Journal: Human molecular genetics

Article Title: Low p14ARF expression in neuroblastoma cells is associated with repressed histone mark status, and enforced expression induces growth arrest and apoptosis.

doi: 10.1093/hmg/ddt020

Figure Lengend Snippet: Figure 1. Genomic loss of p14ARF in neuroblastoma cell lines and tumors. (A) aCGH of chromosomal region 9p21.5 to 9p22.5, which includes the CDKN2A locus, in the SH-EP, LAN6, SK-HDN-33 and SK-N-AS neuroblastoma cell lines. Gains and losses are represented as increased or decreased log2 ratio plotted against the genomic position. Analyses were carried out on NCBI36/hg18 (hg18)-based 385 K arrays (blue) or on GRCh37/hg19 (hg19)-based 730 K arrays (yellow). Gene location according to hg18 and hg19 is shown above the plot. (B) PCR-based CDKN2A deletion analysis in neuroblastoma cell lines. Primers were located in p14ARF exon 1b (upper panel) or upstream of CDKN2A in a deletion-free region, as determined by aCGH (lower panel). NTC, non- template control. (C) Five-color FISH analysis of SK-N-AS and SH-SY5Y cells using probes localized to the CDKN2A locus (BAC clone RP11-149I2; Cy3) or upstream (fosmid G248P82386C9; Cy5, BAC RP11-441I5; Cy3.5) and downstream (fosmid G248P82984G12; FITC, BAC RP11-473O3; DEAC) of the gene. (D) aCGH for 193 primary neuroblastomas of whole chromosome 9 (left image) and the chromosomal region spanning 20.5 to 22.5 Mb on chromosome 9 (right image), which includes the CDKN2A locus (p14ARF and p16INK4a). Heterozygous (red) and homozygous (black) losses and gains (green) are illustrated by horizontal lines. Clinical parameters and molecular marker status are color-coded at the right of each image for each patient: outcome (black, death due to neuro- blastoma; grey, progression/relapse; white, event-free), MYCN status (black, amplified; white, non-amplified), 1p status (black, deleted; white, normal; grey, not available), 11q status (black, deleted; white, normal; grey, not available), age at diagnosis (black ≥1.5 years, white ,1.5 years) and disease stage (black, stage 4; grey, stage 3; yellow, stage 4s; white, stages 1 and 2).

Article Snippet: Immunoblotting was performed using antibodies against p14ARF (sc-53640, Santa Cruz, Santa Cruz, CA, USA), TP53 (sc-126, Santa Cruz), p21 (sc-6246, Santa Cruz), caspase 3 (610323, BD Transduction Laboratories, BD Biosciences, at U niversity of T asm ania L ibrary on M ay 21, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Heidelberg, Germany), cleaved caspase 3 (9661, Cell Signaling, Danvers, MA, USA), cleaved caspase 9 (9501, Cell Signaling), cleaved PARP-1 (AM30, Calbiochem, Merck Millipore, Darmstadt, Germany) and b-actin (Sigma-Aldrich, St Louis, MO, USA) as previously described (44). p14ARF promoter cloning and luciferase assays A 1476 bp fragment upstream of the p14ARF transcription start site was subloned into the pGL4.17 firefly luciferase reporter plasmid (Promega, Manheim, Germany) as previously published (45).

Techniques: Control, Marker, Biomarker Discovery

Journal: Human molecular genetics

Article Title: Low p14ARF expression in neuroblastoma cells is associated with repressed histone mark status, and enforced expression induces growth arrest and apoptosis.

doi: 10.1093/hmg/ddt020

Figure Lengend Snippet: Figure 2. p14ARF suppresses neuroblastoma cell growth. p14ARF-inducible SH-EP (A) and IMR5-75 (B) cells were treated with tetracycline (ON) or solvent control (OFF), then cell viability was analyzed via alamarBluew assays (AbD ser otec) at the indicated time points. Data are means of three replicates +- SD. LacZ-transfected cells were used as negative controls. WB, western blot. (C) p14ARF-inducible SH-EP cells were seeded at low density, treated with tetra- cycline or solvent control, then fixed and Giemsa stained after 7 days. Colonies were counted using the Quantity One software (Bio-Rad) to assess clonogenicity. Bars represent the means of three replicates +SD. LacZ-transfected cells were used as negative controls. (D) p14ARF-inducible IMR5-75 cells were treated with tetracycline (ON) or solvent control (OFF), then cultured 2 weeks in soft agar to assess anchorage-independent growth. Colonies were stained with crystal violet and counted using the Quantity One software. Bars represent the means of three replicates +SD. LacZ-transfected cells were used as negative controls.

Article Snippet: Immunoblotting was performed using antibodies against p14ARF (sc-53640, Santa Cruz, Santa Cruz, CA, USA), TP53 (sc-126, Santa Cruz), p21 (sc-6246, Santa Cruz), caspase 3 (610323, BD Transduction Laboratories, BD Biosciences, at U niversity of T asm ania L ibrary on M ay 21, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Heidelberg, Germany), cleaved caspase 3 (9661, Cell Signaling, Danvers, MA, USA), cleaved caspase 9 (9501, Cell Signaling), cleaved PARP-1 (AM30, Calbiochem, Merck Millipore, Darmstadt, Germany) and b-actin (Sigma-Aldrich, St Louis, MO, USA) as previously described (44). p14ARF promoter cloning and luciferase assays A 1476 bp fragment upstream of the p14ARF transcription start site was subloned into the pGL4.17 firefly luciferase reporter plasmid (Promega, Manheim, Germany) as previously published (45).

Techniques: Solvent, Control, Transfection, Western Blot, Staining, Software, Cell Culture

Journal: Human molecular genetics

Article Title: Low p14ARF expression in neuroblastoma cells is associated with repressed histone mark status, and enforced expression induces growth arrest and apoptosis.

doi: 10.1093/hmg/ddt020

Figure Lengend Snippet: Figure 3. p14ARF activates the TP53 pathway and induces G1 arrest or apoptosis in neuroblastoma cells. IMR5-75 cells stably transfected with a tetracycline-inducible expression vector for p14ARF were treated with tetracycline (ON) or solvent control (OFF), then cells were harvested at the indicated time points (h ¼ hours) for protein quantification via western blotting (A) or after 24 h for qRT–PCR-based mRNA quantification (B). Bars represent means of three replicates +SD. p14ARF-inducible SH-EP (C) and IMR5-75 (D) cells were treated with tetracycline (ON) or solvent control (OFF) for 24 h and then cell cycle profiles were determined using flow cytometry. LacZ-transfected cells served as negative controls. Data are the means of three replicates. (E) Cell cycle data for p14ARF-inducible IMR5-75 cells treated as in D for 48 h were analyzed for the percentage of cells in sub-G1/0. Cells were fixed and stained with Nicoletti buffer for flow cytometry. Data are the means of three replicates +SD. (F) p14ARF-inducible IMR5-75 cells were treated with tetracycline (ON) or solvent control (OFF) for 96 h, then harvested for western blotting to assess apoptotic pathway activity.

Article Snippet: Immunoblotting was performed using antibodies against p14ARF (sc-53640, Santa Cruz, Santa Cruz, CA, USA), TP53 (sc-126, Santa Cruz), p21 (sc-6246, Santa Cruz), caspase 3 (610323, BD Transduction Laboratories, BD Biosciences, at U niversity of T asm ania L ibrary on M ay 21, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Heidelberg, Germany), cleaved caspase 3 (9661, Cell Signaling, Danvers, MA, USA), cleaved caspase 9 (9501, Cell Signaling), cleaved PARP-1 (AM30, Calbiochem, Merck Millipore, Darmstadt, Germany) and b-actin (Sigma-Aldrich, St Louis, MO, USA) as previously described (44). p14ARF promoter cloning and luciferase assays A 1476 bp fragment upstream of the p14ARF transcription start site was subloned into the pGL4.17 firefly luciferase reporter plasmid (Promega, Manheim, Germany) as previously published (45).

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Solvent, Control, Western Blot, Quantitative RT-PCR, Cytometry, Staining, Activity Assay

Journal: Human molecular genetics

Article Title: Low p14ARF expression in neuroblastoma cells is associated with repressed histone mark status, and enforced expression induces growth arrest and apoptosis.

doi: 10.1093/hmg/ddt020

Figure Lengend Snippet: Figure 4. E2F1 and p14ARF expression is not correlated in primary neuroblastomas. E2F1 and p14ARF expression was analyzed in 68 neuroblastomas using qRT–PCR. Scatter plots show the correlation between E2F1 and p14ARF expression (A), and between E2F1 expression and the expression of two established E2F1 transcriptional targets in neuroblastoma, SKP2 (B) and BIRC5 (C), as positive controls. Pearson’s correlation coefficients (r) were calculated from the data using the R statistical package.

Article Snippet: Immunoblotting was performed using antibodies against p14ARF (sc-53640, Santa Cruz, Santa Cruz, CA, USA), TP53 (sc-126, Santa Cruz), p21 (sc-6246, Santa Cruz), caspase 3 (610323, BD Transduction Laboratories, BD Biosciences, at U niversity of T asm ania L ibrary on M ay 21, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Heidelberg, Germany), cleaved caspase 3 (9661, Cell Signaling, Danvers, MA, USA), cleaved caspase 9 (9501, Cell Signaling), cleaved PARP-1 (AM30, Calbiochem, Merck Millipore, Darmstadt, Germany) and b-actin (Sigma-Aldrich, St Louis, MO, USA) as previously described (44). p14ARF promoter cloning and luciferase assays A 1476 bp fragment upstream of the p14ARF transcription start site was subloned into the pGL4.17 firefly luciferase reporter plasmid (Promega, Manheim, Germany) as previously published (45).

Techniques: Expressing, Quantitative RT-PCR

Journal: Human molecular genetics

Article Title: Low p14ARF expression in neuroblastoma cells is associated with repressed histone mark status, and enforced expression induces growth arrest and apoptosis.

doi: 10.1093/hmg/ddt020

Figure Lengend Snippet: Figure 5. Epigenetic activity patterns reflect p14ARF expression in neuroblastoma cells. (A) ChIP-chip analyses were conducted on the 244K oligonucleotide promoter microarray for p14ARF in the neuroblastoma cell lines, Be(2)-C, IMR5-75, SH-SY5Y and SJNB-12. Genomic position and enrichment ratio are dis- played on the X and Y axes, respectively. Smoothed local enrichment ratios are shown for H3K4me3 (green), H3K27me3 (red) and H3K36me3 (blue). The red line indicates the median enrichment ratio for the histone marks versus the input, as calculated for all probes for chromosome 9. Arrows indicate transcriptional start site and direction of transcription. AB, antibody. (B) mChIP-qPCR was used to validate H3K4me3 and H3K27me3 enrichment in cell lines used for ChIP- chip. Bars represent the percentage of DNA recovered in the ChIP in relation to the amount of input DNA. The qRT–PCR assays for the p14ARF gene positions 1–3 are also indicated as vertical bars above the plots in section A. (C) Bars represent p14ARF expression (qRT–PCR) in the cell lines used for ChIP-chip ana- lyses. Data are the means of three replicates +SD. (D) Bars represent p14ARF promoter activity in the cell lines used for ChIP-chip analyses. Cells were trans- fected with the p14ARF promoter luciferase reporter (p14-pGL4.17-Luc), and promoter activity was analyzed after 24 h using the dual luciferase assay protocol (Promega).

Article Snippet: Immunoblotting was performed using antibodies against p14ARF (sc-53640, Santa Cruz, Santa Cruz, CA, USA), TP53 (sc-126, Santa Cruz), p21 (sc-6246, Santa Cruz), caspase 3 (610323, BD Transduction Laboratories, BD Biosciences, at U niversity of T asm ania L ibrary on M ay 21, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Heidelberg, Germany), cleaved caspase 3 (9661, Cell Signaling, Danvers, MA, USA), cleaved caspase 9 (9501, Cell Signaling), cleaved PARP-1 (AM30, Calbiochem, Merck Millipore, Darmstadt, Germany) and b-actin (Sigma-Aldrich, St Louis, MO, USA) as previously described (44). p14ARF promoter cloning and luciferase assays A 1476 bp fragment upstream of the p14ARF transcription start site was subloned into the pGL4.17 firefly luciferase reporter plasmid (Promega, Manheim, Germany) as previously published (45).

Techniques: Activity Assay, Expressing, ChIP-chip, Microarray, Quantitative RT-PCR, Luciferase

Journal: Autophagy

Article Title: Lactucopicrin promotes the autophagic degradation of MAP2K4/MKK4 by mediating CCDC50 palmitoylation to alleviate osteoarthritis progression

doi: 10.1080/15548627.2025.2601041

Figure Lengend Snippet: Lactucopicrin (LCP) inhibits OA progression and cartilage degeneration in mice . ( A ) Representative images of mouse articular cartilage stained with H&E, Safranin O/Fast Green; CC: calcified cartilage, and HC: hyaline cartilage. ( B ) Quantification of OA severity using the OARSI histological scoring system . ( C–E ) qRT-PCR analysis of core OA molecular markers: Col10a1/ColX (collagen, type x, alpha 1), Mmp13 , and Adamts5 . ( F ) WB analysis of OA markers: COL2A1 (collagen type ii, alpha 1) and ADAMTS5. ( G–I ) qRT-PCR detection of senescence-associated genes: Cdkn1a/p21, Cdkn2a/p16 INK4a , and Gadd45 . ( J ) WB analysis of senescence-related proteins: CDKN2A/p16 INK4a , CDKN1A/p21, TRP53/p53, and γH2AX. ( K-L ) Immunohistochemical staining of γH2AX in cartilage tissue and quantification of γH2AX-positive cells (%). Scale bar: 100 µm. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Anti-CDKN2A/p16 INK4A , Abclonal , A0262.

Techniques: Staining, Quantitative RT-PCR, Immunohistochemical staining

Journal: Autophagy

Article Title: Lactucopicrin promotes the autophagic degradation of MAP2K4/MKK4 by mediating CCDC50 palmitoylation to alleviate osteoarthritis progression

doi: 10.1080/15548627.2025.2601041

Figure Lengend Snippet: LCP inhibits chondrocyte senescence in vitro . ( A-K ) In the exogenous oxidative stress model induced by hydrogen peroxide (H 2 O 2 ), LCP treatment inhibited chondrocyte senescence in a dose-dependent manner, as evidenced by ( A-B ) SA-GLB1/β-gal staining and corresponding quantification, ( C-H ) downregulation of senescence-associated markers ( Cdkn2a/p16, Cdkn1a/p21, Gadd45 ) and SASP-related genes ( Il6, Il1α, Cxcl1 ) as detected by qRT-PCR, ( I ) decreased protein levels of CDKN2A/p16 INK4a , CDKN1A/p21, TRP53/p53, and γH2AX detected via WB, and ( J-K ) reduced CDKN2A/p16 INK4a expression shown by immunofluorescence staining and quantified fluorescence intensity. ( L-P ) In the natural senescence model induced by long-term in vitro culture (passage 7, P7), LCP also suppressed chondrocyte senescence, as demonstrated by: ( L-M ) SA-GLB1/β-gal staining and corresponding quantification, ( N ) reduced protein levels of CDKN2A/p16 INK4a , CDKN1A/p21, TRP53/p53, and γH2AX in WB analysis, and ( O-P ) decreased CDKN2A/p16 INK4a expression in immunofluorescence staining and its quantification. Scale bar: 100 µm. Data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. ns = not significant.

Article Snippet: Anti-CDKN2A/p16 INK4A , Abclonal , A0262.

Techniques: In Vitro, Staining, Quantitative RT-PCR, Expressing, Immunofluorescence, Fluorescence

Journal: Autophagy

Article Title: Lactucopicrin promotes the autophagic degradation of MAP2K4/MKK4 by mediating CCDC50 palmitoylation to alleviate osteoarthritis progression

doi: 10.1080/15548627.2025.2601041

Figure Lengend Snippet: LCP inhibits chondrocyte senescence via suppression of the MAPK/JNK signaling pathway . ( A ) Heatmap showing hierarchical clustering of differentially expressed genes in chondrocytes treated with DMSO or LCP. ( B ) KEGG pathway enrichment analysis of differentially expressed genes (DEGs) identified from RNA-seq, indicating that the MAPK/JNK signaling pathway was significantly enriched and is highlighted in red, suggesting its potential involvement in the anti-senescent effects of LCP. ( C ) WB analysis showing reduced levels of phosphorylated MAPK/JNK (p-MAPK/JNK) following LCP treatment in H 2 O 2 -induced senescent chondrocytes, with total MAPK/JNK and ACTB/β-actin as loading controls. ( D ) WB analysis of senescence-associated markers (CDKN2A/p16 INK4a , CDKN1A/p21, TRP53/p53, and γH2AX) in H 2 O 2 -induced chondrocytes, with or without LCP and/or the MAPK/JNK inhibitor JNK-IN-8 (2 μM). ( E-F ) Representative images of SA-GLB1/β-gal staining and corresponding quantification of SA-GLB1/β-gal-positive cells. ( G-H ) qRT-PCR analysis of senescence-related genes Gadd45 and Il6 showing reduced expression in response to LCP or JNK-IN-8 (2 μM) treatment. ( I-J ) Immunofluorescence staining of CDKN2A/p16 INK4a in H 2 O 2 -treated chondrocytes and corresponding quantification of fluorescence intensity. Scale bar: 100 µm. Data are expressed as mean ± SD. *** p < 0.001. ns = not significant.

Article Snippet: Anti-CDKN2A/p16 INK4A , Abclonal , A0262.

Techniques: RNA Sequencing, Staining, Quantitative RT-PCR, Expressing, Immunofluorescence, Fluorescence

Journal: Autophagy

Article Title: Lactucopicrin promotes the autophagic degradation of MAP2K4/MKK4 by mediating CCDC50 palmitoylation to alleviate osteoarthritis progression

doi: 10.1080/15548627.2025.2601041

Figure Lengend Snippet: LCP promotes MAP2K4/MKK4 degradation via the autophagy-lysosome pathway to inhibit MAPK/JNK signaling and chondrocyte senescence . ( A ) WB analysis of MAP3K7/TAK1, MAP3K11/MLK3, MAP2K4/MKK4, and MAP2K7/MKK7 in H 2 O 2 -induced chondrocytes treated with LCP, revealing that only MAP2K4/MKK4 protein levels were significantly downregulated. ( B ) qRT-PCR analysis showing no significant change in Map2k4/Mkk4 mRNA levels following LCP treatment in chondrocytes. ( C ) WB analysis of exogenously expressed MAP2K4/MKK4 in HEK293T cells treated with LCP. ( D ) qRT-PCR analysis of MAP2K4/MKK4 mRNA in HEK293T cells following LCP treatment. ( E-F ) Cycloheximide (CHX) chase assay showing accelerated degradation of endogenous MAP2K4/MKK4 in chondrocytes by LCP, with corresponding quantification. ( G-H ) CHX chase assay demonstrating enhanced degradation of FLAG-MAP2K4/MKK4 in HEK293T cells by LCP, with quantification. ( I ) Effects of MG132 (proteasome inhibitor, 10 μM), 3-MA (autophagy inhibitor, 10 mM), and chloroquine (CQ, lysosomal inhibitor, 50 μM) on LCP-induced MAP2K4/MKK4 degradation in chondrocytes were evaluated. ( J-K ) WB analysis of MAP2K4/MKK4 degradation in autophagy-deficient HEK293T cells ( ATG5 or BECN1/Beclin 1 knockout ) treated with LCP. ( L ) WB analysis of MAPK/JNK phosphorylation in H 2 O 2 -induced senescent chondrocytes with or without map2k4/mkk4 knockdown and LCP treatment. ( M-N ) SA-GLB1/β-gal staining and quantification in H 2 O 2 -induced chondrocyte senescence with or without map2k4/mkk4 silencing and LCP treatment. ( O-P ) qRT-PCR analysis of Gadd45 and Il6 expression, ( Q-R ) immunofluorescence staining and quantification of CDKN2A/p16 INK4a , and ( S ) WB analysis of senescence markers (CDKN2A/p16 INK4a , TRP53/p53, and γH2AX) under the same conditions. Scale bar: 100 µm. Data are expressed as mean ± SD. * p < 0.05, *** p < 0.001. ns = not significant.

Article Snippet: Anti-CDKN2A/p16 INK4A , Abclonal , A0262.

Techniques: Quantitative RT-PCR, Knock-Out, Phospho-proteomics, Knockdown, Staining, Expressing, Immunofluorescence

Journal: Autophagy

Article Title: Lactucopicrin promotes the autophagic degradation of MAP2K4/MKK4 by mediating CCDC50 palmitoylation to alleviate osteoarthritis progression

doi: 10.1080/15548627.2025.2601041

Figure Lengend Snippet: LCP targets ZDHHC4 to catalyze CCDC50 palmitoylation . ( A ) Knockdown of zdhhc4 abolished palmitoylation (palm) of endogenous CCDC50 in chondrocytes. ( B ) ZDHHC4 knockout blocked LCP-induced palmitoylation of CCDC50 in HEK293T cells. (C) The zdhhc4 knockdown blocked LCP-induced palmitoylation of CCDC50 in chondrocytes. ( D ) WB analysis showed that ZDHHC4 knockout blocked LCP-induced MAP2K4/MKK4 degradation in HEK293T cells. ( E ) WB analysis showed that zdhhc4 knockdown prevented LCP-induced MAP2K4/MKK4 degradation in H 2 O 2 -induced chondrocytes. ( F ) Co-IP analysis revealed that the ZDHHC4 knockout disrupted the interaction between exogenous CCDC50 and MAP2K4/MKK4. ( G ) Co-IP analysis demonstrated that knockdown of zdhhc4 impaired the interaction between endogenous CCDC50 and MAP2K4/MKK4 in chondrocytes treated with LCP. ( H ) WB analysis showed that zdhhc4 knockdown abolished the inhibitory effect of LCP on MAPK/JNK phosphorylation. ( I-J ) SA-GLB1/β-gal staining ( I ) and quantification ( J ) showed that zdhhc4 knockdown reversed the anti-senescent effects of LCP. ( K ) WB analysis revealed that zdhhc4 knockdown reversed the LCP-mediated downregulation of senescence markers CDKN2A/p16 INK4a and CDKN1A/p21 in chondrocytes. Scale bar: 100 μm. Data are expressed as mean ± SD. *** p < 0.001. ns = not significant.

Article Snippet: Anti-CDKN2A/p16 INK4A , Abclonal , A0262.

Techniques: Knockdown, Knock-Out, Co-Immunoprecipitation Assay, Phospho-proteomics, Staining

Journal: Advanced Healthcare Materials

Article Title: A Human Kidney Tubuloid Model of Repeated Cisplatin‐Induced Cellular Senescence and Fibrosis for Drug Screening

doi: 10.1002/adhm.202501795

Figure Lengend Snippet: Tubuloids exhibit cellular senescence and release inflammatory cytokines following repeated cisplatin treatment. A) Experimental design for repeated cisplatin treatment. B) Representative images of SA‐β‐GAL staining of tubuloids exposed to cisplatin (0.2 µg mL −1 ) 5 times repeatedly or control. Scale bars: 100 µm. C) Immunostaining of p16 in tubuloids exposed to cisplatin (2.0 µg mL −1 ) 5 times repeatedly or control. Scale bars: 100 µm. D) Western blotting of p16 in tubuloids exposed to cisplatin (0.2 and 2.0 µg mL −1 ) 5 times repeatedly or control. E) Statistical analysis of p16 expression (n = 4). F) Western blotting of p21 in tubuloids exposed to cisplatin (0.2 µg mL −1 ) 5 times repeatedly or control. G) Statistical analysis of p21 expression (n = 5). H) Western blotting of p53 and Lamin B1 in tubuloids exposed to cisplatin (2.0 µg mL −1 ) 5 times repeatedly or control. I) Statistical analysis of p53 expression (n = 3). J) Statistical analysis of Lamin B1 expression (n = 3). K) Cell Cycle Analysis in tubuloids exposed to cisplatin (0.2 µg mL −1 ) 5 times repeatedly or control. L) Immunostaining of an inflammatory cytokine, IL‐1β, in tubuloids exposed to cisplatin (2.0 µg mL −1 ) 5 times repeatedly or control. Scale bars: 100 µm. M,N) Quantitative PCR of IL‐1β (M) and IL‐6 (N) in tubuloids exposed to cisplatin (0.2 and 2.0 µg mL −1 ) 5 times repeatedly or control (n = 3). Statical analysis was applied to delta‐delta Ct value, not fold increase. All data are presented as means ± standard errors (SEs). Statistical significance is presented by * p < 0.05, ** p < 0.01.

Article Snippet: Primary antibodies were also used, including rabbit anti‐p16‐INK4A polyclonal antibody (1:200) (Proteintech), mouse anti‐p21 antibody (1:100) (sc‐6247, Santa Cruz Biotechnology, TX, USA), rabbit anti‐Lamin B1 polyclonal antibody (1:1000) (12987‐1‐AP, Proteintech), rabbit anti‐p53 polyclonal antibody (1:1000) (10442‐1‐AP, Proteintech), rabbit anti‐KIM‐1 antibody (1:400) (PA5‐20244, Thermo‐Fisher Scientific, MA, USA), rabbit anti‐caspase‐3 antibody (1:200) (Cell Signaling Technology), and rabbit anti‐Vimentin antibody (1:1000) (#5741, Cell Signaling Technology, MA, USA).

Techniques: Staining, Control, Immunostaining, Western Blot, Expressing, Cell Cycle Assay, Real-time Polymerase Chain Reaction

Journal: Cancer Research

Article Title: p14ARFExpression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

doi: 10.1158/0008-5472.can-03-1045

Figure Lengend Snippet: Fig. 1. Isolation of a p14ARF-inducible clone and detection by Western blot. A, Western blot using NB-200-111 polyclonal p14ARF antibody of 50 g of whole-cell lysate from HT-1080 parental cells, BT-549, or p14JM clone uninduced (DOX) or induced (DOX) for 24 h with 1 g/ml doxycycline (DOX). B, Western blot using NB-200-111 polyclonal p14ARF antibody of p14JM cell lysate from cells that were incubated with various amounts of doxycycline for 24 h. Ponceau S staining revealed equal transfer. Equal intensities of higher Mr nonspecific bands served as loading controls.

Article Snippet: The -tubulin antibody (B-5-1-2) was purchased from Sigma (St. Louis, MO), and the p14ARF antibody (NB 200-111) was purchased from Novus Biologicals (Littleton, CO). mRNA Analysis.

Techniques: Isolation, Western Blot, Incubation, Staining

Journal: Cancer Research

Article Title: p14ARFExpression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

doi: 10.1158/0008-5472.can-03-1045

Figure Lengend Snippet: Fig. 2. Effect of p14ARF on growth and cell cycle state of cells. A, observed number of p14JM or HT-1080 colonies (14 days in / doxycycline- containing medium) after staining with crystal vi- olet solution. The results represent averages of three separate experiments, and the error bars rep- resent the respective SD. B, Western blot using respective antibodies of whole-cell lysate from p14JM cells induced to express p14ARF for various times. C, trypan blue viability assay of p14JM cells or HT-1080 parental cells in the presence or ab- sence of 1 g/ml doxycycline. The average of three separate experiments and their respective SD are shown. D, fluorescence-activated cell sorter analy- sis after propidium iodide staining of cell cycle stage of p14JM and HT-1080 cells at 48 h post- induction. The change in percentage in the amount of induced cells compared with the uninduced ones for each state of the cell cycle is shown in the bar graph (Change in % [(% induced % unin- duced)/% uninduced] 100). The results are the average of three experiments. Error bars represent the SD.

Article Snippet: The -tubulin antibody (B-5-1-2) was purchased from Sigma (St. Louis, MO), and the p14ARF antibody (NB 200-111) was purchased from Novus Biologicals (Littleton, CO). mRNA Analysis.

Techniques: Staining, Western Blot, Viability Assay, Fluorescence

Journal: Cancer Research

Article Title: p14ARFExpression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

doi: 10.1158/0008-5472.can-03-1045

Figure Lengend Snippet: Fig. 3. p14ARF inhibition of DHFR protein by proteosomal degradation. A, 1, Western blot with each respective antibody of p14JM lysates of cells that were allowed to reach confluency, detached, replated, and incubated with (DOX) or without (DOX) 1 g/ml doxycycline (DOX) for various amounts of time. 2, as a control, HT-1080 parental cells treated (DOX) or not treated (DOX) with 1 g/ml doxycycline for 72 h were also subjected to Western analysis using DHFR and tubulin antibodies as described above. 3, the p14JM whole-cell lysates from cells incubated with (DOX) or without (DOX) 1 g/ml doxycycline for 72 h for the TS and TK Western blots were electrophoresed on a separate polyacrylamide gel and blotted simultaneously with the TK and TS primary antibody. B, relative DHFR mRNA levels using TaqMan quantitative reverse transcription-PCR analysis (probe and primers specific for DHFR cDNA and normalized by actin message levels) of p14JM cells that were allowed to reach confluency, replated, incubated in the presence (DOX) or in the absence (DOX) of 1 g/ml doxycycline, and allowed to grow for various amount of time. Average values for three experiments of DHFR/ACTIN ratios are shown with their respective SD. C, p14JM cells incubated with (DOX) or without (DOX) 1 g/ml doxycycline for 24 h were treated with 10 g/ml cycloheximide. Right panel, at the indicated times, cells were lysed and Western blot was performed using DHFR and -tubulin antibodies. Intensity of protein bands at different time points after cycloheximide addition relative to band intensity at the 0-h time point was plotted. Left panel, graph represents average and SDs of at least three separate experiments. D, cells were incubated with () or without () 1 g/ml doxycycline for 48 h, and 100 nM MG-132 or 0.1% DMSO was added for 12 h. The cells were then lysed, and Western blots were performed using the antibodies as described above.

Article Snippet: The -tubulin antibody (B-5-1-2) was purchased from Sigma (St. Louis, MO), and the p14ARF antibody (NB 200-111) was purchased from Novus Biologicals (Littleton, CO). mRNA Analysis.

Techniques: Inhibition, Western Blot, Incubation, Control, Reverse Transcription

Journal: Cancer Research

Article Title: p14ARFExpression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

doi: 10.1158/0008-5472.can-03-1045

Figure Lengend Snippet: Fig. 5. p14ARF alters the balance of thymidine supply. A, p14JM cells were incubated with (DOX) or without (DOX) 1 g/ml doxycycline (DOX) for 72 h, lysed, and protein extracted for TK activity assay (see “Materials and Methods”). TK activity, as measured by radioactivity per 4 g of whole-cell extract every 20 min for 1 h, was plotted against time. The results are the average of three experiments, and the error bars represent the re- spective SD. B, p14JM cells were plated in the presence (DOX) or absence (DOX) of 1 g/ml doxycycline. After 72 h, cells were collected, and extracts were analyzed by high performance liquid chromatography, as described in “Materials and Methods.” The collected samples were then counted for radioactivity and converted to cpm/ 3.0 106 cells. The average and SD of three experiments are shown. C, p14JM cells were incu- bated with (DOX) or without (DOX) 1 g/ml doxycycline for 48 h and then incubated with [5-3H]deoxyuridine for 0, 15, 30, and 45 min, and in situ thymidylate synthesis activity was calcu- lated as described in “Materials and Methods.” In situ thymidylate synthesis activity is expressed as cpm/min/107 cells. The average and SD of four experiments are shown. D, p14JM cells (1,000) were plated in each well of a six-well plate with (DOX) or without (DOX) 1 g/ml doxycycline, with or without TP-treated FBS. After 14 days, the plates were stained with crystal violet solution and colonies counted. The average of three experiments and their respective SDs are shown.

Article Snippet: The -tubulin antibody (B-5-1-2) was purchased from Sigma (St. Louis, MO), and the p14ARF antibody (NB 200-111) was purchased from Novus Biologicals (Littleton, CO). mRNA Analysis.

Techniques: Incubation, Activity Assay, Radioactivity, High Performance Liquid Chromatography, In Situ, Staining

Journal: Journal of Translational Medicine

Article Title: Antitumor efficacy of a recombinant adenovirus encoding endostatin combined with an E1B55KD-deficient adenovirus in gastric cancer cells

doi: 10.1186/1479-5876-11-257

Figure Lengend Snippet: The sequence of primers used in this study

Article Snippet: These gene fragments were amplified with the corresponding primers (Table ) and dl 1520 genomic DNA as the template. pCD-p14 ARF was constructed by subcloning full length of p14 ARF cDNA fragment (clone IMAGE: 6173590) into pcDNA3.1(+) vector. p14 ARF siRNA (sc-37622) was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA).

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Cloning

Journal: Journal of Translational Medicine

Article Title: Antitumor efficacy of a recombinant adenovirus encoding endostatin combined with an E1B55KD-deficient adenovirus in gastric cancer cells

doi: 10.1186/1479-5876-11-257

Figure Lengend Snippet: dl 1520 inhibited the proliferation of GC cells by selectively replicating in and destroying the cancer cells. A, B) The efficiencies of infection and replication of dl 1520 in GC and normal cells. The infection efficiency (A) of dl 1520 was shown as the dl 1520 DNA copy number relative to β-actin at 0 hours after infection. The replication efficiency (B) of dl 1520 was presented as the fold of the dl 1520 DNA copy number at indicated time relative to that at 0 hours post-infection (One-way ANOVA, *p<0.05, **p<0.01 compared to that at 0 hours post-infection). C~E) The cytopathic effect (CPE) of dl 1520 on GC cells. MTT cell proliferation assays were used to analyze the CPE of dl 1520 on AGS (C) , MGc80-3 (D) and SGC-7901 (E) GC cells. The results are presented as the percentages of viable cells related to the negative control (one-way ANOVA, *p<0.05, **p<0.01 compared to that at 0 MOIs). F) Western blotting analysis of protein levels of p14 ARF in GC cells (Actin was used as the internal control). G) Quantitative RT-PCR analysis of the relative mRNA levels of p14 ARF and p53 (normalized to that of GAPDH). H) The replication of dl 1520 after modifying the p14 ARF levels by knockdown or overexpression. AGS cells were transfected with pCD-p14 ARF plasmid (pcDNA3.1(+) as a negative control), and MGc80-3 cells were transfected with p14 ARF siRNA (si-p14 ARF ) (scrambled siRNA as a negative control). The cells were analyzed p14 ARF expression by Western blotting after 48 hours post-transfection (H upper). Or the cells were infected with 10 MOIs of dl 1520 after 24 hours post-transfection, and dl 1520 DNA copy numbers were analyzed in GC cells after 48 hours post-infection (normalized against that at 0 hours) (H lower). (Student’s t test, *p< 0.05 compared with their respective control).

Article Snippet: These gene fragments were amplified with the corresponding primers (Table ) and dl 1520 genomic DNA as the template. pCD-p14 ARF was constructed by subcloning full length of p14 ARF cDNA fragment (clone IMAGE: 6173590) into pcDNA3.1(+) vector. p14 ARF siRNA (sc-37622) was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA).

Techniques: Infection, MTT Cell Proliferation, Negative Control, Western Blot, Control, Quantitative RT-PCR, Knockdown, Over Expression, Transfection, Plasmid Preparation, Expressing

Journal: Aging Cell

Article Title: FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

doi: 10.1111/acel.13226

Figure Lengend Snippet: FOXO3 and p16 INK4a oppositely modulate the vulnerability of human HD NSCs. Significance was tested using two‐way ANOVA (panels a–c), paired t test (panels d) and Mann‐Whitney test (panel g). ns: not significant. (A) Human HD NSCs show reduced rates of cell growth. Data are mean ± SEM . (b) Reducing FOXO3 does not alter the growth of C116 NSCs (left panel) and strongly reduces the growth of HD NSCs (right panel), with no change detected in HTT mRNA levels (see Figure , left panel). Data are mean ± SEM . (c) Reducing p16 INK4a slightly increases the growth of C116 (left panel) and HD (right panel) NSCs. Reducing p16 INK4a does not alter HTT mRNA levels in HD NSCs (see Figure , right panel). Data are mean ± SEM . (d) Reducing FOXO3 increases the mortality of HD NSCs with no effect detected in C116 NSCs (left: * p < .05). Reducing p16 INK4a decreases the mortality of HD NSCs with no effect detected in C116 NSCs (right: * p < .05). (e) Lenti‐myc‐ p16 INK4a transduction promotes nuclear release of HMGB1 in cytoplasm of HD and corrected (C116) MSNs. HD and C116 MSNs transduced for 4 days with lenti‐myc‐ p16 INK4a (red) were immunostained with HMGB1 (green). NT: transduction without myc‐ p16 INK4a . HMGB1 co‐localizes with the nucleus (DAPI), with low level in cytoplasm. The transduction with lenti‐myc‐ p16 INK4a significantly relocates HMGB1 into cytoplasm of HD and C116 MSNs (arrowhead). Scale bars: 100 µm. (f) Upper panel: The quantification of cytoplasmic HMGB1 pixel intensity shows a significant increase of nuclear HMGB1 release in HD vs. C116 MSNs and in HD vs. C116 MSNs following p16 INK4a overexpression (Wilcoxon ranked‐sum test: C116‐ p16 INK4a vs. HD‐ p16 INK4a , p = 6.1e‐22; C116‐ p16 INK4a vs. HD‐NT, p = 3.4e‐06; C116‐ p16 INK4a vs. C116‐NT, p = 1.8e‐23; HD‐ p16 INK4a vs. HD‐NT, p = 2.3e‐7; HD‐NT vs. C116‐NT, p = 5.6e‐37). Lower panel: data normalized against Myc‐p16 INK4a levels using the ratio (sum of HMGB1 intensity in ‘cells’ MOI 1/number of cells detected in cells MOI 1)/(sum of myc‐tag intensity in ‘cells’ MOI 1/number of cells detected in ‘cells’ MOI 1). The ratios show that HMGB1 relocalization is CAG‐repeat‐length‐dependent. C116‐p16 INK4a : 1604 cells; C116‐NT: 1403 cells; HD‐p16 INK4a : 879 cells; HD‐NT: 1792 cells

Article Snippet: Lentivirus transduction was performed with myc‐p16 INK4a from Origene (RC220937L1V) using a multiplicity of infection (MOI) of 1 and transduction without myc‐p16 INK4a as a control.

Techniques: MANN-WHITNEY, Transduction, Over Expression

Journal: Aging Cell

Article Title: FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

doi: 10.1111/acel.13226

Figure Lengend Snippet: Gene expression analyses in human NSCs. The mRNA levels are normalized to cells treated with nontargeting control (NTC) siRNAs (siRNA tests) or to C116 cells or cells without growth factor (GF) deprivation (other experiments). ns, not significant. (a) ETS2 mRNA levels are increased by FOXO3 reduction in HD NSCs subjected to GF deprivation with no effect detected in basal conditions nor in normal HTT cells (left panel: * p < .05). ETS2 mRNA levels are decreased in HD NSCs (middle panel: ** p < .01). GF deprivation does not change ETS2 mRNA levels in C116 and decreases ETS2 mRNA levels in HD NSCs (right panel: * p < .05). (b) p16 INK4a mRNA levels are decreased by ETS2 reduction in HD NSCs in basal conditions and in cells subjected to stress with no effect detected in normal HTT cells (left panel: * p < .05, ** p < .01). p16 INK4a mRNA levels are increased in HD NSCs (middle left panel: *** p < .001). GF deprivation does not change p16 INK4a mRNA levels in C116 NSCs and decrease p16 INK4a mRNA levels in HD NSCs (middle right panel: * p < .05). p16 INK4a mRNA levels tend to be increased by FOXO3 knockdown in HD NSCs subjected to GF deprivation (right panel: not significant with p = .0736). (c) ETS2 and p16 INK4a mRNA levels are decreased by overexpression of FOXO3, but not that of FOXO3‐TM, in human HD NSCs subjected to GF deprivation. The mRNA levels are normalized to cells treated with empty vector. * p < .05 and ** p < .01. (d) p16 INK4a mRNA levels are decreased by ETS1 reduction in C116 NSCs in basal conditions and in HD NSCs in both basal and stress conditions (upper panel: * p < .05, ** p < .01). ETS1 mRNA levels are unchanged in HD compared with C116 NSCs (lower left panel). GF deprivation does not change ETS1 mRNA levels in C116 NSCs and decreases ETS1 mRNA levels in HD NSCs (lower right panel: * p < .05). (d) Working model for effect of FOXO3 target reprogramming on the ETS2‐p16 INK4a pathway

Article Snippet: Lentivirus transduction was performed with myc‐p16 INK4a from Origene (RC220937L1V) using a multiplicity of infection (MOI) of 1 and transduction without myc‐p16 INK4a as a control.

Techniques: Gene Expression, Control, Knockdown, Over Expression, Plasmid Preparation

Journal: Aging Cell

Article Title: FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

doi: 10.1111/acel.13226

Figure Lengend Snippet: Human HD prepatterned NSCs show increase of p16 INK4a and of SA‐β‐gal activity. (a) p16 INK4a mRNA levels are increased in HD prepatterned NSCs. Data are mean ± SD (** p < .01), N = 3. (b) Representative images for modest p16 INK4a increase in HD NSCs. Scale bar in all panels: 100 µm. (c) Quantification of nuclear p16 INK4a pixel intensity for 532 C116‐NSCs and 1000 HD NSCs. Data are mean ± SD (** p < .01). (d) Representative images for increase of SA‐ß‐gal activity in HD NSCs. Scale bar in all panels: 200 µm. (e) Quantification of SA‐ß‐gal activity for 547 C116‐NSCs and 645 HD NSCs. Data are mean ± SD (**** p < .0001). (f) Frequency distribution of SA‐ß‐gal signals for data shown in panel (e)

Article Snippet: Lentivirus transduction was performed with myc‐p16 INK4a from Origene (RC220937L1V) using a multiplicity of infection (MOI) of 1 and transduction without myc‐p16 INK4a as a control.

Techniques: Activity Assay

Journal: Aging Cell

Article Title: FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

doi: 10.1111/acel.13226

Figure Lengend Snippet: p16 INK4a expression is elevated in human HD MSNs. (a) Representative images of human NSC‐derived MSNs using defined enhanced media (Synaptojuice medium). (b) RT‐PCR analysis of p16 INK4a , FOXO3, and Ryk in C116 and HD MSNs reveals modest increase of FOXO3 mRNA levels and robust increase of p16 INK4a and Ryk mRNA levels in HD MSNs. Data are mean ± SD (* p < .05, *** p < .001). N = 3. (c) Immunofluorescence analysis reveals dramatic increase of p16 INK4a in HD MSNs. Scale bar in all panels: 100 µm. (d) Quantification of nuclear p16 INK4a pixel intensity for N = 596 C116 NSCs and N = 609 HD NSCs. Data are mean ± SD (**** p < .0001). (e) Frequency distribution of nuclear p16 INK4a signals for data shown in Panel (d). (e) RT‐PCR analysis of CDKN2AIP, MMP3, SELL, IGFBP7, EST1, and EST2 show increased mRNA levels in HD MSNs compared with C116 MSNs. Data are mean ± SD (* p < .05, ** p < .01, *** p < .001). N = 3

Article Snippet: Lentivirus transduction was performed with myc‐p16 INK4a from Origene (RC220937L1V) using a multiplicity of infection (MOI) of 1 and transduction without myc‐p16 INK4a as a control.

Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence