Journal: Cancer Cell International

Article Title: Silencing lncRNA ZFAS1 or elevated microRNA-135a represses proliferation, migration, invasion and resistance to apoptosis of osteosarcoma cells

doi: 10.1186/s12935-019-1049-x

Figure Lengend Snippet: Down-regulated ZFAS1 or up-regulated miR-135a restrains colony formation ability and proliferation of MG63 cells. A The colony formation ability of MG63 cells in each group. B Quantification results in A . C MG63 cell proliferation tested by MTT assay. D EdU assay for detecting proliferation of MG63 cells. E Quantification results in D . F Ki-67 and CyclinD1 protein band and expression in each group of MG63 cells. The data in the figure were all measurement data, in the form of mean ± standard deviation; a vs the sh-NC group, P

Article Snippet: The protein was transferred onto polyvinylidene difluoride membrane and the membrane was sealed with 5% bovine serum albumin for 1 h. The membrane was added with primary antibody Ki-67 (1:1000), APEX1 (1:5000), MMP9 (1:1000) (Abcam, Cambridge, UK), CyclinD1 (1:1000), Bax (1:1000), Bcl-2 (1:1000), MMP2 (1:500) (Santa Cruz Biotechnology, Santa Cruz, California, USA), and GAPDH (1:2000) (Jackson Immuno Research, Grove, Pennsylvania, USA).

Techniques: MTT Assay, EdU Assay, Expressing, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Bidirectional promoters link cAMP signaling with irreversible differentiation through promoter-associated non-coding RNA (pancRNA) expression in PC12 cells

doi: 10.1093/nar/gkw113

Figure Lengend Snippet: Knockdown (KD) of pancNusap1 mimicked irreversible differentiation of PC12 cells induced by cAMP ( A ) The effect of pancNusap1 KD on the expression level of Nusap1 in Ndiff cells. Gapdh was used as a control. Empty vector-introduced PC12 cells were used as a negative control. ( B ) Histone modification status of the Nusap1 promoter in pancNusap1- KD PC12 cells under reversible differentiation condition. The expression of sh-pancNusap1 was induced by Dox at day 5. Empty vector-infected PC12 cells were used as a control. ( C ) The effects of pancNusap1 KD on cell proliferation in Ndiff cells. The numbers of living cells were counted. ( D ) Proportion of the EdU+ and the Ki67+ cells in pancNusap1 -KD PC12 cells after NGF deprivation. After 7 days of differentiation, NGF was removed to allow proliferation. White arrowheads indicate locations of proliferating infected cells (triple-positive for Ki67, EdU and GFP). In (A–C), viral vectors were introduced at day 3. In (A–D), values are mean ± SD ( n = 3). Statistical analysis was performed using Student's two-tailed t -tests, or the Tukey-Kramer multiple comparison test. N.S. = not significant. * P

Article Snippet: As primary antibodies, chicken anti-GFP (AVES Labs), mouse anti-Ki67 (BD Biosciences) and rabbit anti-active Caspase 3 (R & D Systems) were used.

Techniques: Expressing, Plasmid Preparation, Negative Control, Modification, Infection, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Bidirectional promoters link cAMP signaling with irreversible differentiation through promoter-associated non-coding RNA (pancRNA) expression in PC12 cells

doi: 10.1093/nar/gkw113

Figure Lengend Snippet: pancNusap1 regulates irreversibility of PC12 differentiation through transcriptional regulation of Nusap1 . ( A ) Proportion of the EdU+ and the Ki67+ cells in pancNusap1 -KD and Nusap1 -OE Undiff cells. White arrowheads indicate locations of the proliferating infected cells (triple-positive for Ki67, EdU and GFP). ( B ) The effect of Nusap1 -OE on cell proliferation in the pancNusap1- KD cells. Number of living cells was counted. In (A and B), values are mean ± SD ( n = 3). Statistical analysis was performed using the Tukey-Kramer multiple comparison test. N.S. = not significant. * P

Article Snippet: As primary antibodies, chicken anti-GFP (AVES Labs), mouse anti-Ki67 (BD Biosciences) and rabbit anti-active Caspase 3 (R & D Systems) were used.

Techniques: Infection

Journal: Nucleic Acids Research

Article Title: Bidirectional promoters link cAMP signaling with irreversible differentiation through promoter-associated non-coding RNA (pancRNA) expression in PC12 cells

doi: 10.1093/nar/gkw113

Figure Lengend Snippet: Combination of NGF and cAMP stimulations caused cell cycle arrest in differentiated PC12 cells. ( A ) Morphologies of PC12 cells that grew during 7 days of differentiation in the presence of NGF (upper) or NGF/cAMP (lower). ( B ) Relative number of living cells after removal of NGF or of NGF and cAMP. Upper diagram shows experimental scheme. After 7 days of differentiation of PC12 cells, NGF and cAMP were removed to allow proliferation. Number of living cells was evaluated by counting trypan blue staining-negative cells. Number of cells in each differentiation state at day 7 was set as 1. ( C ) Cell proliferation monitored by EdU incorporation and Ki67 staining in undifferentiated (Undiff), NGF-differentiated (Ndiff) and NGF/cAMP-differentiated (NcAdiff) cells, and in Ndiff and NcAdiff cells after 4 days of incubation in proliferative condition (Ndiff + reverse 4 day, NcAdiff + reverse 4 day, respectively). ( D ) Proportion of apoptotic cells before/after removal of NGF or NGF and cAMP, detected by active Caspase 3 (aCas3) staining. In ( B – D ), values are mean ± SD ( n = 3). Statistical analysis was performed using Student's two-tailed t -tests, or the Tukey-Kramer multiple comparison test. N.S. = not significant. * P

Article Snippet: As primary antibodies, chicken anti-GFP (AVES Labs), mouse anti-Ki67 (BD Biosciences) and rabbit anti-active Caspase 3 (R & D Systems) were used.

Techniques: Staining, Incubation, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Bidirectional promoters link cAMP signaling with irreversible differentiation through promoter-associated non-coding RNA (pancRNA) expression in PC12 cells

doi: 10.1093/nar/gkw113

Figure Lengend Snippet: pancNusap1 overexpression (OE) caused cells subjected to the irreversibly differentiation condition to have the morphological and epigenetic characters of reversibly differentiated cells. ( A ) Genomic structure of the rat Nusap1 promoter. Bold-lined boxes show the region homologous to mouse Oip5 . Gray boxes indicate predicted locations composing the Oip5 open reading frame (ORF). Lower thick arrows indicate the region and transcribed strands used for OE-experiments of ncRNA. Upper and lower vertical lines show the positions of methionines (Met) or stop codons (Stop), respectively, in each translational frame. ( B ) The effect of Dox-induced sense or antisense pancNusap1 OE on the Nusap1 expression level in NcAdiff cells. Gapdh was used as a control. Empty vector-introduced PC12 cells were used as a negative control. ( C ) Histone modification status of the Nusap1 promoter in pancNusap1- overexpressing PC12 cells under irreversible differentiation condition. ( D ) The effect of Dox-induced sense or antisense pancNusap1 OE on cell proliferation in NcAdiff cells. Upper diagram shows experimental scheme. Number of living cells was counted. After 7 days of differentiation of PC12 cells, NGF/cAMP were removed to allow proliferation. ( E ) Proportion of Ki67+ cells in antisense pancNusap1 -overexpressing PC12 cells after NGF/cAMP deprivation. In (B–E), values are mean ± SD ( n = 3). Statistical analysis was performed using Student's two-tailed t -tests or the Tukey-Kramer multiple comparison test. N.S. = not significant. * P

Article Snippet: As primary antibodies, chicken anti-GFP (AVES Labs), mouse anti-Ki67 (BD Biosciences) and rabbit anti-active Caspase 3 (R & D Systems) were used.

Techniques: Over Expression, Expressing, Plasmid Preparation, Negative Control, Modification, Two Tailed Test