Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 1 The expression of DEF6 is increased in hypertrophic hearts and cardiomyocytes. A mRNA levels of DEF6 in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 5). B Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 4). C mRNA levels of DEF6 in NRCMs administrated with PBS or 24 h of PE (50 μM) (n = 5). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in NRCMs administrated with PBS or 24 h of PE (n = 4). *P < 0.05, ***P < 0.001 vs. sham or PBS. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Student’s t test (A, C) or Mann–Whitney U test (B, D).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Expressing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 2 Ablation of DEF6 mitigates TAC-induced cardiac hypertrophy. A Strategy to construct KO mice and the sequencing results of WT and KO mice. B Protein levels of cardiac DEF6 in WT and KO mice (n = 5). C Comparisons of HW, HW/BW, LW/BW, and HW/TL in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). D Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area from groups (n = 6). E RT-PCR analyses of the hypertrophic markers in the indicated groups (n = 4). F–H Comparisons of the LVEDd, LVESd, LVPWd, FS, and EF values in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). I Left, PSR-stained LV sections in WT and KO mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). J RT-PCR analysis of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by One-way ANOVA (C, D, F–I) or Kruskal–Wallis test (E, J).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Construct, Sequencing, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 3 Overexpression of DEF6 aggravates TAC-induced cardiac hypertrophy. A mmunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the hearts of mice injected with AAV9-vector or AAV9-DEF6 (n = 4). B Comparisons of HW, HW/BW, LW/ BW, and HW/TL in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). C Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area between groups (n = 6). D RT-PCR analyses of the hypertrophic markers in each groups (n = 4). E–G Comparisons of LVEDd, LVESd, LVPWd, FS, and EF in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). H Left, PSR-stained LV sections in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). I RT-PCR analyses of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AAV9- vector or AAV9-vector sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AAV9-vector TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by Mann–Whitney U test (A) or One-way ANOVA (B, C, E–H) or Kruskal–Wallis test (D, I).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Over Expression, Expressing, Injection, Plasmid Preparation, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 4 DEF6 exacerbates PE-induced cardiomyocyte hypertrophy. A Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCMs infected with AdshRNA or AdshDEF6 (n = 3). B Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with AdshRNA or AdshDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). C RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCVs infected with Advector or AdDEF6 (n = 3). E Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with Advector or AdDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). F RT-PCR analyses of the hypertrophic markers in cultured NRCVs of each groups (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AdshRNA or AdshRNA PBS or Advector or Advector PBS, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AdshRNA PE or Advector PE. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Mann–Whitney U test (A, D) or One-way ANOVA (B, E) or Kruskal–Wallis test (C, F).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Western Blot, Expressing, Cell Culture, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 6 Prohypertrophic effect of DEF6 depends on Rac1-MEK-ERK signaling. A Co-IP of DEF6 was performed with anti-Flag and probed by Western blots with anti-HA (left); Co-IP of Rac1 was performed with anti-HA and probed by Western blots with anti-Flag (right). B In vitro GST pulldown assays for the interaction of purified Flag-DEF6 and GST-HA-Rac1 (left), as well as Flag-Rac1 and GST-HA-DEF6 (right). C The activity of Rac1 changes in the same direction as the DEF6 expression. D Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with Advector or AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM) (n = 3). E Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with Advector and AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM). (n ≥48 cells per group). F RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). G Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with AdshRNA or AdshDEF6 and with Adcontrol or AdRac1(G12V) under PE 24 h of PE treatment (50 μM) (n = 3). H Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with the indicated adenovirus and administrated with 24 h of PE (50 μM) (n ≥48 cells per group). I RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). •P < 0.05, P < 0.01,•P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, *P < 0.05, **P < 0.01, ***P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, ###P < 0.001 vs. AdDEF6 PBS PE or AdshDEF6 Adcontrol PE, and n.s. indicates no significance. Data are displayed as mean ± SD. Statistical analysis were conducted by Kruskal–Wallis test.

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Vitro, Activity Assay, Expressing, Cell Culture, Infection, Staining, Comparison, Reverse Transcription Polymerase Chain Reaction

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Mmu-miR-151-5p cleaves E2f6 in the absence of a seed match. (a) Genomic locus of mmu-miR-151 encoded by a LINE2 repeat element. (b) Schematic of the binding site of mmu-miR-151-5p to E2f6 3′UTR. (c) Dual-luciferase reporter assay for the wildtype E2f6 3′UTR (wt) or other mutants (per 5p and mut 5p) in presence of miR-151-5p overexpression (sh-151-5p). (d) Western blot for E2f6in presence of sh-151-5p or a scrambled control (sh-scr). Actin serves as a loading control. Uncropped blot in . (e) E2f6 qPCR on miR-151-5p overexpression. Error bars, s.e.m. (n = 3 replicates). (f) Dual-luciferase reporter assay for E2f6 3′UTR or control ( Sox4 3′UTR) in presence of an increasing dosage of miR-151-5p inhibitor. (g) Dual-luciferase reporter assay in Ago2 −/− cells for E2f6 3′UTR in presence of sh-151-5p and a functional copy of Ago2 or cleavage deficient Ago2 (D597A) or Ago1. (h) 5′-RACE of E2f6 . Arrowhead in the E2f6 3′UTR sequence (schematic) indicates the 5′ end of majority of E2f6 cleavage products in mouse lung tissue. Agarose gel showing E2f6 cleaved products (shown by an asterisk) is shown in the top gel (uncropped gel in ). The bottom gel serves as a RACE reaction control to detect the presence of E2f6 and ARHGDIA cDNAs. (c,f, g) For reporter assays, normalization was done with respect to sh-scr. Error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates), ns denotes not significant,*** P = 0.001 by two-tailed Student′s t test.

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Binding Assay, Luciferase, Reporter Assay, Over Expression, Western Blot, Control, Functional Assay, Sequencing, Agarose Gel Electrophoresis, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: miR-151-3p suppresses E2f6 expression by binding to E2f6 3′UTR adjacent to where miR-151-5p binds. (a) Schematic of a putative binding site of the miR-151-3p to the E2f6 3′UTR region adjacent to where 5p strand binds, in both mice and humans. The seed regions (nucleotides 2-8) are indicated for both the 5p and 3p arms. (b) Dual-luciferase reporter assay for the wildtype E2f6 3′UTR (wt) or other mutants (mut 3p and seed 3p as shown in the schematic) in presence of a miR-151-3p overexpression (sh-151-3p) or a scrambled control(sh-scr). A reporter construct with deletion of the entire miR-151-3p binding site in E2f6 3′UTR was also included. (c) Dual-luciferase reporter assay in wildtype MEF and Ago2 −/− cells for E2f6 3′UTR in presence of sh-151-3p. For comparision, dual-luciferase reporter assay for E2f6 3′UTR in presence of sh-151-5p is also shown. (b,c) For reporter assays, normalization was done with respect to sh-scr. Error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates).

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Over Expression, Control, Construct

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Precursor miR-151 competes with the mature miR-151-5p for binding to E2f6 3′UTR. (a) Thermodynamics of pre-miR-151 binding to E2f6 . (b) Schematic of the stem-loop structure of the pre-miR-151 with the 5p arm (blue), 3p arm (purple) and two adenosines (green) substituted to guanosines (orange). (c) Northern analysis of miR-151 processing from pre-miR-151 overexpression plasmid (pEZX-151) or the double mutant form of pre-miR-151 (pEZX-DM). Let-7a serves as a loading control. (d) Dual-luciferase reporter assay for E2f6 3′UTR in presence of only the mature miR-151-5p (sh-miR-151-5p), or both the pre-miR-151 and mature miR-151-5p (pEZX-151 and pEZX-DM). (e) Schematic of the binding site of pre-miR-151 in E2f6 3′UTR and its modifications ( E2f6 3p del and E2f6 3p-5p swap). (f) In-vitro gel shift assay with radiolabed (denoted by an asterisk) synthetic pre-miR-151(I) or a control pre-miR-122 and increasing molar concentrations of wildtype E2f6 3′UTR (1, 10 and 100 nM) or its modified forms. (g) Dual-luciferase analysis for E2f6 3′UTR (wt), 3p del or 3p-5p swap reporters with pEZX-151 or pEZX-DM. (h) In-vitro gel shift assay of E2f6 3′UTR bound to miR-151-5p with increasing molar concentrations of a synthetic pre-miR-151 or a control pre-miR-122. Bands below the blue star and orange star represent radiolabeled pre-miR-151 and pre-miR-122 respectively. “*” denotes radiolabeled oligos. (d, g) For reporter assays, normalization was done with respect to a scrambled control (sh-scr). Error bars, s.e.m. (n = 3 biological replicates, each with 3 technical replicates). * P = 0.05, ** P = 0.01 by two-tailed Student′s t test.

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Binding Assay, Northern Blot, Over Expression, Plasmid Preparation, Mutagenesis, Control, Luciferase, Reporter Assay, In Vitro, Gel Shift, Modification, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Pre-miR-151 binds to E2f6 in vivo and may protect the E2f6 transcript in quiescent tissues. (a) Schematic of the ChIRP method used to pull-down E2f6 mRNA from mouse brain. Quantitative PCR of (b) E2f6 mRNA and a control Ctdnep1 mRNA, (c) pre-miR-151 and a control pre-miR-124, (d) mature miR-151-5p and a control miR-124, pulled down by the biotinylated tiling oligonucleotides against the E2f6 3′UTR or a control lacZ mRNA. Quantitative PCR of (e) E2f6 mRNA, (f) pri-miR-151, (g) mature miR-151-5p in quiescent and non-quiescent tissues. In each case ( e — g ), the data are presented as fold induction after normalization to the liver sample (value = 1). (h) Northern analysis of miR-151 processing in various tissues. The blot on the left was probed with a LNA probe against mature miR-151-5p as shown by the schematic above the blot. The primary or intermediate product in the miR-151 biogenesis pathway is indicated by an arrowhead (→) and the mature miR-151-5p is indicated by a circle (○). U6 serves as a loading control. The blot on the right was probed (sequence is provided in ) for a region just outside the annotated stem loop structure of mmu-miR-151 (as shown by the schematic above the blot). (i) Quantitative PCR analyses of E2f6 , pri-miR-151 and miR-151-5pduring differentiation of muscle cells (C2C12) ( b — g, i ) For qPCR data, error bars, s.e.m. (n = 2 biological replicates, each with 3 technical replicates).

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: In Vivo, Real-time Polymerase Chain Reaction, Control, Northern Blot, Sequencing

Journal: EMBO Molecular Medicine

Article Title: Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

doi: 10.15252/emmm.201708483

Figure Lengend Snippet: Variant identification pipeline for WES in P1 and P2 (see also Appendix Tables S1 and ). Unrelated familial trees showing affected children in black and healthy individuals in white. Sanger sequencing of the region corresponding to mutations in ALPI in both families. Location of ALPI on chromosome 2 and diagrams featuring ALPI gene with its nine exons and ALPI protein with its N‐terminal signal peptide, phosphatase domain and C‐terminal recognition signal for the transamidase complex (GPI‐anchor attachment site), which removes the GPI signal sequence and replaces it by a preformed GPI precursor glycolipid. Arrows point to mutations identified in P1 and P2. Numbers indicate amino acid position.

Article Snippet: Total RNA (5 μg) was reverse transcribed using M‐MLV reverse transcriptase (Invitrogen). qRT–PCR was performed with an Assays‐on‐Demand probe (Applied Biosystems, Thermo Fisher Scientific) specific for ALPI‐FAM (Hs00357579_g1) and RPLPO‐FAM (Large Ribosomal Protein; Hs99999902_m1), which was used for normalization. qPCR was performed with a ABI PRISM 7900 (Applied Biosystems).

Techniques: Variant Assay, Sequencing, Recognition Signal

Journal: EMBO Molecular Medicine

Article Title: Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

doi: 10.15252/emmm.201708483

Figure Lengend Snippet: Confirmatory Sanger sequencing for siblings in Family 2. Multiple alignments of ALPI orthologs from different species using the Clustal Omega software. Residues altered by mutations in P1 and P2 are boxed in red. Conserved residues are indicated as follow: full identity (*), similar characteristics (:) (> 0.5 in the Gonnet PAM 250 matrix), weak similarities (.) (< 0.5 in the Gonnet PAM 250 matrix).

Article Snippet: Total RNA (5 μg) was reverse transcribed using M‐MLV reverse transcriptase (Invitrogen). qRT–PCR was performed with an Assays‐on‐Demand probe (Applied Biosystems, Thermo Fisher Scientific) specific for ALPI‐FAM (Hs00357579_g1) and RPLPO‐FAM (Large Ribosomal Protein; Hs99999902_m1), which was used for normalization. qPCR was performed with a ABI PRISM 7900 (Applied Biosystems).

Techniques: Sequencing, Software

Journal: EMBO Molecular Medicine

Article Title: Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

doi: 10.15252/emmm.201708483

Figure Lengend Snippet: A, B Analysis with ALPI antibody of HEK293T cells (lacking endogenous ALPI expression) after mock transduction or transduction with lentiviral particles encoding indicated mutants. (A) Western blot of whole‐cell lysates. (B) Flow cytometry analysis of surface‐labelled cells. C–E 3D modelling of ALPI calculated on the basis of the crystal structure of ALPP. ALPI is represented with one monomer in yellow, and the second in blue. Alanine 350, alanine 360 and catalytic Ser111 are represented in stick in violet, cyan and red, respectively. Deletion of the C‐terminal from Gln439 is shown in pink. Top and bottom views shown in (D) correspond to a 90° rotation. F pNPP phosphatase activity of WT and ALPI mutants measured by the OD405 nm of the reaction supernatants. n = 5, error bars indicate SD. ** P = 0.0079, nonparametric, unpaired two‐tailed Mann–Whitney test. G IL‐8 inhibition of LPS‐induced transcription in THP1 cells by WT and ALPI mutants. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; EV, empty vector. n = 3, error bars indicate SD. ** P = 0.022, nonparametric, unpaired two‐tailed Mann–Whitney test. Source data are available online for this figure.

Article Snippet: Total RNA (5 μg) was reverse transcribed using M‐MLV reverse transcriptase (Invitrogen). qRT–PCR was performed with an Assays‐on‐Demand probe (Applied Biosystems, Thermo Fisher Scientific) specific for ALPI‐FAM (Hs00357579_g1) and RPLPO‐FAM (Large Ribosomal Protein; Hs99999902_m1), which was used for normalization. qPCR was performed with a ABI PRISM 7900 (Applied Biosystems).

Techniques: Expressing, Transduction, Western Blot, Flow Cytometry, Activity Assay, Two Tailed Test, MANN-WHITNEY, Inhibition, Plasmid Preparation

Journal: EMBO Molecular Medicine

Article Title: Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

doi: 10.15252/emmm.201708483

Figure Lengend Snippet: WT ALPI and mutant expression by qRT‐PCR. Data are representative of three experiments and are expressed as mean ± SD.

Article Snippet: Total RNA (5 μg) was reverse transcribed using M‐MLV reverse transcriptase (Invitrogen). qRT–PCR was performed with an Assays‐on‐Demand probe (Applied Biosystems, Thermo Fisher Scientific) specific for ALPI‐FAM (Hs00357579_g1) and RPLPO‐FAM (Large Ribosomal Protein; Hs99999902_m1), which was used for normalization. qPCR was performed with a ABI PRISM 7900 (Applied Biosystems).

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

doi: 10.15252/emmm.201708483

Figure Lengend Snippet: A, B Immunofluorescence microscopy of duodenum (A) and ileum (B) sections from Patient 1 (A) and Patient 2 (B) compared to normal or IBD controls. Sections were stained with DAPI (4′,6‐diamidino‐2‐phenylindole) for DNA (blue) and antibodies against ALPI alone (second column, red) or ALPI and E‐cadherin (green) (first column, merge staining). Magnification 20× (scale bar = 100 μm). C Effect of AP inhibitors (L‐phe, L‐arg) on AP activity in stools of non‐inflamed controls ( n = 20). Data are expressed as mean ± SD. **** P < 0.0001, nonparametric, unpaired two‐tailed Mann–Whitney test. D ALPI activity in stools of non‐inflamed ( n = 20) or IBD controls ( n = 14) and of P1. AP values are expressed as units of ALPI/g stool and as medians with interquartiles; * P = 0.0122, nonparametric, unpaired two‐tailed Mann–Whitney test.

Article Snippet: Total RNA (5 μg) was reverse transcribed using M‐MLV reverse transcriptase (Invitrogen). qRT–PCR was performed with an Assays‐on‐Demand probe (Applied Biosystems, Thermo Fisher Scientific) specific for ALPI‐FAM (Hs00357579_g1) and RPLPO‐FAM (Large Ribosomal Protein; Hs99999902_m1), which was used for normalization. qPCR was performed with a ABI PRISM 7900 (Applied Biosystems).

Techniques: Immunofluorescence, Microscopy, Staining, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

doi: 10.15252/emmm.201708483

Figure Lengend Snippet: A, B Immunofluorescence microscopy of duodenum (A) and ileum (B) sections from P1 (A) and P2 (B) compared with normal or IBD controls. Sections were counterstained with blue RedDot2 dye for DNA and antibodies against ALPI alone (red), TNAP alone (green) or TNAP and ALPI (first column, merge staining). Magnification 20× (Scale bars: 100 μm).

Article Snippet: Total RNA (5 μg) was reverse transcribed using M‐MLV reverse transcriptase (Invitrogen). qRT–PCR was performed with an Assays‐on‐Demand probe (Applied Biosystems, Thermo Fisher Scientific) specific for ALPI‐FAM (Hs00357579_g1) and RPLPO‐FAM (Large Ribosomal Protein; Hs99999902_m1), which was used for normalization. qPCR was performed with a ABI PRISM 7900 (Applied Biosystems).

Techniques: Immunofluorescence, Microscopy, Staining

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: RNA sequencing reveals that tomatidine modulates ATF4-dependent ER stress genes in pancreatic cancer cells Human and murine PDAC cell lines were treated for 40 h with 6.4 μg/mL tomatidine, RNA was isolated, and RNA sequencing was performed to analyze differences in gene regulation. N = 3 biological separate experiments. (A–D) (A) Heatmap of all genes and how they change in tomatidine-treated vs. untreated cells for Panc1 cells (FDR<0.05). Volcano plot highlighting ER stress-related genes in (B) Panc1 and (C) MT5 cells. (Upregulated genes on the right of the central axis and vice versa with higher fold change as we go away from the origin on y axis) (D) IPA upstream analysis of ATF4-related genes in Panc1 cells. (E) Heatmap elucidating targeting of ATF4-related genes in treated vs. untreated Panc1 cells. (Fold change: +2.5 to −1.5; FDR<0.03). (F) Top 10 pathways focusing on UPR, ATF4, and ER stress via Reactome analysis of the RNA-sequencing data of treated vs. untreated samples for Panc1 cells. (FDR< 0.03).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: RNA Sequencing, Isolation

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: ATF4 expression in PDAC (A) KM plotter was utilized to examine ATF4 expression in human PDAC specimens examining high vs. low expression compared to overall survival. (B) Data distribution elucidating significance of the overall survival curve prepared by KM Plotter. (C and D) (C) scRNA-seq datasets of pancreatic cancer tissue from metastatic patients were obtained from NIH dbGAP (accession phs002045.v1.p1) and (D) analyzed for ATF4 expression. (See also Table S1 ). (E) Pancreatic tumor tissue from KPC mice were stained by multiplex IF and imaged using Akoya Vectra Polaris and the Phenochart software to analyze ATF4 in multiple cellular compartments. Markers used to determine ATF4 (white), epithelial cells (CK19; red), stroma (αSMA; orange and PDGFRβ; Green), CD8 T+ cells (yellow), and macrophages (F4/80; turquoise). Scale bar = 100 μm.

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Staining, Multiplex Assay, Software

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine inhibits ATF4-dependent signaling in PDAC (A and B) (A) MiaPaca-2 tumor cells were treated with tomatidine for 72 h and cell lysates were immunoblotted for ATF4, 4EBP1, and phospho-4EBP1(p-4EBP1) protein expression and (B) p-4EBP1/4EBP1 levels quantified by densitometry. (C) Immunofluorescence (IF) was performed on vehicle (DMSO) or tomatidine-treated Panc1 cells to track ATF4 (FITC-Green) translocation from nucleus (DAPI-Blue) to cytoplasm. Scale bar = 50 μm. (D–G) (D) Nuclear to cytoplasmic translocation was quantified. Panc1 cells were treated with vehicle (DMSO) or tomatidine for 40 h and ATF4 transcriptional activity was analyzed by chromatin immunoprecipitation (ChIP) qPCR evaluating binding of ATF4 to the downstream promoter regions of (E) eIF4EBP1 (F) CHOP- B site and (G) ASNS. Data are reported as the means + SEMs. n = 3 or more independent biological replicates (4B, One-way ANOVA with Tukey’s test for pairwise comparisons was used to analyze the data; 4D-G, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Immunofluorescence, Translocation Assay, Activity Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: In vivo tomatidine treatment inhibits pancreatic tumor growth (A–C) (A) MT5 tumor-bearing C57BL/6 mice (5 mice/group) were treated with 5 mg/kg daily i.p. injections of tomatidine or vehicle control (40% HPBCD) and monitored for tumor growth. (n = 5/group). RNA isolated from the tumor tissues of MT5 tumor-bearing C57BL/6 mice treated with vehicle or 5 mg/kg daily i.p. injections of tomatidine were assessed for (B) ATF4 and (C) eIF4EBP1 expression via qPCR. Data are reported as the means + SEMs. n = 5 mice per group. (5A, mixed between-within subjects ANOVA shows a significant interaction between days of treatment and group; 5B-C, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: In Vivo, Control, Isolation, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine mediated inhibition of ATF4 signaling can increase sensitivity to ferroptotic cell death in PDAC (A) Panc-1 tumor cells were treated with tomatidine (6.4 μg/mL) and analyzed by RNA sequencing. Ingenuity pathway analysis of the regulated genes suggested ferroptosis as a top hit for tomatidine-treated cells. (B–D) (B) Pancreatic cancer cells were treated with vehicle (DMSO), erastin (to induce ferroptosis), ferrostatin-1 (to inhibit ferroptosis), tomatidine, or in different combinations and lipid peroxidation of (C) Panc-1 and (D) MiaPaca-2 was analyzed by flow cytometry using Bodipy-11. (E) Panc-1 and MiaPaca-2 cells treated with vehicle (DMSO) or tomatidine and lysates collected after 24 h were immunoblot for GPX4 expression. (F) Panc1 cells were plated overnight and treated with tomatidine for 6 h and then assayed by Seahorse assay to analyze mitochondrial fitness. (G) Schematic showing how tomatidine can regulate ATF4-dependent signaling to induce ferroptosis in pancreatic cancer. Data are reported as the means + SEMs. n = 3 or more independent biological replicates. (7C-D, Two-tailed independent student’s test was used to analyze the data; 7F, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05, ∗∗p < 0.007).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Inhibition, RNA Sequencing, Flow Cytometry, Western Blot, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet:

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Control, Recombinant, Viability Assay, Luciferase, ChIP-qPCR, Gene Expression, Software

Journal: Journal of Neuroscience

Article Title: SOCS3-Mediated Blockade of JAK/STAT3 Signaling Pathway Reveals Its Major Contribution to Spinal Cord Neuroinflammation and Mechanical Allodynia after Peripheral Nerve Injury

doi: 10.1523/jneurosci.5007-09.2010

Figure Lengend Snippet: Figure 5. Effects of transducing primary glial cells or BV2 microglia with LV–SOCS3t on JAK/STAT3 pathway activity and inflammatory state markers. A, Stimulation of primary glia with IL-6 (50 ng/ml) resulted in rapid (15 min) pSTAT3 accumulation (i.e., JAK/STAT3 activation, Western blot). This effect was prevented in cells transduced 48 h earlier with LV–SOCS3t (350 or 35 ng/ml p24). Data are shown as mean SEM of three independent experiments. #p 0.001, IL-6-treated versus untreated cell cultures;*p 0.001,IL-6-treatedLV–SOCS3t-transducedcellsversusIL-6-treateduninfectedcells.B,C,Inbothprimaryglialcells (B) and BV2 microglial cell line (C), IL-6-induced production (after 3 h incubation with IL-6) of inflammatory markers (IL-6, CCL2, TNF) was efficiently inhibited in cells transduced 48 h before with LV–SOCS3t. IL-6 can also induce ATF3 production in BV2 microglia, this effect being significantly prevented in LV–SOCS3t-transduced cells. Each bar is the mean SEM (n 4 for each group). #p 0.05, IL-6-treated cells versus control cell cultures; *p 0.05, IL-6-treated LV–SOCS3t-infected cells versus IL-6- treated uninfected cells. R.Q., Relative quantification; A.U., arbitrary unit.

Article Snippet: Assay-onDemand Gene TaqMan PCR probes (Applied Biosystems) were used for target genes: SOCS3 (GenBank accession number Rn00585674_s1), integrin alpha M (ITGAM) (GenBank accession number Rn00709342_ m1), GFAP (GenBank accession number Rn01460868_m1), IL-6 (Rn00561420_m1), IL-1 (GenBank accession number Rn00580432_ m1), ATF3 (GenBank accession number Rn00563784_m1), CCL2 (GenBank accession number Rn00580555_m1), tumor necrosis factor- (TNF ) (GenBank accession number Rn99999017_m1), GAPDH (GenBank accession number Rn99999916_s1), and ribosomal subunit 18S (RS 18S) (GenBank accession number Hs 99999901_s1).

Techniques: Activity Assay, Activation Assay, Western Blot, Incubation, Control, Infection, Quantitative Proteomics

Journal: EMBO Molecular Medicine

Article Title: Benchmarking porcine pancreatic ductal organoids for drug screening applications

doi: 10.1038/s44321-025-00330-3

Figure Lengend Snippet: ( A , B ) UMAPs showing the cell types/states ( A ) and sample composition ( B ) of the integrated scRNA-Seq data from two PPDO lines (1 LPN and 1 Ad) at passage 4 and 7. ( C ) Dot plot showing expression of the top 5 marker genes of each PPDO cluster. ( D ) Dot plot showing expression of key pancreatic gene markers for major cell types of the HPDO scRNA-Seq dataset including ductal ( KRT19 - SLC4A4 - BMPR1A - MUC1 - SOX9 ), endocrine ( INS - CHGB ), acinar/pancreatic progenitor ( GP2 ), acinar ( RBPJL - CELA1 ) endothelial ( PECAM1 - FLT1 ), stellate ( COL1A1 ), immune ( PTPRC ), and schwann ( CDH19 ) cell markers. ( E ) UMAP showing the cell types/states of scRNA-Seq data from HPDO. ( F ) UMAP showing the cross-species comparison of the integrated PPDO-HPDO scRNA-Seq datasets. ( G – L ) Dot plots of expression of important genes from the WNT ( G , H ), NOTCH ( I , J ) and Hippo ( K , L ) signaling pathways in the scRNA-Seq dataset of the integrated PPDO ( G , I , K ), and HPDO ( H , J , L ).

Article Snippet: RT-PCR was performed with 0.5 μl of initial RNA using the TaqMan Fast Advanced Mix and the following TaqMan probes coupled with 6-carboxyfluorescein (FAM): INS (Ss03386682_u1), GCG (Ss03384069_u1), NEUROD1 (Ss03373333_s1), SST (Ss03391856_m1), ACTB (Ss03376563_Uh), KRT7 (Ss06890421_Mh), and CFTR (Ss03389420_m1).

Techniques: Expressing, Marker, Comparison, Protein-Protein interactions

Journal: EMBO Molecular Medicine

Article Title: Benchmarking porcine pancreatic ductal organoids for drug screening applications

doi: 10.1038/s44321-025-00330-3

Figure Lengend Snippet: ( A , B ) Brightfield microscopy images of PPDO in complete ( A ) or porcine serum supplemented ( B ). Scale bar 500 µm. ( C , D ) Brightfield microscopy images of PPDO at the end of differentiation in complete ( C ) or at the end of the differentiation using S5 + S6 combination ( D ) media. Scale bar 500 µm. ( E –J ) Bar plots showing the fold change of gene expression analysis at the end of the differentiation after treatment with porcine serum. Gene expression was measured for CFTR ( E ), KRT7 ( F ), NEUROD1 ( G ), GCG ( H ), INS ( I ), and SST ( J ). n = 3 independent PPDO lines (1 Em and 2 EPN). Absence of samples from the plots indicate non-detectable amplification following the RT-PCR. Data are shown as mean ± SEM. Unpaired Student’s t-test was used to assess significance with * P = 0.0196 for NEUROD1 . ( K , L ) Single-plane confocal images of PPDO in growth media ( K ) or in differentiation media from Loomans et al ( L ) (see Methods). PPDO were immunostained against INS-KRT7-CHGA and counterstained with DAPI. Experiment was repeated with n = 3 biological replicates (1 Em-1 EPN-1 Ad). Scale bar: 50 µm. ( M , N ) Brightfield microscopy images of HPDO in differentiation media ( M ) or differentiation media supplemented with DAPT/DEAB small molecules ( N ). Experiment was repeated with n = 3 biological replicates. Scale bar 500 µm.

Article Snippet: RT-PCR was performed with 0.5 μl of initial RNA using the TaqMan Fast Advanced Mix and the following TaqMan probes coupled with 6-carboxyfluorescein (FAM): INS (Ss03386682_u1), GCG (Ss03384069_u1), NEUROD1 (Ss03373333_s1), SST (Ss03391856_m1), ACTB (Ss03376563_Uh), KRT7 (Ss06890421_Mh), and CFTR (Ss03389420_m1).

Techniques: Microscopy, Gene Expression, Amplification, Reverse Transcription Polymerase Chain Reaction

Journal: EMBO Molecular Medicine

Article Title: Benchmarking porcine pancreatic ductal organoids for drug screening applications

doi: 10.1038/s44321-025-00330-3

Figure Lengend Snippet: ( A ) Volcano plot indicating the number of significantly changed genes following the 7 days of culturing in the basal differentiation medium. Genes important for developmental processes are highlighted in the graph. Bulk RNA-Seq was performed on 4 biological replicate PPDO from 1 Em, 2 EPN and 1 LPN samples. Statistical analysis was employed as described in DESEQ2 package using a Wald test with Benjamin and Hochberg adjustment for multiple testing. ( B , C ) Bar plots showing the fold enrichment of significantly affected biological processes for the upregulated and downregulated genes of the RNA-Seq dataset shown in ( A ). ( B ) shows selected enriched GO terms for the significantly upregulated genes and ( C ) shows selected GO terms for the significantly downregulated genes. ( D – G ) Plots showing the fold change of gene expression for KRT7 ( D ), NEUROD1 ( E ), INS ( F ) and GCG ( G ) genes between differentiation medium alone or supplemented with DAPT-DEAB-BMS754807. Data are shown as mean ± SEM. Mann–Whitney test was used to assess significance with ** P = 0.0043 for NEUROD1 , ns P = 0.1508 for INS and ns ns P = 0.3095 for GCG . n = 6 different PPDO lines were assayed (2 Em, 3 EPN, 1 Ad). ( H – K ) Single-plane confocal images of PPDO derived from Em pig pancreas from passage 2 ( H , I ) or passage 6 ( J , K ) cultures and treated with differentiation only ( H – J ) or differentiation media supplemented with the NOTCH inhibitor DAPT and the aldehyde dehydrogenase inhibitor DEAB ( I – K ). PPDO were immunostained against KRT7 (gray)-INS/GCG (magenta) and counterstained with DAPI. Arrowheads in I indicate the insulin positive cells. Similar staining patterns were obtained from n = 4 different biological replicate PPDO lines (1 Em, 2 EPN, 1 Ad). Scale bar: 50 µm. .

Article Snippet: RT-PCR was performed with 0.5 μl of initial RNA using the TaqMan Fast Advanced Mix and the following TaqMan probes coupled with 6-carboxyfluorescein (FAM): INS (Ss03386682_u1), GCG (Ss03384069_u1), NEUROD1 (Ss03373333_s1), SST (Ss03391856_m1), ACTB (Ss03376563_Uh), KRT7 (Ss06890421_Mh), and CFTR (Ss03389420_m1).

Techniques: RNA Sequencing, Gene Expression, MANN-WHITNEY, Derivative Assay, Staining

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) Primary HKs were transduced with pLXSN HPV38 E6/E7 or pLSXN. mRNA levels were measured by RT-qPCR and normalized to GAPDH . Error bars represent standard deviations from 3 biological replicates of 2 different donors ( n = 6). ***, p <0.001; ****, p <0.0001; ns, not significant. (B) Total RNA levels of HKs expressing or not expressing HPV38 E6 and E7 were analyzed by TaqMan PCR. Commercial probes for ITGA1 and GAPDH were used. Results were normalized to GAPDH . Data shown are the means of 3 independent experiments for 2 different donors ( n = 6). ****, p <0.0001. (C) Proteins extracts from HKs expressing or not expressing HPV38 E6 and E7 were analyzed by immunoblotting (IB) with the indicated antibodies. (D) The TaqMan assay was also performed as previously described in primary HKs previously retrovirally transduced with the hTERT gene and expressing E6 and/or E7 from HPV38 ( n = 3). Results were normalized to GAPDH . ****, p <0.0001. (E) Proteins extracts from hTERT pLXSN or hTERT HPV38 E6/E7 cells were analyzed by IB with the indicated antibodies. Images shown are representative examples of 2 different experiments. (F) hTERT pLXSN or hTERT HPV38 E6/E7 cells were plated on coverslips and after 24 h were probed for ITGA1 using anti-ITGA1 antibody followed by secondary Alexa Fluor 488-conjugated antibody. Nuclei were stained with DAPI (pseudocoloured red), and cells were analyzed under a microscope. Images were merged using ImageJ software.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Transduction, Quantitative RT-PCR, Expressing, Western Blot, TaqMan Assay, Staining, Microscopy, Software

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) Electromobility shift assay performed with 38HK nuclear protein extracts and biotinylated probes containing p53RE WT or mutated sequences. Probes were incubated and cross-linked with protein extracts. Unlabeled WT or mutant p53RE2 probes were used as a control. Images shown are representative examples of 2 different experiments. (B) 38HK were cross-linked and chromatin was processed for ChIP using p53 antibody. Results were analyzed by qPCR with primers spanning p53RE1, p53RE2, p53RE3, or the intergenic region of chromosome 22 as a negative control (nc). Error bars represent standard deviations of 3 independent experiments performed in triplicate. **, p <0.01. (C) HKs or 38HK were cross-linked and chromatin was processed for ChIP using p53 or IgG antibodies. Results were analyzed by qPCR using primers spanning for p53 REs of the ITGA1 promoter and normalized to IgG enrichment (negative control). Error bars represent the standard deviation of 2 independent experiments performed in 2 different HKs donors. *, p <0.05, ns, not significant. (D) Cell lysate was incubated with WT biotinylated probe containing p53 REs of the ITGA1 promoter. Incubation without a probe was used as a control. DNA-associated proteins were recovered by precipitation with streptavidin beads and analyzed by IB. Images shown are representative examples of 3 independent experiments. Signals of 3 different IBs were quantified with Image Lab software (right panel). Data shown are the means of 3 independent experiments. *, p <0.05. (E) Chromatin from 38HK was processed for ChIP experiments using p53 or DNMT1 antibodies. Results were obtained by qPCR with primers spanning p53RE2 or the intergenic region of chromosome 22 (nc). Error bars indicate standard deviations from 3 independent experiments performed in duplicate. **, p <0.01; ***, p <0.001. (F) 38HK were cultured in medium containing cyclic pifithrin-α hydrobromide or DMSO as a control. Chromatin was processed for ChIP using p53 or DNMT1 antibodies. Results were obtained by qPCR using primers spanning p53RE2. Data shown are the means of 2 independent experiments performed in triplicate. *, p <0.05, **, p <0.01. (G) Chromatin was processed for a ChIP-reChIP assay in which p53-immunoprecipitated DNA was re-immunoprecipitated by DNMT1. Enrichment of p53RE2 or the intergenic region of chromosome 22 (nc) was obtained by qPCR. Data shown are the means of 3 independent experiments performed in triplicate. **, p <0.01. (H) Nuclear protein extracts from 38HK were processed for IP. Agarose beads were conjugated with IgG or p53 antibodies. Conjugated beads were incubated with protein lysate overnight. IgG was used as a control. Results were obtained by IB using the indicated antibodies. (I) 38HK were transfected with DNMT1 siRNA or control siRNA (Scramble). After 72 h, a ChIP assay was performed with p53 or DNMT1 antibodies. Results were obtained by qPCR using p53RE2 primers. Error bars represent standard deviations from 3 independent experiments. *, p <0.05; **, p <0.01. DNMT1 protein levels in different cells were determined by IB with the indicated antibodies (right panel).

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Electro Mobility Shift Assay, Incubation, Mutagenesis, Control, Negative Control, Standard Deviation, Software, Cell Culture, Immunoprecipitation, Transfection

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) mRNA levels of 38HK treated with cyclic pifithrin-α hydrobromide or DMSO for 6 h were analyzed by RT-qPCR and normalized to GAPDH . Histograms represent the mean of at least 3 independent experiments. ****, p <0.0001. (B and C) ITGA1 and p53 mRNA and protein levels from 38HK expressing WT p53 (Scramble) or with CRISPR/Cas9-mediated p53 deletion (CRISPR-p53) were measured by RT-qPCR (B) and IB (C). (B) Gene expression was normalized to GAPDH . (C) Protein quantification was normalized to β-actin. Signals of 4 different IBs were quantified with Image Lab software (right panel). Data shown are the means of 4 independent experiments. *, p <0.05. (D and E) 38HK N-HA-p53 or p53-C-HA cells were generated by retroviral transduction with WT p53 tagged at the N- or C-terminus. As a control, 38HK were transduced with the corresponding empty plasmid. Protein extracts and total mRNA levels were processed for IB and RT-qPCR analysis, respectively. (D) IB images shown are representative examples of 3 independent experiments. (E) ITGA1 mRNA levels were normalized to GAPDH . Error bars indicate standard deviations of 4 independent experiments. ***, p <0.001. (F) 38HK were transfected with control siRNA (Scramble) or with DNMT1 siRNA (siDNMT1). After 72 h, cells were collected for RNA extraction and RT-qPCR analysis. Error bars indicate standard deviations of 3 independent experiments. *, p <0.05; ***, p <0.001. (G) ITGA1 expression was evaluated by RT-qPCR after 24 h of treatment with 5-aza-2′- deoxycytidine (Aza) or DMSO at 30 μM final concentration. Error bars represent standard deviations of 3 independent experiments. **, p <0.01. (H) H3K9ac at the ITGA1 promoter was evaluated by ChIP assay after treatment with Aza or DMSO as previously described ( n = 4). Results were obtained by qPCR using primers for p53RE2. *, p <0.05.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, CRISPR, Gene Expression, Software, Generated, Retroviral, Transduction, Control, Plasmid Preparation, Transfection, RNA Extraction, Concentration Assay

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) Protein extracts from 38HK were processed for oligonucleotide pulldown as previously described. Images shown are representative examples of 3 independent experiments. (B) HK and 38HK were processed for protein extraction and IB with the indicated antibodies. After incubation with p446PKR antibody, the membrane was stripped and incubated with total PKR antibody. (C) Proteins extracts from U2OS cells retrovirally transduced with E6 and/or E7 from HPV38 were analyzed by IB with the indicated antibodies. (D) 38HK were treated with PKR inhibitor, 2AP, or PBS:glacial acetic acid (200:1) as a control for 4 h at 10 mM final concentration. p-p53 S392 and p53 band intensities were quantified and normalized to total p53 (central panel) or β-actin (right panel). Membranes were first incubated with p446PKR, then stripped and incubated with total PKR. Data shown are the means of 3 independent experiments. *, p <0.05, **, p <0.01. (E and F) 38HK were treated with 2AP, and ITGA1 mRNA (D) and protein levels (E) were determined by RT-PCR and IB, respectively. (D) Data shown are the means of 3 independent experiments (**, p <0.01). (E) Images shown are representative examples of 3 independent experiments. (G and H) ChIP assay using p53 or DNMT1 antibodies was performed in 38HK treated with or PBS:glacial acetic acid (200:1) as a control (CTR) or 2AP. Data shown are the means of 2 independent experiments performed in duplicate by qPCR using p53RE2 primers. *, p <0.05.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Protein Extraction, Incubation, Membrane, Transduction, Control, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) 38HK were transfected with ITGA1 cDNA (ITGA1) or empty plasmid control (pcDNA). After zeocin selection, 38HK were fixed with crystal violet (left panel) and total colony number per well was counted (right panel). Data shown are the means of 3 independent experiments. ****, p <0.0001. (B) 38HK transfected with ITGA1 cDNA (ITGA1) or empty plasmid control (pcDNA) were fixed and stained with propidium iodide for flow cytometry analysis. The histograms (right) represent the means of the sub-G0 population of 3 independent experiments. **, p <0.01. (C and D) Total protein and mRNA extracts from transfected 38HK were analyzed by IB and RT-qPCR. (C) Protein band intensity was quantified and normalized to β-actin. (D) ITGA1 , EGFR , and CCND1 mRNA levels were normalized to GAPDH . For (C) and (D), data shown are the means of 4 independent experiments *, p <0.05; **, p <0.01; ***, p <0.001; ns, not significant.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Control, Selection, Staining, Flow Cytometry, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) Skin keratinocytes were isolated from WT animals ( n = 4) and K14 HPV38 E6/E7 transgenic mice ( n = 3). After 30 weeks of UV irradiation, cSCC samples (cSCC1–3) were isolated from HPV38 E/E7 transgenic mice. Total RNA extraction was performed and Itga1 mRNA levels were determined by quantitative RT-PCR by normalizing to Gapdh . Whole-exome sequencing of the same mice was also performed. Mutational analysis of the Trp53 , Itga1 , and Egfr genes was performed as described in Materials and Methods. (B) Genomic position of the exonic mutations and the corresponding amino acid change are represented for the Itga1 and Egfr genes. WT exons are represented as blue boxes, and mutated exons are represented in red boxes. Text boxes describe the cSCC sample, the genomic position of the nucleotide change on the GRCm38/mm10 mouse reference genome, the type of mutation, and the corresponding amino acid change.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Isolation, Transgenic Assay, Irradiation, RNA Extraction, Quantitative RT-PCR, Sequencing, Mutagenesis

Journal: eLife

Article Title: Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62

doi: 10.7554/eLife.17290

Figure Lengend Snippet: ( A ) Scramble, ATG7 and UFM1 sgRNAs were introduced into H4 Cas9 clone 4 by lentiviral infection. 7 days post-infection cells were treated with 0.1% DMSO or 50 nM Bafilomycin A1 for 2, 4 or 8 hr and protein lysates were collected and probed by immunoblot. ( B ) Representative images of H4 cells stably expressing mCherry-GFP-LC3B or mCherry-GFP-UFM1. Scale bar corresponds to 20 µm. Note that the absence of mCherry-positive puncta in mCherry-GFP-UFM1 expressing cells suggests that UFM1 is not delivered to endolysosomal compartments. ( C ) GFP-SQSTM1 clearance in ATG7- and UFM1-depleted cells. Scramble, ATG7 and UFM1 sgRNAs were introduced into H4 Cas9 GFP-SQSTM1 cells by lentiviral infection. 7 days post-infection cells were treated with 0.1% DMSO or 0.5 µM AZD8055 for 8 hr or 24 hr, fixed, and subjected to high content imaging. GFP fluorescence was quantified as ratio of GFP-SQSTM1 puncta area in DMSO- versus AZD8055-treated cells and is shown as mean +/- SD from three independent experiments. Statistical significance was assessed via a two-tailed paired t-test (*p<0.05, **p<0.01). ( D ) UFM1 knockout induces ER stress and increases SQSTM1 mRNA levels. Scramble and UFM1 sgRNAs were introduced into H4 Cas9 cells by lentiviral infection. 7 days post-infection total RNA was extracted and subjected to RNA sequencing. The volcano plot visualizes genes differentially expressed in UFM1-depleted versus cells infected with a scramble sgRNA. Selected ER stress markers and SQSTM1 are highlighted and entire dataset is reported in . ( E ) Time course analysis of ER stress and SQSTM1 induction. Indicated sgRNAs were introduced into H4 Cas9 cells by lentiviral infection, protein lysates collected 4, 6 and 8 days post-infection and probed by immunoblot. ( F ) Total RNA was collected from H4 Cas9 cells treated with 0.1% DMSO or 10 µg/ml tunicamycin for 24 hr, SQSTM1 mRNA was quantified by RT-qPCR analysis and normalized to HPRT1. The mean +/- SD of biological triplicates is shown. ( G ) Indicated sgRNAs were introduced into Huh-1 Cas9 or HepG2 Cas9 cells by lentiviral infection, protein lysates collected 7 days post-infection and probed by immunoblot. DOI: http://dx.doi.org/10.7554/eLife.17290.010 10.7554/eLife.17290.011 Figure 4—source data 1. Fold change and significance of differentially regulated genes by RNA sequencing in UFM1-depleted versus scramble sgRNA-infected cells. DOI: http://dx.doi.org/10.7554/eLife.17290.011

Article Snippet: Cells were lysed and processed using the RNeasy Plus Kit (Qiagen) according to the manufacturer’s instructions. cDNA synthesis was performed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA). qPCR was performed with the TaqMan Gene Expression Master Mix and the following TaqMan probes from Applied Biosystems: HPRT1 (Hs02800695_m1), SQSTM1 (Hs01061917_g1).

Techniques: Infection, Western Blot, Stable Transfection, Expressing, Imaging, Fluorescence, Two Tailed Test, Knock-Out, RNA Sequencing, Quantitative RT-PCR