Fig 1: LINC00511 silenced restricted tumor growth in vivo in GC cells a Tumor images. b The effect of LINC00511 knockdown was examined on tumor growth. c The result of tumor volume affected by sh-LINC000511#1. d Tumor weight in cells transfected with sh-LINC00511#1 and sh-NC. e The expression of Ki67 was tested by immunohistochemistry. f QRT-PCR assays and western blot were used to measure NFIX and protein. *P < 0.05, **P < 0.01
Fig 2: Nfix deletion results in germ cell arrest prior to round spermatid formation in the first round of spermatogenesis. Cross sections through testis samples collected from P20 Nfix+/+ (A–C) and Nfix-/- mice (D–F). Paraffin sections were stained with hematoxylin and eosin. The white lines in panels A and C show the measurements used to calculate the diameter of the seminiferous tubules. Panels B and E reveal a seminiferous tubule in a wild-type (B) and a Nfix-null mutant (E), respectively; the dashed boxes in these panels are shown in C and F, respectively. Histological analysis of Nfix+/+ mice revealed normal progression through the cycle of the seminiferous epithelium, with round spermatids (double arrowhead in C) being the most developed spermatogenic cells present at this age. (D–F) In contrast, very few germ cells progressed to round spermatids in the testis of Nfix-/- mice. Moreover, numerous multi-nucleated cells were present in the mutant seminiferous tubule (arrows in F). Quantitative analysis showed that Nfix-/- mice had a significant reduction in the number of round spermatids (G) and in seminiferous tubule diameter (H) in comparison to controls. n = 4 for each genotype. Scale bars: A, D = 400 µm; B, E = 100 µm; C, F = 50 µm. Error bars indicate SD, Students t-test, **P < 0.01, ***P < 0.001.
Fig 3: Regional gene signatures exhibit universally conserved transcription factor motif enrichment. (A) Significant transcription factor motifs enriched in all regional DEGs. (B–D) Motif sequence and normalized expression of top 3 most significantly enriched motifs across surveyed brain regions (B) Nkx2-2, (C) Maz, (D) NFIA/NFIB/NFIX. (E) Frequency of motif occurrence in each region DEGs (F) Statistically significant Gene Ontologies associated with each transcription factor. p-value < 0.05. (G) Representative images of immunofluorescence staining of Aldh1l1-eGFP (green) and NFIB or NFIX (red). Yellow arrows indicate double positive cells. (H) Quantification of immunofluorescence staining. n = 3 scale bar = 50 µm.
Fig 4: Identification of the NFIX binding site in Ezrin.a Location and sequences of NFIX putative response elements (REs) identified in the promoter region of Ezrin gene. b PGL3 reporter plasmids encode luciferase under the control of human Ezrin gene promoter. The putative NFIX REs within the promoter region were mutated as Mut1, 2, and 3. c Measurement of firefly-luciferase activity normalized with the renilla-luciferase activity (n = 6). d U87 cells were subjected to chromatin immunoprecipitation using anti-NFIX or anti-IgG antibody, and followed with PCR amplification using primers specific to NFIX RE2 of Ezrin promoter region or distal region of Ezrin as negative control. All data are represented as the mean ± s.e.m. *p < 0.05 (Student’s t test).
Fig 5: NFIX is upregulated in human GBM.a–c Human GBM tissues and normal brain tissues were used. a Valcano plot of transcription factors identified by oligonucleotide array-based transcription factor assay (n = 5). The vertical lines correspond to 1.5-fold up and down, respectively, and the horizontal line stands for a p-value of 0.05. b Relative mRNA levels of NFIA, NFIB, NFIC, and NFIX normalized with GAPDH in human GBM tissues and normal brain tissues (n = 5). c Immunoblotting analysis of NFIX and GAPDH in human GBM tissues and normal brain tissues. Representative images are shown. The bar chart is a relative expression level of NFIX normalized with GAPDH (n = 5). d NFIX mRNA level in human normal brain tissues and GBM (GSE4290; n = 23 for normal brain tissue and n = 77 for GBM). e IHC staining with the NFIX antibody in human glioma tissue microarray (n = 8 for normal brain tissue, n = 25 for WHO II, n = 26 for WHO III and n = 19 for GBM). All data are represented as the mean ± s.e.m. *p < 0.05, Normal vs. GBM group (Student’s t test).
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