Journal: JCI Insight

Article Title: FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium

doi: 10.1172/jci.insight.174888

Figure Lengend Snippet: ( A ) Representative immunoblot images and bar graphs showing densitometric analyses for p16, p21, and BCL-xL in CF ΔF508 and non-CF donors at ALI ( N = 5). ( B ) Representative images for SA-β-gal staining using brightfield imaging of the same CF ΔF508 and non-CF donor ALI cultures including quantification of β-gal staining using the ratio of SA-β-gal–positive cells per brightfield by ImageJ (NIH) ( N = 3); arrows show β-gal–positive cells (scale bar = 100 μm, original magnification, ×40). ( C ) Bar graphs demonstrating relative mRNA levels of SASP ( IL1B , IL6 , and IL8 ) markers normalized to GAPDH. ( D ) Bar graphs indicating relative mRNA levels of FGFR1–4 normalized to GAPDH in the same 2 groups. Statistical analysis was done using unpaired Student’s t test showing means ± SEM with * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 from 3–5 different donors per group with experiments repeated 3 times.

Article Snippet: Membranes were blocked with either 5% BSA or 5% low-fat milk depending on antibody manufacturer recommendations for 30 minutes, then incubated overnight with the following primary antibodies: rabbit anti-p21 (catalog 29478), rabbit anti–BCL-xL (catalog 2764S), rabbit anti-FGFR4 (catalog 8562S), rabbit total and phospho–anti-ERK1/2 (catalog 4695S and 9101S), rabbit total and phospho–anti-p38 MAPK (catalog 9212S and 4511S), rabbit total and phospho–anti-PLCγ1 (catalog 2822S and 8713S) (Cell Signaling Technology), mouse anti–β-actin–peroxidase (MilliporeSigma A1978), and rabbit anti-p16 (Proteintech 10883-1-AP) diluted according to the manufacturer’s recommendations.

Techniques: Western Blot, Staining, Imaging

Journal: JCI Insight

Article Title: FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium

doi: 10.1172/jci.insight.174888

Figure Lengend Snippet: ( A ) Representative immunoblot images and densitometric analyses of the cellular senescence markers p16, p21, and BCL-xL from CFBEs, which were treated with AZD4547 0.1 μM or BLU9931 0.1 μM for 24 hours. ( B ) Representative images of SA-β-gal staining in CFBEs treated with AZD4547 and BLU9931 and quantification by capturing 3 images from different regions of the cell culture plates and counting total cells and β-gal–positive cells from the 3 images to make a ratio of β-gal–positive cells to total cells; arrows indicate β-gal–positive cells (scale bar = 100 μm, original magnification, ×40). ( C ) Representative immunoblots and densitometric analysis for p-ERK/ERK, p-PLC/PLCγ, and p-p38/p38 MAPK in CFBEs treated with AZD4547 and BLU9931 for 24 hours with β-actin loading control. ( D ) Representative immunoblot images and densitometric analyses from primary bronchial epithelial ALI cultures of CF (ΔF508) donors and non-CF control donors for p-p38/p38 MAPK expression. Statistical analysis was done using unpaired Student’s t test showing means ± SEM with * P < 0.05, ** P < 0.01, and *** P < 0.001; 3 independent experiments were done in triplicates.

Article Snippet: Membranes were blocked with either 5% BSA or 5% low-fat milk depending on antibody manufacturer recommendations for 30 minutes, then incubated overnight with the following primary antibodies: rabbit anti-p21 (catalog 29478), rabbit anti–BCL-xL (catalog 2764S), rabbit anti-FGFR4 (catalog 8562S), rabbit total and phospho–anti-ERK1/2 (catalog 4695S and 9101S), rabbit total and phospho–anti-p38 MAPK (catalog 9212S and 4511S), rabbit total and phospho–anti-PLCγ1 (catalog 2822S and 8713S) (Cell Signaling Technology), mouse anti–β-actin–peroxidase (MilliporeSigma A1978), and rabbit anti-p16 (Proteintech 10883-1-AP) diluted according to the manufacturer’s recommendations.

Techniques: Western Blot, Staining, Cell Culture, Control, Expressing

Journal: JCI Insight

Article Title: FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium

doi: 10.1172/jci.insight.174888

Figure Lengend Snippet: ( A ) Representative immunoblot images and densitometric analyses showing p16, p21, and BCL-xL expression of CFBEs treated with SB203580 at 20 μM for 24 hours compared with controls. ( B ) Bar graphs showing protein levels of IL-6 and IL-8 in CFBE supernatant after treatment with SB203580 for 24 hours. ( C ) Representative images of SA-β-gal staining in control and SB203580-treated CFBEs including quantification; arrows indicate β-gal–positive cells (scale bar = 100 μm, original magnification, ×40). Statistical analysis was done using unpaired Student’s t test showing means ± SEM with * P < 0.05, ** P < 0.01 with n = 3–5 experiments.

Article Snippet: Membranes were blocked with either 5% BSA or 5% low-fat milk depending on antibody manufacturer recommendations for 30 minutes, then incubated overnight with the following primary antibodies: rabbit anti-p21 (catalog 29478), rabbit anti–BCL-xL (catalog 2764S), rabbit anti-FGFR4 (catalog 8562S), rabbit total and phospho–anti-ERK1/2 (catalog 4695S and 9101S), rabbit total and phospho–anti-p38 MAPK (catalog 9212S and 4511S), rabbit total and phospho–anti-PLCγ1 (catalog 2822S and 8713S) (Cell Signaling Technology), mouse anti–β-actin–peroxidase (MilliporeSigma A1978), and rabbit anti-p16 (Proteintech 10883-1-AP) diluted according to the manufacturer’s recommendations.

Techniques: Western Blot, Expressing, Staining, Control

Journal: JCI Insight

Article Title: FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium

doi: 10.1172/jci.insight.174888

Figure Lengend Snippet: ( A ) Immunohistochemical staining for p16, p21, and BCL-xL in Cftr –/– rat lung tissue compared to controls demonstrating an increased signal in the bronchial epithelium (scale bar = 100 μm, original magnification, ×10; for insets, ×20). ( B ) SA-β-gal stain and nuclear counterstain (DAPI) in lung tissue from Cftr –/– rats and littermate controls; arrows indicate areas of airway epithelial β-gal staining (scale bar = 100 μm, original magnification, ×40). ( C ) Relative mRNA levels of Cdkn2a (p16), Cdkn1a (p21), Bcl2l1 , and SASP markers ( Il1b , Il6 , and Cxcl2 [IL-8]) normalized to GAPDH, from total lung tissue of control and Cftr –/– rats. Statistical analysis was done using unpaired Student’s t test showing means ± SEM with * P < 0.05, ** P < 0.01 with n = 5–10 rats per group and experiments done in triplicates.

Article Snippet: Membranes were blocked with either 5% BSA or 5% low-fat milk depending on antibody manufacturer recommendations for 30 minutes, then incubated overnight with the following primary antibodies: rabbit anti-p21 (catalog 29478), rabbit anti–BCL-xL (catalog 2764S), rabbit anti-FGFR4 (catalog 8562S), rabbit total and phospho–anti-ERK1/2 (catalog 4695S and 9101S), rabbit total and phospho–anti-p38 MAPK (catalog 9212S and 4511S), rabbit total and phospho–anti-PLCγ1 (catalog 2822S and 8713S) (Cell Signaling Technology), mouse anti–β-actin–peroxidase (MilliporeSigma A1978), and rabbit anti-p16 (Proteintech 10883-1-AP) diluted according to the manufacturer’s recommendations.

Techniques: Immunohistochemical staining, Staining, Control

Journal: JCI Insight

Article Title: FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium

doi: 10.1172/jci.insight.174888

Figure Lengend Snippet: ( A ) Representative images of immunohistochemical staining for p16 and p21 in Cftr –/– and control rat lungs ± AZD4547 treatment (scale bar = 100 μm, original magnification, ×20). ( B ) Representative immunoblot images and bar graphs demonstrating densitometric analyses of p21 and BCL-xL protein expression in Cftr –/– rat lungs ± AZD4547 treatment. ( C ) Representative immunoblot images of phosphorylated and total p38 MAPK and densitometric analysis. ( D ) IL-8 protein levels in Cftr –/– rat lung tissue ± AZD4547 treatment. ( E ) Representative images showing mucociliary transport (MCT) (cross-sectional arrow in blue indicates the velocity of the mucus particle via the slope), along with representative µOCT images of the trachea of Cftr –/– rats (yellow line representing airway surface liquid [ASL] depth and the red line representing periciliary liquid depth; ep, epithelial layer; lp, lamina propria). ( F ) Bar graphs indicating analysis of µOCT images quantifying ASL, ciliary beat frequency (CBF), periciliary liquid depth (PCL), and MCT from Cftr –/– rat trachea after treatment for 5 days with AZD4547 (12.5 mg/kg) or sham. Statistical analysis was done using unpaired Student’s t test showing means ± SEM with * P < 0.05, ** P < 0.01, with n = 3–4 rats per group.

Article Snippet: Membranes were blocked with either 5% BSA or 5% low-fat milk depending on antibody manufacturer recommendations for 30 minutes, then incubated overnight with the following primary antibodies: rabbit anti-p21 (catalog 29478), rabbit anti–BCL-xL (catalog 2764S), rabbit anti-FGFR4 (catalog 8562S), rabbit total and phospho–anti-ERK1/2 (catalog 4695S and 9101S), rabbit total and phospho–anti-p38 MAPK (catalog 9212S and 4511S), rabbit total and phospho–anti-PLCγ1 (catalog 2822S and 8713S) (Cell Signaling Technology), mouse anti–β-actin–peroxidase (MilliporeSigma A1978), and rabbit anti-p16 (Proteintech 10883-1-AP) diluted according to the manufacturer’s recommendations.

Techniques: Immunohistochemical staining, Staining, Control, Western Blot, Expressing

Journal: JCI Insight

Article Title: FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium

doi: 10.1172/jci.insight.174888

Figure Lengend Snippet: ( A ) Immunohistochemistry of p16 and p21 from ex vivo Cftr –/– rat tracheae treated with BLU9931 at 0.1 M for 24 hours compared with vehicle-treated “control” Cftr –/– rat tracheae. ( B ) mRNA levels of Cdkn2a (p16), Cdkn1a (p21), Bcl2 , and Bcl2l1 (BCL-xL) and SASP markers ( C ) Il1b , Il6 , and Cxcl2 (IL8) from the ex vivo Cftr –/– rat tracheae +/– BLU9931. ( D ) Representative µOCT images (ep, epithelial layer; lp, lamina propria) and ( E ) bar graphs showing µOCT quantification of periciliary liquid depth (PCL), mucociliary transport (MCT), ciliary beat frequency (CBF), and airway surface liquid (ASL) depth both from BLU9931 and vehicle control treated ex vivo Cftr –/– rat tracheae. Statistical analysis was done using unpaired Student’s t test showing means ± SEM with * P < 0.05, ** P < 0.01, with n = 3–4 rat trachea per group.

Article Snippet: Membranes were blocked with either 5% BSA or 5% low-fat milk depending on antibody manufacturer recommendations for 30 minutes, then incubated overnight with the following primary antibodies: rabbit anti-p21 (catalog 29478), rabbit anti–BCL-xL (catalog 2764S), rabbit anti-FGFR4 (catalog 8562S), rabbit total and phospho–anti-ERK1/2 (catalog 4695S and 9101S), rabbit total and phospho–anti-p38 MAPK (catalog 9212S and 4511S), rabbit total and phospho–anti-PLCγ1 (catalog 2822S and 8713S) (Cell Signaling Technology), mouse anti–β-actin–peroxidase (MilliporeSigma A1978), and rabbit anti-p16 (Proteintech 10883-1-AP) diluted according to the manufacturer’s recommendations.

Techniques: Immunohistochemistry, Ex Vivo, Control

Journal: JCI Insight

Article Title: FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium

doi: 10.1172/jci.insight.174888

Figure Lengend Snippet: ( A ) Immunohistochemistry of Cdkn2a (p16) and Cdkn1a (p21) of Cftr –/– rat tracheae, which were treated with 100 nM dasatinib and 2 μM quercetin (D+Q) for 24 hours, compared with vehicle-treated Cftr –/– rat tracheae. Scale bar is 100 µm. ( B ) Representative µOCT images of the different Cftr –/– rat trachea groups (ep, epithelial layer; lp, lamina propria), including representative images to assess mucociliary transport (MCT). ( C ) Bar graphs showing quantification of all regions of interest from µOCT images for assessment of ASL, MCT, CBF, and PCL from the vehicle- and D+Q-treated groups. ( D ) mRNA levels of SASP markers ( Il1b , Il6 , and Cxcl2 [IL-8]) along with ( E ) senescence markers Cdkn2a (p16), Cdkn1a (p21), Bcl2 , and Bcl2l1 (BCL-xL) from Cftr –/– rat tracheae ± D+Q. Statistical analysis was done using unpaired Student’s t test showing means ± SEM with * P < 0.05, ** P < 0.01 with n = 3–4 rat tracheae per group.

Article Snippet: Membranes were blocked with either 5% BSA or 5% low-fat milk depending on antibody manufacturer recommendations for 30 minutes, then incubated overnight with the following primary antibodies: rabbit anti-p21 (catalog 29478), rabbit anti–BCL-xL (catalog 2764S), rabbit anti-FGFR4 (catalog 8562S), rabbit total and phospho–anti-ERK1/2 (catalog 4695S and 9101S), rabbit total and phospho–anti-p38 MAPK (catalog 9212S and 4511S), rabbit total and phospho–anti-PLCγ1 (catalog 2822S and 8713S) (Cell Signaling Technology), mouse anti–β-actin–peroxidase (MilliporeSigma A1978), and rabbit anti-p16 (Proteintech 10883-1-AP) diluted according to the manufacturer’s recommendations.

Techniques: Immunohistochemistry

Journal: JCI Insight

Article Title: FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium

doi: 10.1172/jci.insight.174888

Figure Lengend Snippet: ( A ) Representative µOCT images and bar graph of CF primary human bronchial epithelial cells ( n = 3 donors per group) treated with VX-661/VX-445/VX-770 (ETI) for 72 hours showing a significant increase in ASL depth in CF cells treated with ETI; yellow line is ASL depth. ep, epithelial layer; F, filter. ( B ) Representative immunoblots and densitometric analysis showing no significant difference in protein expression of cellular senescence markers p16, p21, and BCL-xL in CF primary human bronchial epithelial cells treated with ETI for 72 hours when compared with untreated CF primary human bronchial epithelial cells ( n = 4 donors per group). ( C ) Relative expression of SASP markers ( Il1b , Il6 , and Cxcl2 [IL-8]) from CF primary human bronchial epithelial cells demonstrates no significant change when compared with untreated CF primary human bronchial epithelial cells. ( D ) Immunoblots and densitometric analysis of p21 and BCL-xL in hG551D rats and hG551D rats treated with VX-770 for 14 days (hG551d = 3 rats, hG551d+VX-770 = 5 rats). ( E ) Relative expression of SASP markers Il1b , Il6 , and Cxcl2 (IL-8) in hG551D rats and hG551D rats treated with VX-770 for 14 days (hG551d = 3 rats, hG551d+VX-770 = 5 rats). Statistical analysis was done using unpaired Student’s t test showing means ± SEM with * P < 0.05.

Article Snippet: Membranes were blocked with either 5% BSA or 5% low-fat milk depending on antibody manufacturer recommendations for 30 minutes, then incubated overnight with the following primary antibodies: rabbit anti-p21 (catalog 29478), rabbit anti–BCL-xL (catalog 2764S), rabbit anti-FGFR4 (catalog 8562S), rabbit total and phospho–anti-ERK1/2 (catalog 4695S and 9101S), rabbit total and phospho–anti-p38 MAPK (catalog 9212S and 4511S), rabbit total and phospho–anti-PLCγ1 (catalog 2822S and 8713S) (Cell Signaling Technology), mouse anti–β-actin–peroxidase (MilliporeSigma A1978), and rabbit anti-p16 (Proteintech 10883-1-AP) diluted according to the manufacturer’s recommendations.

Techniques: Western Blot, Expressing

Journal: Oncology Letters

Article Title: MicroRNA-2053 overexpression inhibits the development and progression of hepatocellular carcinoma

doi: 10.3892/ol.2019.10501

Figure Lengend Snippet: miR-2053 inhibits the PI3K signaling pathway. (A) Protein expression levels of total and p-AKT, total and p-mTOR, cyclin D1 and p70. (B) Ratios of p-AKT/AKT and p-mTOR/mTOR, and the expression of cyclin D1 and p70. n=3, *P<0.05 vs. pNC control group. pNC, cells transfected with pCMV-MIR empty vector; miR-2053, cells transfected with pCMV-MIR- miR-2053 vector, miR-2053 overexpression group; p, phosphorylated; AKT, serine/threonine protein kinase; mTOR, Mammalian target of rapamycin.

Article Snippet: The following primary antibodies were used for analysis: Anti-caspase-9 (cat. no. ab219590; 1:1,000), anti-AKT (cat. no. ab32505, 1:1,000), anti-phosphorylated AKT (cat. no. ab81283, 1:1,000), anti-mTOR (cat. no. ab2732, 1:1,000), anti-phosphorylated mTOR (cat. no. ab131538, 1:100), anti-Wnt3 (cat. no. ab32249, 1:10), anti-β-catenin (cat. no. ab32572, 1:1,000, all Abcam, Cambridge, UK), anti-E-cadherin (cat. no. 3195; 1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-Cyclin D1 (cat. no. 60186-1-Ig, 1:1,000), anti-p70 (cat. no. 14485-1-AP, 1:1,000), anti-B-cell lymphoma 2 (BCL2; cat. no. 60178-1-Ig, 1:1,000), anti-BCL-2-associated X protein (BAX; cat. no. 60267-1-Ig, 1:1,000) and anti-cleaved caspase-3 (cat. no. 25546-1-AP, 1:1,000) and GAPDH (cat. no. 60004-1-Ig, 1:5,000; all Proteintech Group, Inc.), which served as the loading control.

Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression

Journal: British Journal of Cancer

Article Title: CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner

doi: 10.1038/s41416-021-01269-1

Figure Lengend Snippet: a , b The protein levels of CREPT and p-STAT3 in human breast cancers ( a ) and colon cancers ( b ). N refers to paired normal tissue and T refers to tumour tissue from the same patient. The bands were quantified using ImageJ and presented as a value normalised according to the moderate level of a band in each blot. c A graph presentation of CREPT/actin and p-STAT3/STAT3 in adjacent normal and tumour tissues. The grey values in a , b were used to calculate the ratio of CREPT/actin or p-STAT3/STAT3. Each sample was dotted by closed circle (normal tissue for CREPT/actin), square (tumor tissue for CREPT/actin), triangle (normal tissue for p-STAT3/STAT3) and inverted triangle (tumor tissue for p-STAT3/STAT3), and the average was subject to a statistic analysis between normal and tumour tissues (* p < 0.05). d , e Representative images for CREPT and p-STAT3 levels using immunohistochemical staining in breast ( d ) and colon ( e ) cancers with an antibody against CREPT or p-STAT3(Y705). f , g Representative images for CREPT and p-STAT3 in breast ( f ) and colon ( g ) tumour tissue and adjacent normal tissue.

Article Snippet: Anti-p300 (D2X6N), anti-c-MYC, anti-cyclinD1 (SP4), anti-pY705 STAT3 (D3A7), anti-pS727 STAT3, anti-acK685 STAT3, anti-Bcl-XL (54H6), anti-H3K18ac (D8Z5H) and anti-H3K27ac (D5E4) antibodies were purchased from Cell Signaling Technology.

Techniques: Immunohistochemical staining, Staining

Journal: British Journal of Cancer

Article Title: CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner

doi: 10.1038/s41416-021-01269-1

Figure Lengend Snippet: a – d Overexpression of CREPT promotes the APRE-luciferase activity. Luciferase assay was performed in wild-type or CREPT transiently overexpressing cells in the absence or presence of LIF (20 ng/ml) (** p < 0.01; *** p < 0.001). e – h Depletion of CREPT using a mixture of siRNA#1 and siRNA#2 resulted in a decreased transcriptional activity of STAT3 (* p < 0.05; ** p < 0.01; *** p < 0.001). i CREPT promotes the expression of STAT3-targeted genes. Flag-CREPT plasmid was transfected into MCF7 cells with or without LIF (20 ng/ml) for 4 h. The mRNA levels of STAT3-targeted genes were examined by qRT-PCR. j Depletion of CREPT using a mixture of siRNA#1 and siRNA#2 impairs the expression of STAT3-targeted genes in response to LIF. k Overexpression of CREPT enhanced the protein levels of STAT3-targeted genes. Flag-CREPT plasmid was transfected into MCF7 (left) or SW480 (right) cells with or without LIF (20 ng/ml) for 12 h. The protein levels of STAT3-targeted genes were examined by Western blots. l Depletion of CREPT decreased the protein levels of STAT3-targeted genes.

Article Snippet: Anti-p300 (D2X6N), anti-c-MYC, anti-cyclinD1 (SP4), anti-pY705 STAT3 (D3A7), anti-pS727 STAT3, anti-acK685 STAT3, anti-Bcl-XL (54H6), anti-H3K18ac (D8Z5H) and anti-H3K27ac (D5E4) antibodies were purchased from Cell Signaling Technology.

Techniques: Over Expression, Luciferase, Activity Assay, Expressing, Plasmid Preparation, Transfection, Quantitative RT-PCR, Western Blot

Journal: British Journal of Cancer

Article Title: CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner

doi: 10.1038/s41416-021-01269-1

Figure Lengend Snippet: a Myc-CREPT interacts with Flag-STAT3 in response to LIF. b The interaction of CREPT and STAT3 occurs in the nucleus. The nuclear marker JunB and the cytoplasmic marker tubulin were used to demonstrate the purity of fractions. c Interaction of CREPT with STAT3 depends on STAT3 phosphorylation rather than its nuclear localisation. d , e CREPT interacts with STAT3 physically. A GST pull-down assay was performed with purified GST or GST-CREPT protein and Flag-STAT3 from HEK293T cell lysates ( d ). A reciprocal GST pull-down assay was performed with purified GST or GST-STAT3 protein and Flag-CREPT from HEK293T cell lysates ( e ). f , g Endogenous interaction between CREPT and STAT3. Nuclear extracts from MCF7 ( f ) or SW480 ( g ) cells were immunoprecipitated with an anti-CREPT antibody. h The RPR domain, but not CCT domain of CREPT interacts with Myc-STAT3. i CREPT preferentially interacts with the SH2-CT domain of STAT3. j CREPT co-localises with STAT3 upon LIF treatment. MCF7 cells were treated with LIF (20 ng/ml) or a control medium for 30 min, followed with immunostaining using an anti-CREPT antibody and an anti-STAT3 antibody. k CREPT and STAT3 co-occupied at the promoter region of STAT3-targeted genes. ChIP assay was performed with the CREPT or STAT3 antibody in MCF7 cells with or without the treatment of LIF (20 ng/ml) for 30 min.

Article Snippet: Anti-p300 (D2X6N), anti-c-MYC, anti-cyclinD1 (SP4), anti-pY705 STAT3 (D3A7), anti-pS727 STAT3, anti-acK685 STAT3, anti-Bcl-XL (54H6), anti-H3K18ac (D8Z5H) and anti-H3K27ac (D5E4) antibodies were purchased from Cell Signaling Technology.

Techniques: Marker, Pull Down Assay, Purification, Immunoprecipitation, Immunostaining

Journal: British Journal of Cancer

Article Title: CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner

doi: 10.1038/s41416-021-01269-1

Figure Lengend Snippet: a , b Deletion of CREPT decreases the level of ac-H3K27 ( a ) or ac-H3K18 ( b ) on the APRE region. c – e Loss of CREPT decreases chromatin accessibility at the promoter region of STAT3. ATAC-seq peaks at the promoter regions of the STAT3-targeted genes from wild-type (blue) or CREPT depletion (green) MCF7 cells were shown. The STAT3 binding consensus sequence was labelled at the corresponding locus. f , g The endogenous interaction between CREPT and p300. Nuclear extracts from MCF7 ( f ) or SW480 ( g ) cells were immunoprecipitated with an anti-CREPT antibody. h CREPT, STAT3 and p300 formed a ternary complex in MCF7 cells. Cell lysates were precipitated by an anti-CREPT antibody. i , j CREPT enhances the interaction of STAT3 and p300. Flag-STAT3 was co-expressed with Myc-CREPT ( I ) or siRNAs against endogenous CREPT ( j ) in MCF7 cells. k Overexpression of CREPT hardly affected the interaction between STAT3 and GCN5. l , m Deletion of CREPT attenuates p300 ( l ) or RNAPII ( m ) occupancy on the promoters of STAT3-targeted genes. The quantity indicating the presence of p300 ( l ) or RNAPII ( m ) on APRE was normalised with respective inputs. (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Anti-p300 (D2X6N), anti-c-MYC, anti-cyclinD1 (SP4), anti-pY705 STAT3 (D3A7), anti-pS727 STAT3, anti-acK685 STAT3, anti-Bcl-XL (54H6), anti-H3K18ac (D8Z5H) and anti-H3K27ac (D5E4) antibodies were purchased from Cell Signaling Technology.

Techniques: Binding Assay, Sequencing, Immunoprecipitation, Over Expression

Journal: British Journal of Cancer

Article Title: CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner

doi: 10.1038/s41416-021-01269-1

Figure Lengend Snippet: a , b Depletion of p300 impaired the enhanced effect of CREPT on STAT3 transcriptional activity. c – g Depletion of p300 impaired the STAT3-targeted gene expression promoted by CREPT both at mRNA ( c – e ) and protein ( f , g ) levels examined by QRT-PCR and western blot. h – k CREPT promotes colony formation dependent on p300. Colony formation assays were performed in MCF7 ( h , i ) or SW480 ( j , k ) cells. A representative colony is shown in ( h , J ) and the quantitative numbers are shown in ( i , k ). l A model indicating that highly expressed CREPT in tumour cells facilitates the recruitment of p300 to the promoter of STAT3-targeted genes (* p < 0.05; ** p < 0.01; *** p < 0.001; n.s., no statistical difference).

Article Snippet: Anti-p300 (D2X6N), anti-c-MYC, anti-cyclinD1 (SP4), anti-pY705 STAT3 (D3A7), anti-pS727 STAT3, anti-acK685 STAT3, anti-Bcl-XL (54H6), anti-H3K18ac (D8Z5H) and anti-H3K27ac (D5E4) antibodies were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Western Blot