Journal: International Journal of Molecular Sciences

Article Title: Expression of the Calcium-Binding Protein CALB1 Is Induced and Controls Intracellular Ca 2+ Levels in Senescent Cells

doi: 10.3390/ijms23169376

Figure Lengend Snippet: CALB1 expression is increased in HMECT in response to diverse senescence inducers. ( A ) Transcriptomics analysis in HMECT-Mek:ER reveals that CALB1 expression is induced after oncogene activation by 4-OHT. PVALB, CALB2, and CALB1 mean expression level from a biological triplicate is indicated at 0 (−4-OHT), 24 and 96 h after 4-OHT treatment. An unpaired t -test with Welch correction was used. ( B – E ). CALB1 mRNA level ( left panels) and protein level and subcellular localization ( right panels) were assessed in HMECT either overexpressing 4-OHT-inducible oncogenes Mek:ER ( B ) or Raf:ER ( C ) or treated with bleomycin ( D ) or nutlin ( E ). CALB1 mRNA level was measured by RT-qPCR and mean +/- SEM of independent experiments are shown (( B ), n = 6; ( C ), n = 8; ( D ), n = 3; ( E ), n = 4). Unpaired t -test ( B , D , E ) or Mann–Whitney test ( C ) were performed, and p -values are indicated. CALB1 protein level and localization were assessed six days after treatment by immunofluorescence with CALB1 antibody, nuclei were stained with Hoechst.

Article Snippet: A retroviral vector encoding a constitutively active NFATc1 ([ ], Addgene_11102) and a lentiviral vector encoding CALB1 (pLV-CALB1, VectorBuilder) were used, as well as the corresponding control vectors (empty or encoding the GFP).

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, MANN-WHITNEY, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Expression of the Calcium-Binding Protein CALB1 Is Induced and Controls Intracellular Ca 2+ Levels in Senescent Cells

doi: 10.3390/ijms23169376

Figure Lengend Snippet: The calcium-dependent calcineurin/NFAT pathway induces CALB1 expression upon oncogene activation in HMECT. ( A ) CALB1 mRNA level was measured by RT-qPCR in HMECT overexpressing 4-OHT-inducible Mek:ER and treated (+) or not (−) with FK-506, a calcineurin inhibitor (treated every day with 2 µM during six days), or/and with 4-OHT (100 nM). The first three days, cells were co-treated with 4-OHT and FK-506 (first treatment 4 h prior 4-OHT), and the three last days only with FK-506. Mean +/− SEM of four independent experiments are shown. An unpaired t -test was performed and p -value is indicated. ( B ) NFATc1 ( left panel) and CALB1 ( right panel) mRNA levels were measured by RT-qPCR in HMECT 24 h after infection with a vector encoding NFATc1 or control empty vector. Mean +/− SEM of six independent experiments are shown. Unpaired t -tests were performed, and p -values are indicated.

Article Snippet: A retroviral vector encoding a constitutively active NFATc1 ([ ], Addgene_11102) and a lentiviral vector encoding CALB1 (pLV-CALB1, VectorBuilder) were used, as well as the corresponding control vectors (empty or encoding the GFP).

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Infection, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Expression of the Calcium-Binding Protein CALB1 Is Induced and Controls Intracellular Ca 2+ Levels in Senescent Cells

doi: 10.3390/ijms23169376

Figure Lengend Snippet: CALB1 overexpression buffers increased Ca 2+ levels induced by oncogene activation in HMECT. ( A ) CALB1 protein level was assessed by Western Blot in HMECT-Mek:ER (without 4-OHT) infected with a vector encoding CALB1 or the corresponding GFP control vector. α-Tubulin protein level was assessed as a loading control. ( B ) Ca 2+ levels were measured with the ratiometric probe Fura2 in HMECT-Mek:ER inducible oncogene and CALB1 or the corresponding GFP control vector, with or without 4-OHT treatment. Resting cytosolic Ca 2+ concentration was evaluated as a stable fluorescent ratio before stimulation ( left panel), and the size of Ca 2+ intracellular stocks was estimated as the Ca 2+ peak amplitude obtained after ionomycin stimulation ( right panel). Means +/− SEM of 3 independent experiments are presented. Kruskal–Wallis test was performed, and p -values are indicated. Three days after 4-OHT treatment, GFP- 4-OHT n = 85 or + 4-OHT n = 126, CALB1- 4-OHT n = 99 or + 4-OHT n = 77.

Article Snippet: A retroviral vector encoding a constitutively active NFATc1 ([ ], Addgene_11102) and a lentiviral vector encoding CALB1 (pLV-CALB1, VectorBuilder) were used, as well as the corresponding control vectors (empty or encoding the GFP).

Techniques: Over Expression, Activation Assay, Western Blot, Infection, Plasmid Preparation, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Expression of the Calcium-Binding Protein CALB1 Is Induced and Controls Intracellular Ca 2+ Levels in Senescent Cells

doi: 10.3390/ijms23169376

Figure Lengend Snippet: Expression of calbindins is induced in many senescence contexts. ( A ) Additional contexts where CALB1 expression is upregulated in HMECT in response to senescence inducers. CALB1 mRNA level was measured by RT-qPCR in HMECT overexpressing 4-OHT-inducible ER:Ras oncogene (day 6 after 100 nM 4-OHT treatment, n = 6) or treated with H 2 O 2 (at 250 µM for 1 h and RNA were collected six days later, n = 3), TGFβ (at 0.5 ng/mL for three days, n = 6) or KCl (at 65 mM for 24 h, n = 5). ( B , C ) CALB2 expression is increased in several senescence contexts. ( B ) CALB2 mRNA level measured in melanocytes overexpressing B-RAF V600E oncogene (transcriptome data ). ( C ) CALB2 mRNA level measured in normal human embryonic lung fibroblasts MRC5 overexpressing 4-OHT-inducible Raf:ER oncogene (RT-qPCR at day 3 after 100 nM 4-OHT treatment, n = 4), IMR90 overexpressing 4-OHT-inducible ER:Ras oncogene (transcriptome data ) or WI38 treated with etoposide (transcriptome data ). For RT-qPCR, mean +/− SEM of independent experiments are shown. Unpaired t -test (for HMECT-ER:Ras, HMEC H 2 O 2 , HMEC TGFβ, and MRC5-Raf:ER) or Mann–Whitney test (for HMECT KCl) were performed and p -values are indicated.

Article Snippet: A retroviral vector encoding a constitutively active NFATc1 ([ ], Addgene_11102) and a lentiviral vector encoding CALB1 (pLV-CALB1, VectorBuilder) were used, as well as the corresponding control vectors (empty or encoding the GFP).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Hepatology Communications

Article Title: Transcriptional Enhancer Factor Domain Family member 4 Exerts an Oncogenic Role in Hepatocellular Carcinoma by Hippo‐Independent Regulation of Heat Shock Protein 70 Family Members

doi: 10.1002/hep4.1656

Figure Lengend Snippet: Modulation of TEAD4 expression level impacts cell proliferation and migration. (A) Proliferation and (B) migration kinetics of TEAD4 ‐overexpressing ( TEAD4ox ) Huh7 and TEAD4 ‐silenced (sh TEAD4 ) HLE cells compared to their respective controls. (C) Wound‐healing assay in TEAD4 ‐overexpressing Huh7 and TEAD4 ‐silenced HLE cells compared to their respective controls at specific time points. Representative pictures at time points 0 and 24 hours after scratch are shown for each condition. Scale bar in (C), 1 cm. Statistical significance was determined by the t test; * P < 0.05,** P < 0.01. Data are mean ± SD. Abbreviations: h, hours; sh, short hairpin.

Article Snippet: For TEAD4 overexpression, pLV[Exp]‐enhanced green fluorescent protein (EGFP)/Neo‐EF1A>hTEAD4 was designed and ordered on the vector builder website ( https://en.vectorbuilder.com/ ), and the empty control vector was plasmid cytomegalovirus (pCMV)‐mir EGFP.

Techniques: Expressing, Migration, Wound Healing Assay

Journal: Hepatology Communications

Article Title: Transcriptional Enhancer Factor Domain Family member 4 Exerts an Oncogenic Role in Hepatocellular Carcinoma by Hippo‐Independent Regulation of Heat Shock Protein 70 Family Members

doi: 10.1002/hep4.1656

Figure Lengend Snippet: TEAD4 overexpression increases tumor growth in vivo . (A) Schematic representation of the CAM assay. (B) Photographs of TEAD4 ‐overexpressing or control Huh7 cells implanted in CAMs and grown for 4 days postimplantation. (C) Volume of tumors derived from the CAM experiment (n ≥ 7 tumors over two independent experiments). Values are normalized to the mean volume of control. (D) TEAD4 expression in Huh7 tumors extracted 4 days postimplantation analyzed by western blot (left) and IHC (right). Tumoral cells were immunostained with the TEAD4 antibody. Scale bars, (B) 1 cm and (D) 50 μm and 20 μm. Statistical significance was determined by the Mann‐Whitney U test; * P < 0.05. Data are mean ± SD. Abbreviations: H&E, hematoxylin and eosin; ox, overexpression.

Article Snippet: For TEAD4 overexpression, pLV[Exp]‐enhanced green fluorescent protein (EGFP)/Neo‐EF1A>hTEAD4 was designed and ordered on the vector builder website ( https://en.vectorbuilder.com/ ), and the empty control vector was plasmid cytomegalovirus (pCMV)‐mir EGFP.

Techniques: Over Expression, In Vivo, Chick Chorioallantoic Membrane Assay, Control, Derivative Assay, Expressing, Western Blot, MANN-WHITNEY

Journal: Hepatology Communications

Article Title: Transcriptional Enhancer Factor Domain Family member 4 Exerts an Oncogenic Role in Hepatocellular Carcinoma by Hippo‐Independent Regulation of Heat Shock Protein 70 Family Members

doi: 10.1002/hep4.1656

Figure Lengend Snippet: TEAD4 regulates cell differentiation and proliferation and the expression of HSP70 family genes. (A) Heatmap showing the top 50 differentially expressed genes in TEAD4 ‐overexpressing Huh7 cells. (B) Volcano plot of –log10 adjusted P value against log2 fold change to illustrate the differentially expressed genes between TEAD4 ‐overexpressing Huh7 cells and control cells. Red dots indicate differentially expressed genes (adjusted P < 0.05). (C‐E) GSEA plots show selected significantly enriched gene sets in TEAD4 ‐overexpressing cells: Hallmark gene sets from (C) MSigDB database, (D) HSP family gene set, and (E) GO gene sets from the MSigDB database. (F) The Huh7 cell line was transiently transfected with a vector overexpressing TEAD4 and the corresponding control. RNA was extracted at different time points. mRNA expression levels of HSPA1A (left) and HSPA6 (right) were measured by qRT‐PCR using GAPDH as the internal control. Data are shown as fold change relative to control. Results represent three independent experiments. Statistical significance was determined by the Mann‐Whitney U test; * P < 0.05, ** P < 0.01, *** P < 0.001. Data are mean ± SD. Abbreviations: E2F, E2 factor; FDR, false discovery rate; GAPDH , glyceraldehyde 3‐phosphate dehydrogenase; LATS1, large tumor suppressor kinase 1; mRNA, messenger RNA; mTORC1, mammalian target of rapamycin complex 1; NFKB, nuclear factor kappa B; ox, overexpression; TJP2, tight junction protein 2; TNFa, tumor necrosis factor alpha; UV, ultraviolet.

Article Snippet: For TEAD4 overexpression, pLV[Exp]‐enhanced green fluorescent protein (EGFP)/Neo‐EF1A>hTEAD4 was designed and ordered on the vector builder website ( https://en.vectorbuilder.com/ ), and the empty control vector was plasmid cytomegalovirus (pCMV)‐mir EGFP.

Techniques: Cell Differentiation, Expressing, Control, Transfection, Plasmid Preparation, Quantitative RT-PCR, MANN-WHITNEY, Over Expression

Journal: Hepatology Communications

Article Title: Transcriptional Enhancer Factor Domain Family member 4 Exerts an Oncogenic Role in Hepatocellular Carcinoma by Hippo‐Independent Regulation of Heat Shock Protein 70 Family Members

doi: 10.1002/hep4.1656

Figure Lengend Snippet: TEAD4 impacts cell proliferation by directly binding HSP70 promoters and associated enhancers. (A) TEAD4 ChIP‐seq peaks at the promoters of HSPA6 and HSPA1A, and regions called as TEAD4 enhancers within 100 kb of HSPA6 and HSPA1A, with TEAD4 binding motifs labeled. Enhancers and promoters are labeled as red and blue blocks, respectively. The TEAD4 binding motif logos are shown below the track, and positions in the HSPA6 and in HSPA1A enhancers harboring this motif are indicated (this motif was also found at the promoter of all three genes). Asterisks indicate peaks validated by ChIP‐qPCR. (B) ChIP‐qPCR showing enrichment for TEAD4 binding at the HSPA6 promoter and the HSPA1A enhancer. CTGF was used as a positive control. (C) Representative immunostain of TEAD4 and HSP70 on human HCCs. Magnification ×40; scale bar 20 μm. (D) Schematic representation of the mechanisms of action of KNK437 on HSP70. (E) Proliferation of TEAD4 ‐overexpressing Huh7 cells treated with 100 μM of KNK437 at various time points, relative to proliferation at 4 hours. DMSO was used as control. Results represent three independent experiments. Statistical significance was determined by the t test; * P < 0.05, ** P < 0.01, *** P < 0.001. Data are mean ± SD. Abbreviations: chr, chromosome; h, hours; mRNA, messenger RNA; ox, overexpression; RLU, relative light unit; TF, transcription factor.

Article Snippet: For TEAD4 overexpression, pLV[Exp]‐enhanced green fluorescent protein (EGFP)/Neo‐EF1A>hTEAD4 was designed and ordered on the vector builder website ( https://en.vectorbuilder.com/ ), and the empty control vector was plasmid cytomegalovirus (pCMV)‐mir EGFP.

Techniques: Binding Assay, ChIP-sequencing, Labeling, ChIP-qPCR, Positive Control, Control, Over Expression

Journal: Hepatology Communications

Article Title: Transcriptional Enhancer Factor Domain Family member 4 Exerts an Oncogenic Role in Hepatocellular Carcinoma by Hippo‐Independent Regulation of Heat Shock Protein 70 Family Members

doi: 10.1002/hep4.1656

Figure Lengend Snippet: The effect of TEAD4 on cell proliferation is independent of expression of cofactors YAP and TAZ. (A) Schematic representation of TEAD4 transcriptional activity associated with YAP/TAZ cofactor expression. (B) Expression levels of YAP and TAZ were measured in stable YAP/TAZ ‐knockdown (sh Y/T ) and control (shCTR) Huh7 and SNU449 cell lines by qRT‐PCR normalized to GAPDH as internal control. (C) TEAD4 expression in stably TEAD4 ‐overexpressing and control Huh7 and SNU449 cell lines with knockdown of YAP/TAZ or control (shCTR). (D) Proliferation kinetics of stable TEAD4 ‐overexpressing and control Huh7 cell lines with YAP/TAZ knockdown or control (shCTR) (left) and in TEAD4 ‐overexpressing and control SNU449 stable cell lines with YAP/TAZ knockdown and control (shCTR) (right). (E) HSPA6 and HSPA1A expression levels in TEAD4‐overexpressing and control Huh7 cell lines with knockdown of YAP/TAZ or control (shCTR) were measured by qRT‐PCR using GAPDH as reference. (F) Expression levels of HSPA6 in TEAD4 ‐overexpressing and control Huh7 cell lines with knockdown of YAP/TAZ or control (shCTR) were measured by western blot at 48 and 72 hours posttransfection. Data are shown as fold change to control. Results represent three independent experiments. Statistical significance was determined by t test; * P < 0.05, ** P < 0.01, *** P < 0.001. Data are mean ± SD. Abbreviations: GAPDH , glyceraldehyde 3‐phosphate dehydrogenase; h, hours; ox, overexpression; sh, short hairpin; VGLL, vestigial‐like protein 1.

Article Snippet: For TEAD4 overexpression, pLV[Exp]‐enhanced green fluorescent protein (EGFP)/Neo‐EF1A>hTEAD4 was designed and ordered on the vector builder website ( https://en.vectorbuilder.com/ ), and the empty control vector was plasmid cytomegalovirus (pCMV)‐mir EGFP.

Techniques: Expressing, Activity Assay, Knockdown, Control, Quantitative RT-PCR, Stable Transfection, Western Blot, Over Expression