Journal: Cancers

Article Title: miR766-3p and miR124-3p Dictate Drug Resistance and Clinical Outcome in HNSCC

doi: 10.3390/cancers14215273

Figure Lengend Snippet: The role of miR124-3p and miR766-3p target genes in HNSCC drug resistance. ( A ) Expression analysis of miR124-3p and miR766-3p direct target genes and downstream target genes by Western blot in HNSCC cell lines (CAL27 and FaDu), with or without transfection with miRNA inhibitors or miRNA mimics. Left: Western blotting showed the expression of miR124-3p target gene (CREBRF) and CREBRF target genes (ATG5 and CREB3). Right: Western blotting showed the expression of miR766-3p target gene (NR3C2) and NR3C2 target genes (β-catenin and c-Myc). Quantitative data (relative expression levels after β-actin-corrected) from three independent experiments are disclosed below each protein band. Data represent the mean ± SD (n = 3). ( B ) Target gene analysis in sensitive (CAL27) vs. resistant (CAL27/FP-R) HNSCC cell lines. Quantitative data (relative expression levels after β-actin-corrected) is shown below each protein band. ( C ) The effect of NR3C2 and/or CREBRF knockdown on drug-induced cytotoxicity in CAL27 and FaDu. Cells were transfected by 10 nM siRNA (single or combined) for 24 h followed by 72 h exposure to the indicated drug. Cytotoxicity was determined by MTT assay. IC 50 values are listed in . ( D ) Measurement of apoptosis in CAL27 or FaDu cells in response to cisplatin or 5-FU ± 24 h prior transfection with 10 nM siRNA. After 24 h of drug treatments, cells were labeled with anti-annexin V-FITC antibody and PI and then analyzed with flow cytometry. A two-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons, and data are presented as means ± SD (n = 3). * p < 0.05, *** p < 0.001 (vs. control siRNA), and ### p < 0.001 (vs. untreated).

Article Snippet: The polyvinylidene difluoride membranes were incubated with specific antibodies, including CREBRF rabbit monoclonal antibody (mAb) (ThermoFisher, PA5-68552; 1:1000), CREB3 rabbit mAb (Proteintech, 11275-1-AP; 1:1000), NR3C2 mouse mAb (Novus, NB300-562; 1:1000), c-Myc rabbit mAb (Novus, NB600-336; 1:1000), ATG5 rabbit mAb (Novus, NB110-53818SS; 1:1000), β-catenin mouse mAb (Novus, NBP1-54467SS; 1:1000), and β-actin mouse mAb (Sigma-Aldrich, A3854; 1:1000).

Techniques: Expressing, Western Blot, Transfection, Knockdown, MTT Assay, Labeling, Flow Cytometry, Control

Journal: Cancers

Article Title: miR766-3p and miR124-3p Dictate Drug Resistance and Clinical Outcome in HNSCC

doi: 10.3390/cancers14215273

Figure Lengend Snippet: Downregulation of CREBRF and NR3C2 increase poor prognosis in HNSCC. ( A ) Histological analysis of miR124-3p and miR766-3p target gene expression (CREBRF-ATG5/CREB3, NR3C2-β-catenin/c-Myc) in Responder vs. Non-Responder HNSCC clinical samples. Magnification, x100. Scale bar, 210 µm. The quantitative data from all specimens are shown in the bar chart. Each dot in the graph represents an individual clinical sample. Two-sided unpaired Student t test was used to analyze comparisons, and data are presented as means ± SEM. * p < 0.05 and *** p < 0.001. ( B ) Histological analysis of tumor morphology in relation to miR124-3p and miR766-3p target gene expression. Representative images of CREBRF, ATG5, CREB3, NR3C2, β-catenin, and c-Myc expression in the serial section of responder and non-responder HNSCC specimens. BV: Blood Vessel. T: Tumor. N: Normal tissue. The invasive cancer cells are indicated by red arrowhead. Magnification, ×200. Scale bar, 100 µm. ( C ) Summary of resistance mechanisms regulated by miR124-3p and miR766-3p. Our data indicated that upon acquired resistance in HNSCC cells or in non-responder HNSCC tumors, the levels of miR124-3p and miR766-3p go up, which in turn down-regulate its direct target genes: CREBRF and NR3C2, and consequently the expression of downstream targets of CREBRF (ATG5/CREB3) and NR3C2 (β-catenin/c-Myc) increased in resistant tumors, which are positively correlated with poor prognosis. Thus, by enhancing the CREBRF-ATG5/CREB3 and NR3C2-β-catenin/c-Myc axis, miR124-3p and miR766-3p support aggressive HNSCC progression.

Article Snippet: The polyvinylidene difluoride membranes were incubated with specific antibodies, including CREBRF rabbit monoclonal antibody (mAb) (ThermoFisher, PA5-68552; 1:1000), CREB3 rabbit mAb (Proteintech, 11275-1-AP; 1:1000), NR3C2 mouse mAb (Novus, NB300-562; 1:1000), c-Myc rabbit mAb (Novus, NB600-336; 1:1000), ATG5 rabbit mAb (Novus, NB110-53818SS; 1:1000), β-catenin mouse mAb (Novus, NBP1-54467SS; 1:1000), and β-actin mouse mAb (Sigma-Aldrich, A3854; 1:1000).

Techniques: Targeted Gene Expression, Expressing

Journal: iScience

Article Title: CREB3L4 promotes hepatocellular carcinoma progression and decreases sorafenib chemosensitivity by promoting RHEB-mTORC1 signaling pathway

doi: 10.1016/j.isci.2024.108843

Figure Lengend Snippet:

Article Snippet: CREB3L4 , Proteintech , Cat No. 13630-1-AP; RRID: AB_2276550.

Techniques: Virus, Recombinant, CCK-8 Assay, Chromatin Immunoprecipitation, Reporter Assay, Expressing, Sequencing, Plasmid Preparation, Software

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Illustration showing the localization of CREB3-FL and CREB3-CF and changes in nuclear morphology. b Western blotting showing the localization of endogenous CREB3-FL and CREB3-CF in the cytosolic and nuclear fractions. c Illustration showing the nuclear membrane localization of CREB3-FL and CREB3-FL-mtS1P. d Left panels: Diagram showing the experimental strategies used to obtain the extracts of the cytosolic and nuclear fractions. Right panels, Western blotting showing nuclear DNA bound to CREB3-FL, CREB3-CF, and CREB3-dTM. e Confocal microscopy image illustrating the nuclear membrane localization of the Myc-CREB3-FL-HA double-tagged protein. f Diagram showing the experimental strategy used to determine the extent of nuclear DNA binding to CREB3-FL, CREB3-CF, and CREB3-FL-mtS1P. g Western blotting showing nuclear DNA binding of CREB3-FL, CREB3-CF, and CREB3-FL-mtS1P in the presence of NaCl. h Illustration of the localization and orientation of CREB3 at the nuclear membrane.

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Western Blot, Membrane, Confocal Microscopy, Binding Assay

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Illustration showing the relationship between the localization of CREB3-FL, CREB3-dTM and CREB3-CF and changes in nuclear morphology. b ICF images showing the relationship between domain deletion of CREB3-CF and changes in nuclear morphology. c ICF images showing the relationship between the serial deletion of CREB3-CF and changes in nuclear morphology. d ICF images showing the effects of CREB3-FL, CREB3-CF, and CREB3-dTM on DDR, DNA herniation, and microsatellite formation. e Illustration showing Myc-CREB-FL-HA at the inner nuclear membrane and nuclear membrane ripping and aggregation via karyoptosis. f Illustration of the frequency of nuclear abnormalities in relation to CREB3-CF expression. The error bars indicate the SEMs. *** P < 0.0001 (Student’s t test).

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Membrane, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Illustration of nuclear membrane deformation-induced nuclear DNA loss. Left panels, Illustration of CREB3-CF-induced nuclear membrane deformity and loss of nuclear DNA. Central panels. Magnification of the boxed area in the left panels. Graphs, Comparison of DAPI intensity between CREB3-CF-negative and CREB3-CF-positive cells ( n = 56–87 cells). The error bars indicate the SEMs. * P < 0.05, *** p < 0.0001 (Student’s t test). b IP/Western blots showing the interaction of Lamin A and B1 with CREB3-CF. c Illustration of the interaction between the bZIP domain of CREB3-CF and Lamin B1. d Transmission electron microscopy images showing nuclear membrane abnormalities in terms of shape and integrity in mock- and CREB3-CF-overexpressing cells. Arrows indicate nuclear membrane invagination and membrane integrity loss. e Illustration of explosive nuclear membrane rupture during karyoptosis. Frame 1, image showing normal nuclear morphology before karyoptosis initiation; Frame 2, image showing initiation of karyoptosis; Frame 3, image of early-stage karyoptosis; Frame 4, image of the moment of karyoptotic nucleus explosion (karyoptosis); Frame 5, image after karyoptotic nucleus explosion. The live and time-lapse videos are in Supplementary Live Images, Supplementary Live Image – .

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Membrane, Comparison, Western Blot, Transmission Assay, Electron Microscopy

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Illustration showing that CREB3-CF-induced karyoptosis evokes DDR, indicating an increase in γH2AX. b Illustration showing that CREB3-CF-induced karyoptosis is decoupled from the p53-dependent DDR signaling pathway. c ICF images showing that loss of CREB3 anchoring at the nuclear membrane induces nuclear lobulation, invagination, and nuclear DNA herniation. d Western blots indicating that CREB3-CF-induced karyoptosis does not induce the cleavage of apoptosis markers. e Western blots indicating that CREB3-CF-induced karyoptosis inhibits autophagy. f ICF analysis showing that CREB3-CF overexpression increased the nuclear accumulation of LC3. g ICF image showing that CREB3-Cf increases the formation of extracellular vesicles containing CREB3-CF, lamin, and genomic DNA. Right panels, TEM images illustrating that CREB3-CF overexpression induces abnormal nuclear morphology and increases extracellular vesicle formation. h Western blots showing that CREB3-CF-induced karyoptosis differs from necroptosis. i Western blots showing that CREB3-CF-induced karyoptosis is distinguishable from pyroptosis.

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Membrane, Western Blot, Over Expression

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Western blot showing that UVB exposure increases the endogenous protein levels of CREB3-FL and CREB3-CF. b Western blot analyses revealing that ER stress, induced by vesicle trafficking inhibitors such as brefeldin A and golgicide A, leads to increased cleavage of CREB3-FL and subsequent production of CREB3-CF. c Western blot results indicating that ER stress, triggered by elevated levels of Ca 2+ and ROS, moderately increases the protein levels of CREB3-FL and CREB3-CF, while the inhibition of glycosylation does not have this effect. d Western blot analyses showing that direct DNA damage caused by cisplatin and doxorubicin, but not oxaliplatin, slightly increases CREB3-CF protein levels without affecting CREB3-FL protein levels. e Western blot analyses illustrating the changes in the protein levels of CREB3-FL and CREB3-CF over time following washout of BFA. f TEM images demonstrating that BFA damages the nuclear membrane. g ICF image revealing that both overexpressed and endogenous S1P are localized not only to the cytoplasm but also to the nuclear membrane.

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Western Blot, Inhibition, Glycoproteomics, Membrane

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Illustration showing the sampling strategies used for proteomic analysis of CREB3-CF-induced karyoptosis. The comparisons used were as follows: analysis 1, mock and CREB3-FL; analysis 2, mock and CREB3-CF; analysis 3 CREB3-CF and mock+UVB, mock+UVB and mock, CREB3-CF and mock, and CREB3-CF/mock+UVB and mock; and analysis 4, mock+UVB and CREB3-FL + UVB. b Illustration of the distances of the protein sets in 5 different cells in Fig. . c Hit map of CREB3-CF-induced karyoptosis according to analysis 2. Red, increase; green, decrease. d Protein frequency of the Cluster 2 proteins in CREB3-CF-induced karyoptotic cells according to analysis 2. e GO analysis of Cluster 2 proteins in CREB3-CF-induced karyoptotic cells according to analysis 2. A detailed list is provided in Supplementary Table . f Hit maps of CREB3-CF, mock+UVB, and mock to compare CREB3-CF and mock+UVB; mock+UVB and mock CREB3-CF and mock, and CREB3-CF/mock+UVB and mock according to analysis 3. Red, increase; green, decrease. g Illustration showing the protein frequency of Cluster 1 and 2 proteins in CREB3-CF-induced karyoptotic cells according to analysis 3. h GO analysis of Cluster 1 and 2 proteins in CREB3-CF-induced karyoptotic cells according to analysis 3. A detailed list is provided in Supplementary Table .

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Sampling

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Graphs showing cellular senescence induction by the expression of CREB3-FL or CREB3-CF. Left, percentage of β-gal-positive cells among more than 200 cells. Right, Fold increase in β-gal-positive cells among cancer cells. The cells stained with β-gal are shown in Supplementary Fig. . b CREB3-CF induces p21 protein expression in SK-MEL-2 and HeLa cells. c Graphs showing that the expression of CREB3-CF or CREB3-dTM, but not CREB3-FL, suppresses the proliferation of SK-MEL-2 cells. The cells stained with crystal violet are shown in Supplementary Fig. . d hat UVB increases CREB3-FL and CREB3-CF expression. e UVB induces DDR via the detection of nuclear puncta of γH2AX. f Illustration of the increase in nuclear DNA bound to endogenous CREB3-FL and -CF by UVB irradiation. g Cell cycle analysis data showed that the increase in UVB dose was not associated with sub-G1 enhancement. h Flow cytometry data showing that the early necrosis/necroptosis population is not associated with the late apoptosis/necrosis/necroptosis population. In contrast, UVB irradiation continuously decreased the live cell population in a dose-dependent manner. i Images of confocal microscopy images showing nuclear membrane ripping and rupture in CREB3-FL-overexpressing cells after UVB treatment.

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Expressing, Staining, Irradiation, Cell Cycle Assay, Flow Cytometry, Confocal Microscopy, Membrane