Journal: Oncogene

Article Title: A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis

doi: 10.1038/onc.2014.46

Figure Lengend Snippet: Mutant p53 and p63 bind to the let-7i promoter to regulate let-7i expression. (a) Schematic representation of let-7i promoter region containing the putative mutant p53-binding site. (b) H1299 cells stably expressing EV or p53R273H were cotransfected with the luciferase promoter plasmid carrying the let-7i promoter (pGL4-let-7i) and pRL-TK (internal control). Luciferase activity was measured after 36 h (left panel). Repression of let-7i promoter by mutant p53 was also analyzed by cotransfecting H1299-p53R273H cells with control (CTL) or p53 siRNAs and pGL4-let-7i for 48 h and measuring luciferase activity (right panel). (c) Parental H1299 cells were cotransfected with the let-7i promoter reporter (pGL4-let-7i), EV or p53R273H plasmids and pRL-TK (internal control). Luciferase activity was measured after 48 h. The effect of stable knockdown of mutant p53 in MDA-MB-231 and MIA-PaCa-2 cells on mature let-7i (d) and primary let-7i (e) levels was assessed by qRT–PCR normalized to U6 or UBC. (f) Mutant p53 co-immunoprecipitates with p63 and p73 in whole-cell lysates prepared from MDA-MB-231 cells. (g) Chromatin immunoprecipitation (ChIP) assays were performed to pulldown mutant p53 or p63 from MDA-MB-231 cells. Fold enrichment of the let-7i promoter region in the mutant p53 or p63 ChIP was determined by qPCR. (h) MDA-MB-231 cells were transfected with CTL or p63 siRNAs for 48 h and the levels of primary let-7i transcript (left panel) or mature let-7i (right panel) was assessed by qRT–PCR normalized to UBC or U6.

Article Snippet: 32 Immunoblotting Immunoblotting was performed using standard protocols using anti-p53 (D0–1, Santa Cruz, 1:5000), anti-p63 (H-137, Santa Cruz, 1:2500), anti-p73 (Bethyl, 1:5000), anti-HA High affinity (Roche, Indianapolis, IN, USA, 1:1000), anti-Ago2 (2A8 1:5000), anti-CPSF1 (Sigma, 1:2500), anti-Myc (9E10 Santa Cruz, 1:2500) and anti-β-actin (Sigma, 1:5000) antibodies. p53 mutational analysis in breast cancer patient samples Freshly frozen tumor specimens were obtained at the time of surgery from breast cancer patients and the p53 mutation status was determined as described previously.

Techniques: Mutagenesis, Expressing, Binding Assay, Stable Transfection, Luciferase, Plasmid Preparation, Control, Activity Assay, Knockdown, Quantitative RT-PCR, Chromatin Immunoprecipitation, Transfection

Journal: Oncogene

Article Title: A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis

doi: 10.1038/onc.2014.46

Figure Lengend Snippet: A mutant p53–p63 complex regulates let-7i transcription through p63. (a) Schematic representation of the let-7i promoter region containing the putative mutant p53/p63-binding site (FL), lacking the mutant p53/p63 binding region (Δ400) or lacking the p63 response element (Δp63RE). (b) MDA-MB-231 cells were cotransfected with pGL4, pGL4-let-7i or pGL4-let-7i-Δ400 or pGL4-let-7i-Δp63RE and CTL, p53 or p63 siRNAs. Luciferase activity was measured after 48 h. (c) ChIP assays were performed to pulldown mutant p53 or p63 from MDA-MB-231 cells transfected with CTL, p53 or p63 siRNAs. Fold enrichment of let-7i promoter region in the mutant p53 or p63 ChIP was determined by qPCR. (d) p63 was immunoprecipitated (IP) from MDA-MB-231 whole-cell lysates in the presence or absence of DNase, and immunoblotting for p53 was performed. (e) Sequential ChIP assays were performed to HA-p63 and then mutant p53 from MDA-MB-231. Enrichment of let-7i promoter region in ChIP reactions was measured by qPCR. (f) Parental H1299 cells were transfected with HA-p63 after 48 h and immunoblotting was performed for using anti-HA. (g) Parental H1299 cells were cotransfected with the let-7i promoter (pGL4-let-7i-FL), EV or HA-p63 or HA-p63 and p53R273H plasmids and pRL-TK (internal control). Luciferase activity was measured after 48 h. Error bars represent mean±s.d. from three independent experiments. *P < 0.05; #P <0.01; **P< 0.005; ##P < 0.001.

Article Snippet: 32 Immunoblotting Immunoblotting was performed using standard protocols using anti-p53 (D0–1, Santa Cruz, 1:5000), anti-p63 (H-137, Santa Cruz, 1:2500), anti-p73 (Bethyl, 1:5000), anti-HA High affinity (Roche, Indianapolis, IN, USA, 1:1000), anti-Ago2 (2A8 1:5000), anti-CPSF1 (Sigma, 1:2500), anti-Myc (9E10 Santa Cruz, 1:2500) and anti-β-actin (Sigma, 1:5000) antibodies. p53 mutational analysis in breast cancer patient samples Freshly frozen tumor specimens were obtained at the time of surgery from breast cancer patients and the p53 mutation status was determined as described previously.

Techniques: Mutagenesis, Binding Assay, Luciferase, Activity Assay, Transfection, Immunoprecipitation, Western Blot, Control

Journal: Oncogene

Article Title: A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis

doi: 10.1038/onc.2014.46

Figure Lengend Snippet: let-7i inhibits the 3′ UTR of multiple oncogenes and RNA post-transcriptional modification genes. MDA-MB-231 cells were reverse-transfected with CTL or let-7i mimics for 36 h and changes in mRNA expression of known let-7 targets (a) and novel targets (b) identified in the microarrays were validated by qRT–PCR normalized to UBC. (c) MDA-MB-231 cells were reverse-transfected with CTL or let-7i mimics for 36 h and the protein levels of CPSF1 and MYC were analyzed by immunoblotting. β-actin was used as loading control. (d) Luciferase assays were performed from H1299-R273H cells cotransfected with psiCHECK2 (Vector), psiCHECK2 containing the 3′UTR of known let-7 targets (E2F5 or NRAS) or psiCHECK2 containing the 3′ UTR of nine RNA post-transcriptional modification genes and CTL or let-7i mimics. (e) Effect of silencing let-7i in H1299 cells with a let-7i sponge (let-7i-Spg) on the expression of let-7i target mRNAs was assessed by qRT–PCR. (f) Expression of CPSF1 and MYC protein from H1299 stably transfected with CTL-spg or let-7i sponge (let-7i-Spg) was measured by immunoblotting. β-actin was used as loading control. (g) mRNA levels of E2F5, LIN28B, MYC and NRAS were measured in breast cancer patient samples that were wild-type for p53 (WTp53) or had a missense mutation in p53 (MTp53) by qRT–PCR relative to GAPDH. (h) Schematic model suggesting a mechanism by which mutant p53 drives cell cycle progression, migration and invasion. Mutant p53 (MTp53) inhibits let-7i expression by inhibiting p63 at the let-7i promoter to upregulate let-7i targets including NRAS, MYC, E2F5, LIN28B, DDX18 and CPSF1 to enhance cellular proliferation, migration and invasion. Error bars represent mean ±s.d. of three independent experiments. *P < 0.05; #P < 0.01; **P < 0.005; ##P < 0.001.

Article Snippet: 32 Immunoblotting Immunoblotting was performed using standard protocols using anti-p53 (D0–1, Santa Cruz, 1:5000), anti-p63 (H-137, Santa Cruz, 1:2500), anti-p73 (Bethyl, 1:5000), anti-HA High affinity (Roche, Indianapolis, IN, USA, 1:1000), anti-Ago2 (2A8 1:5000), anti-CPSF1 (Sigma, 1:2500), anti-Myc (9E10 Santa Cruz, 1:2500) and anti-β-actin (Sigma, 1:5000) antibodies. p53 mutational analysis in breast cancer patient samples Freshly frozen tumor specimens were obtained at the time of surgery from breast cancer patients and the p53 mutation status was determined as described previously.

Techniques: Modification, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Control, Luciferase, Plasmid Preparation, Stable Transfection, Mutagenesis, Migration

Journal: bioRxiv

Article Title: Heterogeneous potential of human airway basal cells: Holoclone-stem cell identification in a clinical-grade system

doi: 10.1101/2023.12.28.573530

Figure Lengend Snippet: (A) IF staining of AT (left) and AB (right) cultures at four different time points of airway epithelial cell lifespan (representative images of n = 3 AT and n = 3 AB primary human independent strains). Scale bars, 50 μm. (B) WB analysis on total cell extracts from six expansion passages (AT2 strain) representative of five consecutive lifespan intervals, immunostained with indicated antibodies. Experiment conducted on n = 3 AT strains (n = 5 technical replicates) and n = 3 AB strains (n = 4 technical replicates). (C) Histograms showing the quantification of the expression levels from top left to bottom right of CK14, Involucrin, p63α, BMI1 (all normalized per GAPDH) and SOX2 (normalized per Vinculin). Average and SD of n = 3 AT and n = 3 AB displayed per each time range (see “Star Methods”). Independent strains are indicated with different shapes. Unpaired, biparametric, two-tailed t test *p < 0.05.; ** p < 0.01 and *** p < 0.001.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p63-alpha (1:5000, customized), rabbit monoclonal anti-BMI1 (1:500, Cell Signalling Technology), rabbit monoclonal anti-SOX2 (1:200, Cell Signalling Technology), mouse monoclonal anti-GAPDH (1:10.000, Abcam), mouse monoclonal anti-Vinculin (1:10.000, Sigma-Aldrich), rabbit polyclonal anti-KERATIN 14 (1:40.000, Biolegend), mouse monoclonal anti-Involucrin (1:10.000, Leica Microsystems), mouse monoclonal anti-Actin (1:5000, Abcam).

Techniques: Staining, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Heterogeneous potential of human airway basal cells: Holoclone-stem cell identification in a clinical-grade system

doi: 10.1101/2023.12.28.573530

Figure Lengend Snippet: (A) Violin plot showing the size measured in mm of AT (left) and AB (right) different clones. AT analysed clones: H, n = 36; EM, n = 86; IM, n = 112; LM, n = 56; P, n = 45 belonging to n = 3 independent AT strains; AB analysed clones: H, n = 35; EM, n = 44; IM, n = 52; LM, n = 13; P, n = 9 belonging to n = 2 independent AB strains. Dots represent single clones. Median, first and third quartiles are displayed. (B) WB analysis on total cell extracts from the progeny of AT2 clones (H, n = 3; EM, n = 2; IM, n = 1; LM, n = 2) immunostained with indicated antibodies (image representative of n = 2 analysis conducted on independent clones). Due to their very limited residual proliferative potential, the amount of material collected by AT2 paraclone’s progenies was not sufficient to carry out a reliable WB analysis. (C) From left to right, histograms showing the quantification of the expression levels of p63α, BMI1 (normalized per GAPDH) and SOX2 (normalized per GAPDH in AT and Actin in AB). Average and SD displayed per each clonal category (see “Star Methods”). AT2 analysed clones: H, n = 3; EM, n = 5; IM, n = 5; LM, n = 3. AB1 analysed clones: H, n = 3; EM, n = 2; IM, n = 3; LM, n = 3. Unpaired, biparametric, two-tailed t test *p < 0.05.; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p63-alpha (1:5000, customized), rabbit monoclonal anti-BMI1 (1:500, Cell Signalling Technology), rabbit monoclonal anti-SOX2 (1:200, Cell Signalling Technology), mouse monoclonal anti-GAPDH (1:10.000, Abcam), mouse monoclonal anti-Vinculin (1:10.000, Sigma-Aldrich), rabbit polyclonal anti-KERATIN 14 (1:40.000, Biolegend), mouse monoclonal anti-Involucrin (1:10.000, Leica Microsystems), mouse monoclonal anti-Actin (1:5000, Abcam).

Techniques: Clone Assay, Expressing, Two Tailed Test

Journal: Scientific reports

Article Title: Assessment of the toxic effect of benzalkonium chloride on human limbal stem cells.

doi: 10.1038/s41598-025-96919-2

Figure Lengend Snippet: Fig. 3. (A) Agarose gel electropherogram of amplification products (passage 1). Lane 1: DNA ladder (M), lanes 2–3: KRT3 (101 bp), lanes 4–5: KRT12 (190 bp), lane 6: DNA ladder (M), lanes 7–8: TP63 (96 bp), lanes 9–10: ABCG2 (144 bp), lanes 11–12: ACTβ (131 bp), lane 13: DNA ladder (M). NC, negative control. (B) Visualization of lack of expression of CK3, and CK12 and expression of positive markers p63 and ABCG2, in LSCs cultured in a medium supplemented with H/I/E. FITC-conjugated specific antibodies (green); DAPI- stained cell nuclei (blue). IC, isotype control. Scale bars = 15 μm.

Article Snippet: After rinsing with PBS, the cells were incubated with blocking buffer for 1 h, rinsed again, and labelled with rabbit anti-ABCG2 polyclonal (1:50; 27286-AP; Proteintech, USA), rabbit anti-p63 polyclonal (1:50; 12143-1-AP; Proteintech, USA), mouse anti-CK3 monoclonal (1:50; ab68260; Abcam, UK), and rabbit anti-CK12 monoclonal (1:50; ab185627; Abcam, UK) primary antibodies at 4 °C overnight.

Techniques: Agarose Gel Electrophoresis, Amplification, Negative Control, Expressing, Cell Culture, Staining, Control

Journal: Circulation

Article Title: Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Cytoskeleton-Associated Protein 4 as a New Modulator of Fibroblasts Activation

doi: 10.1161/circulationaha.117.030742

Figure Lengend Snippet: Figure 6. Ckap4 is specifically expressed in fibroblasts post I/R, and in activated fibroblasts

Article Snippet: Cells were fixed with 4%PFA, quenched with NH4Cl, permeabilized, blocked with 1% fish gelatin (Gelatin from cold water fish skin, Sigma-Aldrich, #G7765) and incubated with rabbit anti-CKAP4 antibody (Proteintech #16686-1-AP) for 25min at RT prior to incubation with the corresponding secondary Alexa FluorTM 568 donkey anti-rabbit IgG (H+L) (Invitrogen, #A10042) together with Alexa FluorTM 488 Phalloidin (Invitrogen, #A12379), to stain for Factin, for 20min at RT.

Techniques:

Journal: Circulation

Article Title: Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Cytoskeleton-Associated Protein 4 as a New Modulator of Fibroblasts Activation

doi: 10.1161/circulationaha.117.030742

Figure Lengend Snippet: Figure 7. CKAP4 is co-expressed with markers of activated fibroblasts in human ischemic

Article Snippet: Cells were fixed with 4%PFA, quenched with NH4Cl, permeabilized, blocked with 1% fish gelatin (Gelatin from cold water fish skin, Sigma-Aldrich, #G7765) and incubated with rabbit anti-CKAP4 antibody (Proteintech #16686-1-AP) for 25min at RT prior to incubation with the corresponding secondary Alexa FluorTM 568 donkey anti-rabbit IgG (H+L) (Invitrogen, #A10042) together with Alexa FluorTM 488 Phalloidin (Invitrogen, #A12379), to stain for Factin, for 20min at RT.

Techniques: