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: Expressing, 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

Journal: The Journal of Clinical Investigation

Article Title: TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts

doi: 10.1172/JCI77321

Figure Lengend Snippet: (A) Representative immunofluorescence image of EdU incorporation in human fibroblasts infected with WT or mutant p16 jTALE-DNMT lentivirus. After 72 hours of infection, cells were incubated with EdU for 1 hour and stained for EdU. Cell nuclei are stained blue (DAPI), and EdU-positive nuclei are stained red. Images were taken at ×10 magnification (n = 7). (B) Percent EdU incorporation of cells infected with p16 jTALE-DNMT WT or mutant lentivirus, with (n = 4) or without (n = 7) coinfection of CMV-p16 lentivirus. Three random images were counted for each biological replicate (mean ± SEM). ***P < 0.001; NS, not significant; 2-tailed t test. (C) Population doubling time of human fibroblasts infected with p16 jTALE-DNMT WT or mutant lentivirus (mean ± SEM; n = 4). *P < 0.05, 2-tailed t test. (D) Transcript levels of cell cycle regulators in human fibroblasts transduced with WT p16 jTALE-DNMT lentivirus relative to mutant lentivirus. Expression was normalized to HPRT1 mRNA levels (mean ± SEM; n = 3). Two-tailed t test. (E) Average percent DNA methylation of CpGs at the nearest CpG island of cell cycle regulators evaluated in D. Average DNA methylation was measured by PCR amplification of bisulfite-converted genomic DNA followed by high-throughput sequencing (mean ± SEM; n = 3). **P < 0.01, ***P < 0.001, 2-tailed t test. CKI, cyclin-dependent kinase inhibitor.

Article Snippet: For p16 re-expression experiments, a lentiviral vector incorporating the human p16 cDNA clone under a CMV promoter was purchased from OriGene Technologies Inc. (catalog RC220937L1).

Techniques: Immunofluorescence, Infection, Mutagenesis, Incubation, Staining, Transduction, Expressing, Two Tailed Test, DNA Methylation Assay, Amplification, Next-Generation Sequencing

Journal: Frontiers in Oncology

Article Title: Positron Emission Tomography-Computed Tomography Parameters Predict Efficacy of Immunotherapy in Head and Neck Squamous Cell Carcinomas

doi: 10.3389/fonc.2021.728040

Figure Lengend Snippet: Baseline data of the enrolled 105 patients.

Article Snippet: From July 2013, immunohistochemical analysis of p16 (Wuhan Boster Biotechnology Co., Ltd., Wuhan, China) was performed for every patient with HNSCC.

Techniques:

Journal: Frontiers in Oncology

Article Title: Positron Emission Tomography-Computed Tomography Parameters Predict Efficacy of Immunotherapy in Head and Neck Squamous Cell Carcinomas

doi: 10.3389/fonc.2021.728040

Figure Lengend Snippet: Univariate analysis of the association between general variables and treatment response in the 105 patients.

Article Snippet: From July 2013, immunohistochemical analysis of p16 (Wuhan Boster Biotechnology Co., Ltd., Wuhan, China) was performed for every patient with HNSCC.

Techniques:

Journal: Frontiers in Oncology

Article Title: Positron Emission Tomography-Computed Tomography Parameters Predict Efficacy of Immunotherapy in Head and Neck Squamous Cell Carcinomas

doi: 10.3389/fonc.2021.728040

Figure Lengend Snippet: Multivariate analysis of the association between general variables and treatment response in the 105 patients.

Article Snippet: From July 2013, immunohistochemical analysis of p16 (Wuhan Boster Biotechnology Co., Ltd., Wuhan, China) was performed for every patient with HNSCC.

Techniques:

Journal: Frontiers in Oncology

Article Title: Positron Emission Tomography-Computed Tomography Parameters Predict Efficacy of Immunotherapy in Head and Neck Squamous Cell Carcinomas

doi: 10.3389/fonc.2021.728040

Figure Lengend Snippet: Univariate analysis and Cox model for progression-free survival (PFS) and overall survival (OS).

Article Snippet: From July 2013, immunohistochemical analysis of p16 (Wuhan Boster Biotechnology Co., Ltd., Wuhan, China) was performed for every patient with HNSCC.

Techniques: