Journal: Circulation research

Article Title: Coexpression of mutant p53 and p193 renders embryonic stem cell-derived cardiomyocytes responsive to the growth-promoting activities of adenoviral E1A.

doi: 10.1161/hh1001.090878

Figure Lengend Snippet: Figure 3. Coexpression of mutant p53 (CB7) and the prosurvival p193 mutant (1152stp) renders ES cell–derived cardiomyocytes responsive to E1A. An ES cell–derived cardiomyocyte colony growth assay was used to monitor the effects of transgene expression on cardiomyocyte growth. All cultures were trans- fected with an MHC-neor/pGK-hygror transgene, as well as with MHC-promoted expression cassettes encoding the proteins indicated. After transfection and subsequent hygromycin selec- tion, the ES cells were pooled and allowed to differentiate. Non- cardiomyocytes were removed by the addition of G418, and the yield of cardiomyocytes was directly visualized via PAS staining. The cultures were processed after 60 days of differentiation. Transfection efficiencies in parallel cultures were calculated by monitoring expression of a cotransfected CMV-bGAL reporter gene at 48 hours after transfection. bGAL activities (relative light units/mg protein, 3103) were as follows: control, 1.260.07; CB7, 1.060.04; 1152stp, 1.160.06; E1A, 1.260.01; E1A1CB7, 1.360.05; E1A11152stp, 1.260.05; CB711152stp, 1.060.04; E1A1CB711152stp, 1.260.06; and T-Ag 1.260.02. There was no significant difference between groups by Kruskal-Wallis non- parametric ANOVA test.

Article Snippet: For Western blots, protein samples were displayed on polyacrylamide gels; transferred to nitrocellulose; and reacted with anti-p53 PC35 (Oncogene), anti-E1A M73 (Santa Cruz Biotechnology), anti–T-Ag PAB-416 (Oncogene), or anti–sarcomeric myosin MF20 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa) as described.17,23,24 For Northern blots, total RNA was purified and processed as described.17,21,25 An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

Techniques: Mutagenesis, Derivative Assay, Growth Assay, Expressing, Transfection, Staining, Control

Journal: Circulation research

Article Title: Coexpression of mutant p53 and p193 renders embryonic stem cell-derived cardiomyocytes responsive to the growth-promoting activities of adenoviral E1A.

doi: 10.1161/hh1001.090878

Figure Lengend Snippet: Figure 6. Confirmation of transgene expression in the ES cell– derived cardiomyocyte cultures. Protein and RNA were prepared from ES cell–derived cardiomyocyte cultures transfected with various MHC-promoted expression cassettes after 60 days of differentiation. Total protein (75 mg) was subjected to Western blot analysis to monitor CB7, E1A, and T-Ag expression, and 10 mg of total RNA was subjected to Northern blot analysis to monitor 1152stp expression. For MHC expression, 1% of the total protein present in culture dishes was analyzed. Note that T-Ag expression stabilizes endogenous p53, which is also detected by the antibody used.

Article Snippet: For Western blots, protein samples were displayed on polyacrylamide gels; transferred to nitrocellulose; and reacted with anti-p53 PC35 (Oncogene), anti-E1A M73 (Santa Cruz Biotechnology), anti–T-Ag PAB-416 (Oncogene), or anti–sarcomeric myosin MF20 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa) as described.17,23,24 For Northern blots, total RNA was purified and processed as described.17,21,25 An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

Techniques: Expressing, Derivative Assay, Transfection, Western Blot, Northern Blot

Journal: OncoTargets and therapy

Article Title: Integrated RNA Sequencing and Single-Cell Mass Cytometry Reveal a Novel Role of LncRNA HOXA-AS2 in Tumorigenesis and Stemness of Hepatocellular Carcinoma

doi: 10.2147/OTT.S272717

Figure Lengend Snippet: Purified Antibodies About the Stem-Like Cells Centric Panel

Article Snippet: p53 , 143Nd , 7F5 , Fluidigm , 3143018A.

Techniques: Purification

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-1323 serves as a biomarker in gestational diabetes mellitus and aggravates high glucose-induced inhibition of trophoblast cell viability by suppressing TP53INP1

doi: 10.3892/etm.2021.9661

Figure Lengend Snippet: miR-1323 directly regulates TP53INP1 level in HTR-8/SVneo and BeWo cells. (A) A putative binding site of miR-1323 at the 3'-UTR of TP53INP1. (B) In two cell lines, luciferase activity was inhibited by miR-1323 overexpression, but was promoted by miR-1323 knockdown in the WT group. (C) No changes were observed in the luciferase activity in MUT group. (D and E) The TP53INP1 level was inhibited by miR-1323-overexpression, but was promoted by miR-1323-knockdown in two cell lines. (F and G) Western blot analysis and quantification of TP53INP1 proteins in two cell lines. * P<0.05. miR, microRNA; UTR, untranslated region; WT, wild-type; MUT, mutant.

Article Snippet: After 4 h of blocking with 5% skimmed milk at 4˚C, the primary antibodies, including anti-TP53INP1 (1:500; cat. no. A04229; Wuhan Boster Biological Technology, Ltd.) and anti-β-actin (dilution, 1:500; cat. no. BA2305; B Wuhan Boster Biological Technology, Ltd.), were incubated with the membranes at 4˚C overnight.

Techniques: Binding Assay, Luciferase, Activity Assay, Over Expression, Western Blot, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-1323 serves as a biomarker in gestational diabetes mellitus and aggravates high glucose-induced inhibition of trophoblast cell viability by suppressing TP53INP1

doi: 10.3892/etm.2021.9661

Figure Lengend Snippet: TP53INP1 reverses the inhibitory effect of miR-1323 on trophoblast viability in HG-treated HTR-8/SVneo and BeWo cells. (A and B) TP53INP1 expression was inhibited by HG treatment in the two cell lines. In the HG-treated two cell lines, the inhibited TP53INP1 expression by miR-1323 overexpression was increased by the pcDNA3.1-TP53INP1. (C and D) In the HG-treated two cell lines, the increase in TP53INP1 expression reversed the inhibitory effect of miR-1323 overexpression on trophoblast cell viability. * P<0.05 vs. normal; # P<0.05 vs. HG; and & P<0.05 vs. mimic. miR, microRNA; HG, high glucose.

Article Snippet: After 4 h of blocking with 5% skimmed milk at 4˚C, the primary antibodies, including anti-TP53INP1 (1:500; cat. no. A04229; Wuhan Boster Biological Technology, Ltd.) and anti-β-actin (dilution, 1:500; cat. no. BA2305; B Wuhan Boster Biological Technology, Ltd.), were incubated with the membranes at 4˚C overnight.

Techniques: Expressing, Over Expression

Journal: Scientific Reports

Article Title: Lysine-specific demethylase 1 (LSD1) suppresses cellular senescence by riboflavin uptake-dependent demethylation activity

doi: 10.1038/s41598-025-91004-0

Figure Lengend Snippet: LSD1 demethylates p53 in response to senescent stress. ( a ) U2OS cells transfected with siRNA for LSD1 and treated with 2 µM etoposide for 5 days were subjected to immunoblot analysis. ( b ) U2OS cells treated with 100 nM ORY-1001 and 2 µM etoposide for 5 days were subjected to immunoblot analysis. ( c ) U2OS cells transfected with p3XFLAG-LSD1 and treated with 2 µM etoposide for 5 days were subjected to immunoblot analysis. Protein levels relative to p53 levels were quantified using NIH ImageJ software. Original blots are presented in Supplementary Information. Data are representative of two independent experiments and values are shown as mean ± s.e.m.

Article Snippet: Anti-FLAG M2 monoclonal antibody (F3165) and anti-γ-tubulin antibody (T6557) were obtained from Sigma Aldrich; anti-Histone H3 (D1H2) antibody (#4499T), anti-Di-Methyl-Histone H3 (Lys4) (C64G9) antibody (#9725 T), anti-Mono-Methyl-Histone H3 (Lys9) (D1P5R) antibody (#14186), anti-Di-Methyl-Histone H3 (Lys9) (D85B4) antibody (#4658T), anti-Sirtuin-4 antibody (#69786) were from Cell Signaling Technology (Beverly, MA); anti-LSD1 antibody (ab69535), anti-H3K4me1 antibody (ab8895) were from Abcam; anti-Di-Methyl-p53 (K370) antibody (STJ90115) was from St John's Laboratory Ltd. (London, UK); anti-p21 antibody (sc-56335), anti-p53 antibody (sc-126), anti-Lamin B1 antibody (sc-6217) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Grb2 antibody (G16720) was from BD Biosciences (Franklin Lakes, NJ); anti-phospho-Histone H2A.X (Ser139) antibody (05-636) was from Merck Millipore (Burlington, MA); AP-conjugated anti-mouse antibody (S372B) and AP-conjugated anti-rabbit antibody (S373B) were from Promega (Madison, WI).

Techniques: Transfection, Western Blot, Software

Journal: Scientific Reports

Article Title: Lysine-specific demethylase 1 (LSD1) suppresses cellular senescence by riboflavin uptake-dependent demethylation activity

doi: 10.1038/s41598-025-91004-0

Figure Lengend Snippet: LSD1 is activated by increased riboflavin uptake. ( a ) Schematic diagram of LSD1 activation by the riboflavin transporter. Riboflavin uptake may lead to increase FAD and activate LSD1. ( b ) U2OS cells treated with 50 µM riboflavin and 2 µM etoposide for 2 days were subjected to measurement of intracellular FAD levels. Data are mean ± s.d. (n = 5 independent cultures). ( c - e ) U2OS cells treated with 50 µM riboflavin and 2 µM etoposide for 2 days were subjected to immunofluorescence ( c ), cell fractionation ( d ), and measurement of FAD levels in each fraction ( e ). Bars, 20 µm. ( f ) U2OS cells treated with 50 µM riboflavin and 2 µM etoposide for 3 days were subjected to immunoblot analysis. Protein levels relative to histone H3 or p53 levels were quantified using NIH ImageJ software. ( g – i ) U2OS cells treated with 50 µM riboflavin and 2 µM etoposide for 4 days were subjected to qPCR ( g and h ) and immunoblot analysis ( i ). Protein levels relative to γ-tubulin levels were quantified using NIH ImageJ software. qPCR and immunoblot data are representative of three ( f ) or two ( g – i ) independent experiments and values are shown as mean ± s.e.m. Original blots are presented in Supplementary Information. Statistical significance is shown using Student’s t -test analysis; * p < 0.05; *** p < 0.005; n.s., not significant ( p > 0.05).

Article Snippet: Anti-FLAG M2 monoclonal antibody (F3165) and anti-γ-tubulin antibody (T6557) were obtained from Sigma Aldrich; anti-Histone H3 (D1H2) antibody (#4499T), anti-Di-Methyl-Histone H3 (Lys4) (C64G9) antibody (#9725 T), anti-Mono-Methyl-Histone H3 (Lys9) (D1P5R) antibody (#14186), anti-Di-Methyl-Histone H3 (Lys9) (D85B4) antibody (#4658T), anti-Sirtuin-4 antibody (#69786) were from Cell Signaling Technology (Beverly, MA); anti-LSD1 antibody (ab69535), anti-H3K4me1 antibody (ab8895) were from Abcam; anti-Di-Methyl-p53 (K370) antibody (STJ90115) was from St John's Laboratory Ltd. (London, UK); anti-p21 antibody (sc-56335), anti-p53 antibody (sc-126), anti-Lamin B1 antibody (sc-6217) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Grb2 antibody (G16720) was from BD Biosciences (Franklin Lakes, NJ); anti-phospho-Histone H2A.X (Ser139) antibody (05-636) was from Merck Millipore (Burlington, MA); AP-conjugated anti-mouse antibody (S372B) and AP-conjugated anti-rabbit antibody (S373B) were from Promega (Madison, WI).

Techniques: Activation Assay, Immunofluorescence, Cell Fractionation, Western Blot, Software

Journal: Scientific Reports

Article Title: Lysine-specific demethylase 1 (LSD1) suppresses cellular senescence by riboflavin uptake-dependent demethylation activity

doi: 10.1038/s41598-025-91004-0

Figure Lengend Snippet: LSD1 is activated by SLC52A1 overexpression. ( a ) U2OS cells transfected with pcDNA3-HA-SLC52A1 and treated with 2 µM etoposide for 24 h were subjected to measurement of intracellular FAD levels. Data are mean ± s.d. (n = 4 independent cultures). ( b ) U2OS cells transfected with pcDNA3-HA-SLC52A1 and treated with 2 µM etoposide for 24 h were subjected to cell fractionation and measurement of FAD levels in each fraction. ( c ) SLC52A1-overexpressing U2OS cells treated with 2 µM etoposide and 100 nM ORY-1001 for 24 h were subjected to immunoblot analysis. Protein levels relative to histone H3 or p53 levels were quantified using NIH ImageJ software. ( d – f ) SLC52A1-overexpressing U2OS cells treated with 2 µM etoposide without ( d and e , left panels) or with ( d and e , right panels) 100 nM ORY-1001 for 24 h was examined by qPCR and immunoblot analysis ( f ). ( g , h ) SLC52A1-overexpressing U2OS cells treated with 2 µM etoposide and 100 nM ORY-1001 for 7 days were subjected to SA-β-gal ( g ) and EdU ( h ) assays. The percentage of SA-β-gal positive cells ( g ) and EdU positive cells ( h ) are shown. Data are mean ± s.d. (n = 3 independent cultures). Representative microscopic images are shown as Fig. c,d. ( i ) U2OS cells transfected with siRNA for SLC52A1 and treated with 2 µM etoposide for 2 days were subjected to qPCR. ( j ) SLC52A1-depleted U2OS cells treated with 2 µM etoposide for 2 days were subjected to immunoblot analysis. Protein levels relative to histone H3 or p53 levels were quantified using NIH ImageJ software. Original blots are presented in Supplementary Information. ( k – m ) SLC52A1-depleted U2OS cells treated with 2 µM etoposide for 2 days were subjected to qPCR ( k and l ) and immunoblot analysis ( m ). ( c – f ) qPCR and immunoblot data are representative of two independent experiments and values are shown as mean ± s.e.m. ( c , f , j , m ) Protein levels relative to histone H3 levels ( c , j ), p53 levels ( c , j ) or γ-tubulin levels ( f , m ) were quantified using NIH ImageJ software. Original blots are presented in Supplementary Information. Statistical significance is shown using Student’s t -test analysis; * p < 0.05; *** p < 0.005; n.s., not significant ( p > 0.05).

Article Snippet: Anti-FLAG M2 monoclonal antibody (F3165) and anti-γ-tubulin antibody (T6557) were obtained from Sigma Aldrich; anti-Histone H3 (D1H2) antibody (#4499T), anti-Di-Methyl-Histone H3 (Lys4) (C64G9) antibody (#9725 T), anti-Mono-Methyl-Histone H3 (Lys9) (D1P5R) antibody (#14186), anti-Di-Methyl-Histone H3 (Lys9) (D85B4) antibody (#4658T), anti-Sirtuin-4 antibody (#69786) were from Cell Signaling Technology (Beverly, MA); anti-LSD1 antibody (ab69535), anti-H3K4me1 antibody (ab8895) were from Abcam; anti-Di-Methyl-p53 (K370) antibody (STJ90115) was from St John's Laboratory Ltd. (London, UK); anti-p21 antibody (sc-56335), anti-p53 antibody (sc-126), anti-Lamin B1 antibody (sc-6217) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Grb2 antibody (G16720) was from BD Biosciences (Franklin Lakes, NJ); anti-phospho-Histone H2A.X (Ser139) antibody (05-636) was from Merck Millipore (Burlington, MA); AP-conjugated anti-mouse antibody (S372B) and AP-conjugated anti-rabbit antibody (S373B) were from Promega (Madison, WI).

Techniques: Over Expression, Transfection, Cell Fractionation, Western Blot, Software