IRX1 promoter methylation (beta-value) and IRX1 expression (2log ps). Methylation level of the three CpG sites of cancer cell lines (GSE68379) was plotted for IRX1 expression (11725275_at) in the corresponding mRNA 1017 of cancer cell lines . Significance of correlation was calculated by the R2 platform. p < 0.05 was considered significant.

Article Snippet: The full length IRX1 coding sequence (BC166635) was obtained from BioCat (Heidelberg, Germany) and cloned into pEYFP (Clontech, Saint-Germain-en-Laye, France).

Techniques: Methylation, Expressing

Journal: Cancers

Article Title: Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis

doi: 10.3390/cancers12123528

Figure Lengend Snippet: Epigenetic inactivation of IRX1 in primary lung cancers compared to matching lung. ( a ) Methylation level of the IRX1 promoter at the nine CpG sites was analyzed in 100 adenocarcinoma (ADC), 100 squamous cell carcinoma (SQCC), and 41 small cell lung cancer (SCLC) samples by bisulfite pyrosequencing. Average CpG methylation levels are depicted by box plot. ( b ) IRX1 methylation of tumors and corresponding matching lung samples were revealed by pyrosequencing. Average methylation was compared to tumor tissue by box plot and p -value was calculated. ( c ) Relative IRX1 expression in primary tumors and corresponding lung samples. mRNA levels were analyzed by qRTPCR normalized to the housekeeping genes ESD and RPS18 (ΔCt). Note that a higher ΔCt value indicates lower IRX1 expression. Median expression levels of IRX1 of NSCLC, ADC, and SQCC and corresponding lung samples are shown and significances ( p -values) were calculated. p < 0.05 was considered significant.

Article Snippet: The full length IRX1 coding sequence (BC166635) was obtained from BioCat (Heidelberg, Germany) and cloned into pEYFP (Clontech, Saint-Germain-en-Laye, France).

Techniques: Methylation, CpG Methylation Assay, Expressing

Journal: Cancers

Article Title: Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis

doi: 10.3390/cancers12123528

Figure Lengend Snippet: Impaired survival probability of ADC patients is associated epigenetic silencing of IRX1 . ( a ) To correlate high and low IRX1 expression with survival probabilities, we performed the Kaplan–Meier estimator for all patients with non-small cell lung cancer (NSCLC) ( n = 416) and adenocarcinoma (ADC) ( n = 226) shown in months. ( b ). Survival probability of NSCLC and ADC patients ( n = 189 and 91, respectively) was correlated with IRX1 methylation level at CpG site 8 (CpG8). ( c ) Correlation analysis of CpG8 methylation and relative IRX1 expression ( p < 0.001). p < 0.05 was considered significant.

Article Snippet: The full length IRX1 coding sequence (BC166635) was obtained from BioCat (Heidelberg, Germany) and cloned into pEYFP (Clontech, Saint-Germain-en-Laye, France).

Techniques: Expressing, Methylation

Journal: Cancers

Article Title: Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis

doi: 10.3390/cancers12123528

Figure Lengend Snippet: IRX1 is regulated by DNA methyltransferase and p300 HAT activity. ( a ) Deletion (Δ) of 5′- and 3′-CTCF binding site in U251 cell by CRISPR/Cas9 system. We generated two control clones, three Δ5′-CTCFBS, four Δ3′-CTCFBS, and two Δ5′+3′-CTCFBS clones and analyzed IRX1 expression for each clone. RNA levels were determined by qRTPCR in triplicates and normalized to ACTB level. IRX1 expression of control clones was set 1 for comparison. ( b ) Deletion of the IRX1 promoter (ΔIRX1) by CRISPR/Cas9 system. Expression of IRX1 was analyzed in 13 ΔIRX1 clones by qRTPCR and normalized to ACTB . p -value was calculated by unpaired t-test. ( c ) A427 and A549 cell lines were treated with the indicated concentration of 5-Aza-2′-deoxycytidine (Aza) and trichostatin A (TSA) for 4 days and RNA was isolated. IRX1 mRNA levels were analyzed in technical triplicates and normalized to ACTB . Then, 0 µM Aza treatment was set 1. ( d ) p300 HAT induced expression of endogenous IRX1 . HeLa cells were transfected with IRX1 guide RNA constructs and p300-dCas9 or pcDNA-dCas (control). IRX1 expression was analyzed by qRTPCR after 1 µM Aza and 0.3 µM TSA treatment for 72 h and normalized to ACTB levels. ( e , f ) Correlation analysis of IRX1 and ( e ) DNMT3A or ( f ) EZH2 expression in primary NSCLC samples (GSE33532 data set, n = 100). Analysis was performed by R2 . ( g ) Impaired survival probability of adenocarcinoma patients is associated with high EZH2 expression. Survival probability of ADC patients ( n = 719) was correlated with EZH2 expression (low and high) through the Kaplan–Meier plotter . p < 0.05 was considered significant.

Article Snippet: The full length IRX1 coding sequence (BC166635) was obtained from BioCat (Heidelberg, Germany) and cloned into pEYFP (Clontech, Saint-Germain-en-Laye, France).

Techniques: Activity Assay, Binding Assay, CRISPR, Generated, Clone Assay, Expressing, Concentration Assay, Isolation, Transfection, Construct

Journal: Cancers

Article Title: Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis

doi: 10.3390/cancers12123528

Figure Lengend Snippet: Significances of hallmarks correlated with IRX1 expression in NSCLC data sets by gene set enrichment analysis (GSEA).

Article Snippet: The full length IRX1 coding sequence (BC166635) was obtained from BioCat (Heidelberg, Germany) and cloned into pEYFP (Clontech, Saint-Germain-en-Laye, France).

Techniques: Expressing

Journal: Cancers

Article Title: Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis

doi: 10.3390/cancers12123528

Figure Lengend Snippet: Nuclear localization of IRX1 and irregular shape of IRX1 expressing nuclei. IRX1 was fused to EYFP and transfected in A549 ( a ) HeLa ( b ) and HEK293T ( c ) cells. Localization of IRX1 in the nucleus was analyzed by DAPI co-staining and fluorescent microscopy. IRX1 deletion construct of homeobox (ΔHomeo), HARE-HTH domain (ΔHare), and IRO box (ΔIro) were generated and transfected. The nuclear shape of transfected cells was analyzed after 24 h in A549 (80–160 cells analyzed), HeLa (200–350 cells), and HEK293 (150–300 cells). Normal nuclei exhibited a round shape and fragmented nuclei showed an irregular, lobed shape. Significance was calculated by Fisher’s exact test. White bar represents the length standard of 5 µM.

Article Snippet: The full length IRX1 coding sequence (BC166635) was obtained from BioCat (Heidelberg, Germany) and cloned into pEYFP (Clontech, Saint-Germain-en-Laye, France).

Techniques: Expressing, Transfection, Staining, Microscopy, Construct, Generated

Journal: Cancers

Article Title: Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis

doi: 10.3390/cancers12123528

Figure Lengend Snippet: IRX1 expression is associated with expression of the apoptotic regulator BAX. ( a ) IRX1 wt and IRX1 deletion construct of homeobox (ΔHomeo), HARE-HTH domain (ΔHare), and IRO box (ΔIro) were transfected in HeLa cells. RNA was isolated after 48 h and BAX mRNA levels were analyzed in technical triplicates and normalized to ACTB . BAX expression was plotted relative to EYFP-control transfected HeLa cells (set = 1). ( b ) BAX expression values (208478_s_at) were analyzed in pcDNA3.1-GFP (control) and pcDNA3.1-IRX1-GFP transfected HEK293T cells in the data set GSE75376 by GEO2R [ , ]. ( c ) Expression of BAX (NM_004324) was analyzed in 143B cells after IRX1 (shIRX1) and control (shCtrl) knock down in technical triplicates in the data set GSE56255 by GEO2R [ , ].

Article Snippet: The full length IRX1 coding sequence (BC166635) was obtained from BioCat (Heidelberg, Germany) and cloned into pEYFP (Clontech, Saint-Germain-en-Laye, France).

Techniques: Expressing, Construct, Transfection, Isolation

Journal: The Journal of Biological Chemistry

Article Title: C6orf106 is a novel inhibitor of the interferon-regulatory factor 3–dependent innate antiviral response

doi: 10.1074/jbc.RA117.001491

Figure Lengend Snippet: Validation of C6orf106 knockdown and overexpression in HeLa cells. a and b, HeLa cells were transfected with siRNAs targeting C6orf106 (siC6orf106) or a nontargeting control (siNEG) and assayed by qRT-PCR (a) or Western blotting (b) for C6orf106 expression levels. c, validation of C6orf106-FLAG expression by Western blotting. d, cell viability in HeLa cells treated with the indicated siRNAs or cDNAs. Values are normalized against cells treated with transfection reagent only (mock). Error bars for all graphs indicate ±1 S.D. of a minimum of three independent experiments; asterisks show significant differences as assessed by one-way ANOVA with Bonferroni post-test (*, p < 0.05; **, p < 0.01).

Article Snippet: The full-length coding sequence of human C6orf106 (accession number {"type":"entrez-protein","attrs":{"text":"NP_077270.1","term_id":"13236514","term_text":"NP_077270.1"}} NP_077270.1 ) was synthesized by GenScript with SacI and XhoI restriction sites at the 5′- and 3′-ends respectively.

Techniques: Biomarker Discovery, Knockdown, Over Expression, Transfection, Control, Quantitative RT-PCR, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: C6orf106 is a novel inhibitor of the interferon-regulatory factor 3–dependent innate antiviral response

doi: 10.1074/jbc.RA117.001491

Figure Lengend Snippet: C6orf106 suppresses antiviral cytokine synthesis. a, HeLa cells were treated with transfected poly(I:C) for 6 h, and the cells were collected and analyzed for mRNA expression of the listed cytokines by qRT-PCR. b, relative cytokine mRNA levels in HeLa cells stimulated with poly(I:C) 48 h post-transfection with siRNAs. c, relative mRNA levels in HeLa cells stimulated with poly(I:C) 24 h post-transfection with cDNAs. d, cell culture supernatants from c were assayed for IFN-β using ELISA. Relative cytokine mRNA levels in HeLa cells infected with SeV (400 hemagglutination units/well) post-transfection with siRNAs (e) or cDNA plasmids (f) are shown. g, HeLa cells were stimulated with 2000 enzyme units/ml IFN-α for 6 h, and ISG15 mRNA expression was determined by qRT-PCR. h, HeLa cells were stimulated with 20 ng/ml TNFα for 6 h, and IκBα mRNA expression was determined by qRT-PCR. i and j, HeLa cells were transfected with siRNAs (i) or cDNA plasmids (j) and then stimulated with poly(I:C) for the times shown. Relative mRNA levels of IFN-β over time were measured by qRT-PCR. k, mock cells were stimulated with poly(I:C) for the times shown, and endogenous C6orf106 and IFN-β mRNA levels were measured by qRT-PCR. Error bars indicate ±1 S.D. of three independent experiments, and asterisks show significant changes compared with controls as measured by one- or two-way ANOVA with Bonferroni post-test (***, p < 0.001; **, p < 0.01; *, p < 0.05 compared with 4 h).

Article Snippet: The full-length coding sequence of human C6orf106 (accession number {"type":"entrez-protein","attrs":{"text":"NP_077270.1","term_id":"13236514","term_text":"NP_077270.1"}} NP_077270.1 ) was synthesized by GenScript with SacI and XhoI restriction sites at the 5′- and 3′-ends respectively.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Infection

Journal: The Journal of Biological Chemistry

Article Title: C6orf106 is a novel inhibitor of the interferon-regulatory factor 3–dependent innate antiviral response

doi: 10.1074/jbc.RA117.001491

Figure Lengend Snippet: C6orf106 predominantly targets IRF-driven transcription. HeLa cells were transfected with siRNAs for 24 h, then transfected with ISRE- (a) or NF-κB-firefly luciferase (luc) (b) and Renilla luciferase vectors for 20 h, and then stimulated with poly(I:C) for 6 h. Alternatively, HeLa cells were transfected with GFP or C6orf106-FLAG and ISRE- (c) or NF-κB-firefly luciferase (d) and Renilla luciferase vectors for 20 h and then stimulated with poly(I:C) for 6 h. All cell lysates were assayed for luciferase activity, normalized to the transfection control Renilla luciferase. Error bars indicate ±1 S.D. of triplicate experiments; asterisks indicate significant differences as determined by Student's t test (*, p < 0.05; **, p < 0.01).

Article Snippet: The full-length coding sequence of human C6orf106 (accession number {"type":"entrez-protein","attrs":{"text":"NP_077270.1","term_id":"13236514","term_text":"NP_077270.1"}} NP_077270.1 ) was synthesized by GenScript with SacI and XhoI restriction sites at the 5′- and 3′-ends respectively.

Techniques: Transfection, Luciferase, Activity Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: C6orf106 is a novel inhibitor of the interferon-regulatory factor 3–dependent innate antiviral response

doi: 10.1074/jbc.RA117.001491

Figure Lengend Snippet: C6orf106 does not impair activation or nuclear translocation of IRF3 and NF-κB. a, HeLa cells transfected with GFP or C6orf106-FLAG for 20 h were stimulated with poly(I:C) for 6 h and then lysed, and cellular proteins were separated into cytosolic and nuclear fractions. Fractions were probed for the transcription factors IRF3 and p65 as well as C6orf106 and the loading controls GAPDH (cytosol) and fibrillarin (nucleus). b, HeLa cells as in a were fixed and labeled for C6orf106-FLAG (green), IRF3 (left panel) or p65 (right panel), and nuclei (blue). White scale bars, 10 μm. c, Fn/c ratios for treatment groups shown in b. Error bars indicate ±1 S.D. of a typical experiment from duplicate experiments; asterisks indicate significant differences as determined by one-way ANOVA with Dunn's multiple comparison test (***, p < 0.001).

Article Snippet: The full-length coding sequence of human C6orf106 (accession number {"type":"entrez-protein","attrs":{"text":"NP_077270.1","term_id":"13236514","term_text":"NP_077270.1"}} NP_077270.1 ) was synthesized by GenScript with SacI and XhoI restriction sites at the 5′- and 3′-ends respectively.

Techniques: Activation Assay, Translocation Assay, Transfection, Labeling, Comparison

Journal: The Journal of Biological Chemistry

Article Title: C6orf106 is a novel inhibitor of the interferon-regulatory factor 3–dependent innate antiviral response

doi: 10.1074/jbc.RA117.001491

Figure Lengend Snippet: C6orf106 does not alter cellular protein levels of IRF3 and NF-κB. HeLa cells were transfected with cDNAs for 20 h (a) or siRNAs for 40 h (b). Cells were then pretreated for 1 h with CHX (20 μg/ml) or MG132 (10 μm) and then stimulated with poly(I:C) for 6 h. Cells were lysed, and cellular levels of IRF3, p65, and C6orf106 (endogenous or FLAG-tagged) were measured by Western blotting. c, immunoblot showing expression levels of cyclin B1 and actin in HeLa cells treated for the indicated times with CHX (20 μg/ml) or MG132 (10 μm).

Article Snippet: The full-length coding sequence of human C6orf106 (accession number {"type":"entrez-protein","attrs":{"text":"NP_077270.1","term_id":"13236514","term_text":"NP_077270.1"}} NP_077270.1 ) was synthesized by GenScript with SacI and XhoI restriction sites at the 5′- and 3′-ends respectively.

Techniques: Transfection, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: C6orf106 is a novel inhibitor of the interferon-regulatory factor 3–dependent innate antiviral response

doi: 10.1074/jbc.RA117.001491

Figure Lengend Snippet: C6orf106 interacts with IRF3 and inhibits binding to its DNA consensus sequence. HeLa cells were transfected with cDNAs and stimulated with poly(I:C), and the nuclear proteins were extracted using a hypotonic lysis method. Nuclear proteins (10 μg) were analyzed for IRF3–DNA (a) or p65–DNA (c) binding. Alternatively, HeLa cells depleted of C6orf106 were stimulated with poly(I:C), and nuclear proteins were analyzed for IRF3–DNA (b) or p65–DNA (d) binding. e, HEK293T cells transfected with IRF3 alone or in combination with C6orf106 were stimulated with poly(I:C), lysed, and subjected to indirect immunoprecipitation with an anti-IRF3 antibody. Immunoprecipitated (IP) samples and input controls were probed with anti-FLAG antibody for Western blotting. An IgG isotype was used as a negative control for the immunoprecipitation experiment. Error bars indicate ±1 S.D. of triplicate experiments; asterisks indicate significant differences as determined by two-way ANOVA with Bonferroni post-test (***, p < 0.001; **, p < 0.01; ns, not significant).

Article Snippet: The full-length coding sequence of human C6orf106 (accession number {"type":"entrez-protein","attrs":{"text":"NP_077270.1","term_id":"13236514","term_text":"NP_077270.1"}} NP_077270.1 ) was synthesized by GenScript with SacI and XhoI restriction sites at the 5′- and 3′-ends respectively.

Techniques: Binding Assay, Sequencing, Transfection, Lysis, Immunoprecipitation, Western Blot, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: C6orf106 is a novel inhibitor of the interferon-regulatory factor 3–dependent innate antiviral response

doi: 10.1074/jbc.RA117.001491

Figure Lengend Snippet: C6orf106 reduces nuclear levels of the transactivator proteins p300 and CBP. Nuclear and cytosolic fractions were isolated from HeLa cells expressing C6orf106-FLAG (a) or knocked down with siRNAs targeting C6orf106 (b) and stimulated with poly(I:C) as shown. Equal amounts of nuclear lysates were probed for members of the enhanceosome complex as shown. c, cytosolic fractions were also probed for the nuclear transactivators CBP/p300.

Article Snippet: The full-length coding sequence of human C6orf106 (accession number {"type":"entrez-protein","attrs":{"text":"NP_077270.1","term_id":"13236514","term_text":"NP_077270.1"}} NP_077270.1 ) was synthesized by GenScript with SacI and XhoI restriction sites at the 5′- and 3′-ends respectively.

Techniques: Isolation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: C6orf106 is a novel inhibitor of the interferon-regulatory factor 3–dependent innate antiviral response

doi: 10.1074/jbc.RA117.001491

Figure Lengend Snippet: Working model of C6orf106 (C6)-mediated inhibition of IRF3-dependent cytokine transcription.

Article Snippet: The full-length coding sequence of human C6orf106 (accession number {"type":"entrez-protein","attrs":{"text":"NP_077270.1","term_id":"13236514","term_text":"NP_077270.1"}} NP_077270.1 ) was synthesized by GenScript with SacI and XhoI restriction sites at the 5′- and 3′-ends respectively.

Techniques: Inhibition