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Addgene inc pmh sfb vectors
Pmh Sfb Vectors, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pmh+sfb+vectors/pm40594913-139-11-16?v=Addgene+inc
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Fig. 1. Loss of DYRK1A suppresses self-renewal and tumorigenic potential of OSCC. (A) Deletion of DYRK1A was performed in two OSCC cell lines (SCC4 and FaDu) by CRISPR/Cas9 genome editing technique. Deletion of DYRK1A was confirmed by Western blot. GAPDH was used as a loading control. WT; Wild-type, KO; DYRK1A knockout. Arrowhead indicates the DYRK1A-specific band, and asterisk indicates a nonspecific cross-reacting band. (B) Effect of DYRK1A deletion on self-renewal capacity was determined by tumorsphere formation assay. Representative images of tumorspheres formed by the WT and DYRK1A KO cells were shown on the right of each bar graph. Bar indicates 200 μm. Data are means ± SD of triplicate experiment. *P < 0.01 compared to WT. (C) Effect of DYRK1A deletion on anchorage- independent growth ability was determined by soft agar assay. *P < 0.01 compared to WT. (D) Effect of DYRK1A deletion on in vivo tumorigenicity was determined by xenograft tumor assay. FaDu WT and FaDu DYRK1A KO were injected subcutaneously into 5 nude mice. Tumor volumes were measured for 3 weeks. (E) Tumor images and tumor incidence of FaDu WT and FaDu DYRK1A KO were obtained 3 weeks after the injections.

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 1. Loss of DYRK1A suppresses self-renewal and tumorigenic potential of OSCC. (A) Deletion of DYRK1A was performed in two OSCC cell lines (SCC4 and FaDu) by CRISPR/Cas9 genome editing technique. Deletion of DYRK1A was confirmed by Western blot. GAPDH was used as a loading control. WT; Wild-type, KO; DYRK1A knockout. Arrowhead indicates the DYRK1A-specific band, and asterisk indicates a nonspecific cross-reacting band. (B) Effect of DYRK1A deletion on self-renewal capacity was determined by tumorsphere formation assay. Representative images of tumorspheres formed by the WT and DYRK1A KO cells were shown on the right of each bar graph. Bar indicates 200 μm. Data are means ± SD of triplicate experiment. *P < 0.01 compared to WT. (C) Effect of DYRK1A deletion on anchorage- independent growth ability was determined by soft agar assay. *P < 0.01 compared to WT. (D) Effect of DYRK1A deletion on in vivo tumorigenicity was determined by xenograft tumor assay. FaDu WT and FaDu DYRK1A KO were injected subcutaneously into 5 nude mice. Tumor volumes were measured for 3 weeks. (E) Tumor images and tumor incidence of FaDu WT and FaDu DYRK1A KO were obtained 3 weeks after the injections.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: CRISPR, Western Blot, Control, Knock-Out, Tube Formation Assay, Soft Agar Assay, In Vivo, Injection

Fig. 2. Loss of DYRK1A diminishes CSC-enriched ALDH1HIGH population and CSC properties in OSCC. (A) Effect of DYRK1A deletion on ALDH1HIGH cell population in SCC4 and FaDu was determined by Aldefluor assay. Cells were labeled with Aldefluor with and without the ALDH inhibitor DEAB and analyzed by flow cytometry. The gate for ALDH1HIGH cells is determined in relation to the DEAB control (+DEAB) and shows the brightly fluorescent ALDH population versus the side scatter, a population that is absent/decreased in the presence of DEAB. The number shown in each panel reflects the percentage of ALDH1HIGH cells in each cell type. A similar result was obtained from an independent experiment (Supplemental Fig. 2). (B) Effect of DYRK1A deletion on migration ability was determined by transwell migration assay. Migration ability was described as number of migrated cells per field with data as mean ± SD for three randomly selected fields. Representative images of migrated cells from each group were shown on the right of each bar graph. Bar indicates 200 μm *P < 0.01 (C) Effect of DYRK1A deletion on chemo resistance was determined by MTT assay. SCC4 WT and SCC4 DYRK1A KO cells were treated with the indicated concentrations of cisplatin for 2 days, and their viability was determined by MTT assay. **P < 0.05.

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 2. Loss of DYRK1A diminishes CSC-enriched ALDH1HIGH population and CSC properties in OSCC. (A) Effect of DYRK1A deletion on ALDH1HIGH cell population in SCC4 and FaDu was determined by Aldefluor assay. Cells were labeled with Aldefluor with and without the ALDH inhibitor DEAB and analyzed by flow cytometry. The gate for ALDH1HIGH cells is determined in relation to the DEAB control (+DEAB) and shows the brightly fluorescent ALDH population versus the side scatter, a population that is absent/decreased in the presence of DEAB. The number shown in each panel reflects the percentage of ALDH1HIGH cells in each cell type. A similar result was obtained from an independent experiment (Supplemental Fig. 2). (B) Effect of DYRK1A deletion on migration ability was determined by transwell migration assay. Migration ability was described as number of migrated cells per field with data as mean ± SD for three randomly selected fields. Representative images of migrated cells from each group were shown on the right of each bar graph. Bar indicates 200 μm *P < 0.01 (C) Effect of DYRK1A deletion on chemo resistance was determined by MTT assay. SCC4 WT and SCC4 DYRK1A KO cells were treated with the indicated concentrations of cisplatin for 2 days, and their viability was determined by MTT assay. **P < 0.05.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Labeling, Flow Cytometry, Control, Migration, Transwell Migration Assay, MTT Assay

Fig. 3. Ectopic DYRK1A expression promotes CSC properties in OSCC. DYRK1A was ectopically overexpressed in SCC9 by transfecting with vector expressing DYRK1A and empty vector (EV) as a control. (A) Ectopic expression of DYRK1A in SCC9 was validated by qPCR (left) and Western blot (right). (B) Effect of DYRK1A overexpression on self-renewal capacity was determined by tumorsphere formation assay. Representative images of tumorspheres formed from each group were shown on the right of the bar graph. *P < 0.01 compared to EV. (C) Effect of DYRK1A overexpression on migration ability was measured by a transwell migration assay. Representative images of migrated cells from each group were shown on the right of the bar graph. *P < 0.05 (D) Effect of DYRK1A overexpression on chemoresistance was determined by MTT assay. The cells were treated with indicated concentrations of cisplatin for 2 days, and their viability was determined. ns, not significant.

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 3. Ectopic DYRK1A expression promotes CSC properties in OSCC. DYRK1A was ectopically overexpressed in SCC9 by transfecting with vector expressing DYRK1A and empty vector (EV) as a control. (A) Ectopic expression of DYRK1A in SCC9 was validated by qPCR (left) and Western blot (right). (B) Effect of DYRK1A overexpression on self-renewal capacity was determined by tumorsphere formation assay. Representative images of tumorspheres formed from each group were shown on the right of the bar graph. *P < 0.01 compared to EV. (C) Effect of DYRK1A overexpression on migration ability was measured by a transwell migration assay. Representative images of migrated cells from each group were shown on the right of the bar graph. *P < 0.05 (D) Effect of DYRK1A overexpression on chemoresistance was determined by MTT assay. The cells were treated with indicated concentrations of cisplatin for 2 days, and their viability was determined. ns, not significant.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Over Expression, Tube Formation Assay, Migration, Transwell Migration Assay, MTT Assay

Fig. 4. DYRK1A is overexpressed in OSCC. (A) Level of DYRK1A mRNA was determined in normal human oral keratinocyte (NHOK) and 9 OSCC cell lines (BapT, FaDu, SCC4, SCC9, SCC15, SCC105, SNU1066, UM6 and YD38) by qPCR. The Ct values of DYRK1A were normalized by the Ct value of GAPDH. (B) Level of DYRK1A protein was determined in NHOK and OSCC cell lines by Western blot analysis. (C) Self-renewal capacity in three groups (high, medium, and low based on their level of DYRK1A protein) was determined by tumorsphere formation assay. (D) ALDH1HIGH cell population in three groups was determined by Aldefluor assay. (E) In vivo DYRK1A expression was determined in normal human oral epithelia (NHOE) and OSCC tissues by immunohistochemical (IHC) staining. (F) Representative examples of DYRK1A IHC staining in NHOE and OSCC tissues in vivo. Bar indicates 100 μm.

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 4. DYRK1A is overexpressed in OSCC. (A) Level of DYRK1A mRNA was determined in normal human oral keratinocyte (NHOK) and 9 OSCC cell lines (BapT, FaDu, SCC4, SCC9, SCC15, SCC105, SNU1066, UM6 and YD38) by qPCR. The Ct values of DYRK1A were normalized by the Ct value of GAPDH. (B) Level of DYRK1A protein was determined in NHOK and OSCC cell lines by Western blot analysis. (C) Self-renewal capacity in three groups (high, medium, and low based on their level of DYRK1A protein) was determined by tumorsphere formation assay. (D) ALDH1HIGH cell population in three groups was determined by Aldefluor assay. (E) In vivo DYRK1A expression was determined in normal human oral epithelia (NHOE) and OSCC tissues by immunohistochemical (IHC) staining. (F) Representative examples of DYRK1A IHC staining in NHOE and OSCC tissues in vivo. Bar indicates 100 μm.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Western Blot, Tube Formation Assay, In Vivo, Expressing, Immunohistochemical staining, Immunohistochemistry

Fig. 5. DYRK1A regulates CSC property via FGF2 expression. (A) Effect of DYRK1A deletion on FGF2 expression was assessed in SCC4 and FaDu by qPCR (upper) and Western blot analysis (lower). (B) Effect of DYRK1A deletion on FGF2 secretion was determined by ELISA. (C) Effect of DYRK1A overexpression on FGF2 expression was assessed in SCC9 by qPCR (upper) and Western blot analysis (lower). (D) Effect of DYRK1A overexpression on FGF2 secretion was determined by determined by ELISA. (E) Effect of FGF2 on self-renewal capacity of SCC4 WT and DYRK1A KO was measured by tumorsphere formation assay. The assays were performed in the absence and presence of FGF2 (50, 100 and 200 ng/ml). *P < 0.05 compared to untreated control cells. (F) Effect of FGF2 on migration ability of SCC4 WT and DYRK1A KO was measured by transwell migration assay. The assays were performed in the absence and presence of FGF2. (G) Effect of FGF2 neutralizing antibody on self-renewal capacity of DYRK1A-overerxpressing SCC9 was determined by tumorsphere assay. The assays were performed in the absence and presence of FGF2 antibody (1, 2.5, and 5 ng/ml). *P < 0.05 and **P < 0.01 compared to untreated control cells. (H) Effect of FGF2 neutralizing antibody on migration ability of DYRK1A-overerxpressing SCC9 was measured by transwell migration assay. The assays were performed in the absence and presence of FGF2 antibody.

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 5. DYRK1A regulates CSC property via FGF2 expression. (A) Effect of DYRK1A deletion on FGF2 expression was assessed in SCC4 and FaDu by qPCR (upper) and Western blot analysis (lower). (B) Effect of DYRK1A deletion on FGF2 secretion was determined by ELISA. (C) Effect of DYRK1A overexpression on FGF2 expression was assessed in SCC9 by qPCR (upper) and Western blot analysis (lower). (D) Effect of DYRK1A overexpression on FGF2 secretion was determined by determined by ELISA. (E) Effect of FGF2 on self-renewal capacity of SCC4 WT and DYRK1A KO was measured by tumorsphere formation assay. The assays were performed in the absence and presence of FGF2 (50, 100 and 200 ng/ml). *P < 0.05 compared to untreated control cells. (F) Effect of FGF2 on migration ability of SCC4 WT and DYRK1A KO was measured by transwell migration assay. The assays were performed in the absence and presence of FGF2. (G) Effect of FGF2 neutralizing antibody on self-renewal capacity of DYRK1A-overerxpressing SCC9 was determined by tumorsphere assay. The assays were performed in the absence and presence of FGF2 antibody (1, 2.5, and 5 ng/ml). *P < 0.05 and **P < 0.01 compared to untreated control cells. (H) Effect of FGF2 neutralizing antibody on migration ability of DYRK1A-overerxpressing SCC9 was measured by transwell migration assay. The assays were performed in the absence and presence of FGF2 antibody.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Over Expression, Tube Formation Assay, Control, Migration, Transwell Migration Assay

Fig. 6. Expression of DYRK1A and FGF2 are elevated in CSC-enriched populations, and their levels are positively correlated in OSCC cells. (A) Cisplatin-resistant SCC4 (CR-SCC4) cells were isolated from SCC4 treated with 25 μM cisplatin for 2 days. The IC50 value for cisplatin in SCC4 and CR-SCC4 was determined by MTT assay. Cells were treated with various concentrations of cisplatin for 48 h, and their viability was determined. (B) Self-renewal capacity of SCC4 and CR-SCC4 was determined by tumorsphere formation assay. *P < 0.05 and **P < 0.01 compared to SCC4. (C) Expression of DYRK1A and FGF2 was assessed in SCC4 and CR- SCC4 by qPCR (left) and Western blot (right). (D) ALDH1HIGH (CSC-enriched population) and ALDH1low (non-CSC population) cell populations were sorted from SCC4 cells by flow cytometry. Expression of DYRK1A and FGF2 was assessed in ALDH1low and ALDH1HIGH cell populations by qPCR. (E) Expression of DYRK1A and FGF2 was assessed in tumorspheres (S) and their corresponding adherent monolayer cells (M) derived from multiple OSCC cell lines by qPCR. (F) Correlation analysis of DYRK1A and FGF2 mRNA was determined based on their expression levels in 14 human SCC cell lines by qPCR.

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 6. Expression of DYRK1A and FGF2 are elevated in CSC-enriched populations, and their levels are positively correlated in OSCC cells. (A) Cisplatin-resistant SCC4 (CR-SCC4) cells were isolated from SCC4 treated with 25 μM cisplatin for 2 days. The IC50 value for cisplatin in SCC4 and CR-SCC4 was determined by MTT assay. Cells were treated with various concentrations of cisplatin for 48 h, and their viability was determined. (B) Self-renewal capacity of SCC4 and CR-SCC4 was determined by tumorsphere formation assay. *P < 0.05 and **P < 0.01 compared to SCC4. (C) Expression of DYRK1A and FGF2 was assessed in SCC4 and CR- SCC4 by qPCR (left) and Western blot (right). (D) ALDH1HIGH (CSC-enriched population) and ALDH1low (non-CSC population) cell populations were sorted from SCC4 cells by flow cytometry. Expression of DYRK1A and FGF2 was assessed in ALDH1low and ALDH1HIGH cell populations by qPCR. (E) Expression of DYRK1A and FGF2 was assessed in tumorspheres (S) and their corresponding adherent monolayer cells (M) derived from multiple OSCC cell lines by qPCR. (F) Correlation analysis of DYRK1A and FGF2 mRNA was determined based on their expression levels in 14 human SCC cell lines by qPCR.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Expressing, Isolation, MTT Assay, Tube Formation Assay, Western Blot, Flow Cytometry, Derivative Assay

Journal: medRxiv

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder

doi: 10.1101/2021.01.20.21250155

Figure Lengend Snippet:

Article Snippet: DYRK1A expression plasmids were generated from the pMH-SFB- DYRK1A vector containing the human DYRK1A cDNA sequence (NM_001396.4) tagged with FLAG peptide at the N-Terminal side (purchased from addgene #101770; Huen lab).

Techniques: Translocation Assay, Sequencing, Hybridization, DNA Methylation Assay

(A) Clinical score out of 20 points established according to the most recurrent clinical features presented by patients (the weight assigned to each symptom being based on its recurrence): clinical symptoms are out of 15 points, while the facial appearance is out of 5 points. EV: enlarged ventricules; CCA/H: corpus callosum agenesis or hypoplasia, CA: cerebral atrophy, CeA: cerebellar atrophy ( B) Clinical scores calculated for individuals carrying pathogenic variants in DYRK1A reported here and for whom photographs were available (n=21)(initial cohort, DYRK1A_I, scores 13-17.5 with a mean of 15.5), the previously published individuals (replication cohort, DYRK1A_R, scores 13.5-18, mean=15.3) and the individuals affected with other frequent monogenic forms of ID, associated to mutations in ANKRD11, MED13L, DDX3X, ARID1B, SHANK3, TCF4 or KMT2A (scores 3-12.5, mean=7). The clinical score for the individuals carrying missense or distal frameshift variants are indicated in yellow (test); The threshold of CS DYRK1A >=13 appeared to be discriminant between individuals with LoF variants in DYRK1A (all ≥13) and individuals suffering from another form of ID (all<13) and a score above this threshold was therefore considered “highly suggestive”. We classified individuals with CS DYRK1A <10 as “poorly evokative” and individuals with a CS DYRK1A comprised between 10 and 13 as “intermediate”. Brown-Forsythe and Welsh ANOVA tests with Dunnett’s T 3 multiple comparisons test were performed. ns: not significant; ** p<0.01; ***< p<0.001, error bars represent SD.

Journal: medRxiv

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder

doi: 10.1101/2021.01.20.21250155

Figure Lengend Snippet: (A) Clinical score out of 20 points established according to the most recurrent clinical features presented by patients (the weight assigned to each symptom being based on its recurrence): clinical symptoms are out of 15 points, while the facial appearance is out of 5 points. EV: enlarged ventricules; CCA/H: corpus callosum agenesis or hypoplasia, CA: cerebral atrophy, CeA: cerebellar atrophy ( B) Clinical scores calculated for individuals carrying pathogenic variants in DYRK1A reported here and for whom photographs were available (n=21)(initial cohort, DYRK1A_I, scores 13-17.5 with a mean of 15.5), the previously published individuals (replication cohort, DYRK1A_R, scores 13.5-18, mean=15.3) and the individuals affected with other frequent monogenic forms of ID, associated to mutations in ANKRD11, MED13L, DDX3X, ARID1B, SHANK3, TCF4 or KMT2A (scores 3-12.5, mean=7). The clinical score for the individuals carrying missense or distal frameshift variants are indicated in yellow (test); The threshold of CS DYRK1A >=13 appeared to be discriminant between individuals with LoF variants in DYRK1A (all ≥13) and individuals suffering from another form of ID (all<13) and a score above this threshold was therefore considered “highly suggestive”. We classified individuals with CS DYRK1A <10 as “poorly evokative” and individuals with a CS DYRK1A comprised between 10 and 13 as “intermediate”. Brown-Forsythe and Welsh ANOVA tests with Dunnett’s T 3 multiple comparisons test were performed. ns: not significant; ** p<0.01; ***< p<0.001, error bars represent SD.

Techniques:

(A) Level of variant DYRK1A proteins expressed in HeLa, HEK293 and COS cells transiently transfected with DYRK1A constructs. Protein levels were normalized on the level of GFP proteins (expressed from a cotransfected pEGFP plasmid). Quantifications were performed on a total of n >=9 series of cells (n>=3 Hela cells, n>= 3 HEK293 and n >= 3 COS cells). One-way ANOVA with multiple comparison test was performed to compare the level of variant DYRK1A proteins to the level of wild[type DYRK1A protein, applying Bonferroni’s correction: ns: not significant; *p < 0.05; **p < 0.01;***p<0.001; error bars represent SEM, standard error of the mean, ( B) DYRK1A’s ability to autophosphorylate on Tyr321 was tested in HEK293 cells (n=3) by immunoprecipitations with anti-DYRK1A followed by an immunoblot using an anti-HIPK2 as described in Widowati et al. DYRK1A phospho-Tyr321 levels were normalized with DYRK1A total level. Variant DYRK1A phospho-Tyr321 levels were normalized with total DYRK1A protein levels and expressed as percentage of wild-type level. One-way ANOVA test was performed to compare variants to wild-type DYRK1A levels. ns: not significant; ***p<0.001; error bars represent SEM, standard error of the mean (C) Immunofluorescence experiment showing that Ser660fs (alias Ser660Profs*43) variant leads to DYRK1A protein aggregation when overexpressed in HeLa cells, using a FLAG-tagged DYRK1A proteins carrying Ser660Profs43. No aggregation was observed for the Ser660* variant.

Journal: medRxiv

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder

doi: 10.1101/2021.01.20.21250155

Figure Lengend Snippet: (A) Level of variant DYRK1A proteins expressed in HeLa, HEK293 and COS cells transiently transfected with DYRK1A constructs. Protein levels were normalized on the level of GFP proteins (expressed from a cotransfected pEGFP plasmid). Quantifications were performed on a total of n >=9 series of cells (n>=3 Hela cells, n>= 3 HEK293 and n >= 3 COS cells). One-way ANOVA with multiple comparison test was performed to compare the level of variant DYRK1A proteins to the level of wild[type DYRK1A protein, applying Bonferroni’s correction: ns: not significant; *p < 0.05; **p < 0.01;***p<0.001; error bars represent SEM, standard error of the mean, ( B) DYRK1A’s ability to autophosphorylate on Tyr321 was tested in HEK293 cells (n=3) by immunoprecipitations with anti-DYRK1A followed by an immunoblot using an anti-HIPK2 as described in Widowati et al. DYRK1A phospho-Tyr321 levels were normalized with DYRK1A total level. Variant DYRK1A phospho-Tyr321 levels were normalized with total DYRK1A protein levels and expressed as percentage of wild-type level. One-way ANOVA test was performed to compare variants to wild-type DYRK1A levels. ns: not significant; ***p<0.001; error bars represent SEM, standard error of the mean (C) Immunofluorescence experiment showing that Ser660fs (alias Ser660Profs*43) variant leads to DYRK1A protein aggregation when overexpressed in HeLa cells, using a FLAG-tagged DYRK1A proteins carrying Ser660Profs43. No aggregation was observed for the Ser660* variant.

Techniques: Variant Assay, Transfection, Construct, Plasmid Preparation, Western Blot, Immunofluorescence

Journal: medRxiv

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder

doi: 10.1101/2021.01.20.21250155

Figure Lengend Snippet: (A) Heatmap showing the hierarchical clustering of discovery DYRK1A LoF cases (n[=[10) and age- and sex-matched neurotypical discovery controls (n[=[24) used to identify the 402 differentially methylated signature sites shown. The color gradient represents the normalized DNA methylation value from -2.0 (blue) to 2.0 (yellow) at each site. DNA methylation at these sties clearly separate discovery cases (grey) from discovery controls (blue). Euclidian distance metric is used for the clustering dendrogram. (B) Principal components analysis (PCA) visualizing the DNAm profiles of the study cohort at the 402 signature sites. Validation of DYRK1A LoF cases (not used to define the signature sites; red) cluster with discovery cases, while missense (yellow) and distal LoF (green) variants cluster with either cases or controls. Ind #33 (Gly486Asp) has an opposite DNAm profile to DYRK1A LoF cases at these sites, suggesting a GoF. (C) Support vector machine (SVM) classification model based on the DNA methylation values in the discovery groups. Each sample is plotted based on its scoring by the model. All samples are clearly positive (>0.5) or negative (<0.5). All DYRK1A validation cases from our cohort (n=6) classified positively, all control validation cases (n=94) classified negatively. Missense variants classified clearly positively or negatively, the distal frameshift variant (Ind #18, c.1978del), analyzed in duplicate, classified positively. Pathogenic ARID1B (Coffin-Siris syndrome) and KMT2A (Wiedemann Steiner syndrome) also classified negatively.

Techniques: Methylation, DNA Methylation Assay, Plasmid Preparation, Variant Assay

Representation of the DYRK1A protein (the kinase domain is indicated in red and the catalytic domain in dark red) with the positions of the different variants tested with the sample #ID of the individuals indicated inside the circles. Number: number of individuals with ID reported with the variant; gAD: variant reported in individuals from gnomAD; CS DYRK1A poorly (white), intermediate (grey) or highly (black) evocative, or unknown (-); CADD below 25 (white), between 25 and 30 (grey) or above 30 (black); conservation: highly conserved V=100%, M>90%, O>80% (black), moderately V=100%, M>90%, O<80% (grey) or midly V=100% M<90%, O<80% (white); Expression or autophosphorylation being normal (white), intermediate decreased (grey), strongly decreased (black); Localization was normal (white), affected (grey) or not tested (-); DNAmethylation positive (black), negative (white), suggestive of a GoF effect (hashed) or not tested (-). Final classification: Pathogenic (P), Benign (B), Unknown significance (U).

Journal: medRxiv

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder

doi: 10.1101/2021.01.20.21250155

Figure Lengend Snippet: Representation of the DYRK1A protein (the kinase domain is indicated in red and the catalytic domain in dark red) with the positions of the different variants tested with the sample #ID of the individuals indicated inside the circles. Number: number of individuals with ID reported with the variant; gAD: variant reported in individuals from gnomAD; CS DYRK1A poorly (white), intermediate (grey) or highly (black) evocative, or unknown (-); CADD below 25 (white), between 25 and 30 (grey) or above 30 (black); conservation: highly conserved V=100%, M>90%, O>80% (black), moderately V=100%, M>90%, O<80% (grey) or midly V=100% M<90%, O<80% (white); Expression or autophosphorylation being normal (white), intermediate decreased (grey), strongly decreased (black); Localization was normal (white), affected (grey) or not tested (-); DNAmethylation positive (black), negative (white), suggestive of a GoF effect (hashed) or not tested (-). Final classification: Pathogenic (P), Benign (B), Unknown significance (U).

Techniques: Variant Assay, Expressing

Down‐regulation of BRCA1 in EBV‐associated NPCs. (A) The expression of BRCA1, ATM and PARP1 proteins in immortalized normal NP (NP69), four NPC cell lines and four NPC patient‐derived xenografts (PDXs) were analysed by immunoblotting. Actin was probed as the loading control. (B) The expression levels of BRCA1 mRNA in the cell lines were measured using RT‐qPCR. The relative BRCA1 mRNA expression was calculated using 2(∆∆−Ct) method, and the expression in NP69 was set as 1 for comparison. The data shown is the mean + SD. (C) The whiskers 10‐90 percentiles plot shows the relative BRCA1 mRNA expression in primary samples. The BRCA1 mRNA was significantly up‐regulated in NPCs (n = 55) when compared with the NPs (n = 22). (D) Immunohistochemistry staining of BRCA1 protein in primary samples (number of NPs = 30 and NPCs = 41). The representative images of negative and positive BRCA1 stain in NP and NPC specimens are shown (original magnification X400). (E) The dot plot shows the total expression levels of miR‐BART2‐3p, BART12, BART17‐5p and BART19‐3p in 20 NPC biopsies, in which the BRCA1 protein expression status was analysed in IHC. The expression of miR‐BARTs was normalized to EBNA1. The median values of each group are shown by the dash line and the Mann‐Whitney test was used for the statistical analysis

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: Down‐regulation of BRCA1 in EBV‐associated NPCs. (A) The expression of BRCA1, ATM and PARP1 proteins in immortalized normal NP (NP69), four NPC cell lines and four NPC patient‐derived xenografts (PDXs) were analysed by immunoblotting. Actin was probed as the loading control. (B) The expression levels of BRCA1 mRNA in the cell lines were measured using RT‐qPCR. The relative BRCA1 mRNA expression was calculated using 2(∆∆−Ct) method, and the expression in NP69 was set as 1 for comparison. The data shown is the mean + SD. (C) The whiskers 10‐90 percentiles plot shows the relative BRCA1 mRNA expression in primary samples. The BRCA1 mRNA was significantly up‐regulated in NPCs (n = 55) when compared with the NPs (n = 22). (D) Immunohistochemistry staining of BRCA1 protein in primary samples (number of NPs = 30 and NPCs = 41). The representative images of negative and positive BRCA1 stain in NP and NPC specimens are shown (original magnification X400). (E) The dot plot shows the total expression levels of miR‐BART2‐3p, BART12, BART17‐5p and BART19‐3p in 20 NPC biopsies, in which the BRCA1 protein expression status was analysed in IHC. The expression of miR‐BARTs was normalized to EBNA1. The median values of each group are shown by the dash line and the Mann‐Whitney test was used for the statistical analysis

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Expressing, Derivative Assay, Western Blot, Control, Quantitative RT-PCR, Comparison, Immunohistochemistry, Staining, MANN-WHITNEY

The BRCA1 is the potential target of miR‐BARTs. (A) The relative luciferase activity of the reporter plasmids harbouring a full length of BRCA1‐3’UTR (sFL‐3’UTR) or a full length of BRCA1‐3’UTR in reversed orientation (asFL‐3’UTR) was co‐transfected together with the indicated miRNAs. The luciferase signal with the co‐transfection of negative miRNA mimic control (miR‐NEG) was set at 1 for comparison. (B) The direct interaction between the putative binding sites on BRCA1 and miR‐BARTs were demonstrated in the reporter assays. The firefly luciferase reporter activity was normalized to the Renilla luciferase control. The data shown is the mean + SD from three independent experiments. The result with the co‐transfection of miR‐NEG and pMIR‐CTL was set at 1. pMIR‐CTL = pMIR‐REPORTTM vectors containing unrelated sequences; pMIR‐B = pMIR‐REPORTTM vector harbouring the predicted miR‐BART binding site, pMIR‐CDS = predicted binding site on CDS (Table ). B2‐3p = BART2‐3p; B12 = BART12; B17‐5p = BART17‐5p; B19‐3p = BART19‐3p. * P < 0.05, ** P < 0.001

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The BRCA1 is the potential target of miR‐BARTs. (A) The relative luciferase activity of the reporter plasmids harbouring a full length of BRCA1‐3’UTR (sFL‐3’UTR) or a full length of BRCA1‐3’UTR in reversed orientation (asFL‐3’UTR) was co‐transfected together with the indicated miRNAs. The luciferase signal with the co‐transfection of negative miRNA mimic control (miR‐NEG) was set at 1 for comparison. (B) The direct interaction between the putative binding sites on BRCA1 and miR‐BARTs were demonstrated in the reporter assays. The firefly luciferase reporter activity was normalized to the Renilla luciferase control. The data shown is the mean + SD from three independent experiments. The result with the co‐transfection of miR‐NEG and pMIR‐CTL was set at 1. pMIR‐CTL = pMIR‐REPORTTM vectors containing unrelated sequences; pMIR‐B = pMIR‐REPORTTM vector harbouring the predicted miR‐BART binding site, pMIR‐CDS = predicted binding site on CDS (Table ). B2‐3p = BART2‐3p; B12 = BART12; B17‐5p = BART17‐5p; B19‐3p = BART19‐3p. * P < 0.05, ** P < 0.001

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Control, Comparison, Binding Assay, Plasmid Preparation

Regulation of BRCA1 expression by miR‐BARTs (A) Western blot of BRCA1 in NPC cell lines. Actin was probed as the protein‐loading control, and the expression level was compared with NP69 (set as 1). (B) The total expression of viral BART2‐3p, BART12, BART17‐5p and BART19‐3p (upper panel) and the expression of cellular miR‐146a (lower panel) in the cell lines were assayed by RT‐qPCR. The expression values of total miR‐BARTs and miR‐146a were calculated using the 2(‐∆Ct) and 2(∆∆‐Ct) methods, respectively. The analysis of each sample was performed in triplicate with mean + SD shown. (C) In the EBV‐negative epithelial cells, the BRCA1 level was suppressed by the transfection of the indicated miRNA mimics, BART2‐3p (B2‐3p), BART12 (BT12), BART17‐5p (BT17‐5p) and BART19‐3p (BT19‐3p). (D) The BRCA1 protein expression in C666‐1 cells was regained by suppressing the endogenous miR‐BARTs activities with specific miR‐BART inhibitors for 48 h. The negative control mimic/inhibitor (Inh‐Ctl) transfection was used for comparison. Either actin or vinculin was probed as the loading control

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: Regulation of BRCA1 expression by miR‐BARTs (A) Western blot of BRCA1 in NPC cell lines. Actin was probed as the protein‐loading control, and the expression level was compared with NP69 (set as 1). (B) The total expression of viral BART2‐3p, BART12, BART17‐5p and BART19‐3p (upper panel) and the expression of cellular miR‐146a (lower panel) in the cell lines were assayed by RT‐qPCR. The expression values of total miR‐BARTs and miR‐146a were calculated using the 2(‐∆Ct) and 2(∆∆‐Ct) methods, respectively. The analysis of each sample was performed in triplicate with mean + SD shown. (C) In the EBV‐negative epithelial cells, the BRCA1 level was suppressed by the transfection of the indicated miRNA mimics, BART2‐3p (B2‐3p), BART12 (BT12), BART17‐5p (BT17‐5p) and BART19‐3p (BT19‐3p). (D) The BRCA1 protein expression in C666‐1 cells was regained by suppressing the endogenous miR‐BARTs activities with specific miR‐BART inhibitors for 48 h. The negative control mimic/inhibitor (Inh‐Ctl) transfection was used for comparison. Either actin or vinculin was probed as the loading control

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Transfection, Negative Control, Comparison

The CDDP and DOX sensitivity in HK1 and NP69 cells. (A) Western blot of p53 and p21 in NPC cell lines were analysed. (B) Transfection of either BRCA1‐specific siRNA, BART17‐5p or BART19‐3p mimics increased CDDP‐ and DOX‐mediated S phase or G2/M phase cell‐cycle arrest in the HK1 and NP69 cells. The transfected cells were incubated with either the control buffer or the indicated chemotherapeutic agent for 24 h. Subsequently, the cells were fixed for DNA content analysis with BD FACSCalibur flow cytometry system. (C) Protein lysate from the treated cells were harvested for phosphor‐CHK1 (p‐CHK1) expression analysis. (D) The suppression of BRCA1 sensitized HK1 cells to CDDP and DOX treatment. HK1 cells were transfected with BRCA1‐specific siRNAs (si‐BRCA1) or siRNA control (si‐NEG) and the protein lysates were collected for BRCA1 expression analysis 24 h after transfection (left panel). The transfected HK1 cells were incubated with different concentrations of CDDP or DOX for 48 h before CCK‐8 analysis. The IC50 value was determined by fitting a sigmoidal dose‐response curve to the data using GraphPad Prism 5 program. Sum‐of‐squares F‐test was used as the comparison method (right panel). (E) Clonogenic survival assays. Approximately 500 or 1000 transfected cells were seeded into the 6‐well plate and treated with CDDP or DOX for 24 h. The cells were cultured for 14‐18 d in normal medium before staining, and colonies containing more than 30 cells were counted. The number of colonies generated from the mock treatment was compared (set as 100%). All the experiments were performed in triplicate and the Student's t ‐test was conducted, compared with the control transfected cells. * P < 0.05; ** P < 0.01

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The CDDP and DOX sensitivity in HK1 and NP69 cells. (A) Western blot of p53 and p21 in NPC cell lines were analysed. (B) Transfection of either BRCA1‐specific siRNA, BART17‐5p or BART19‐3p mimics increased CDDP‐ and DOX‐mediated S phase or G2/M phase cell‐cycle arrest in the HK1 and NP69 cells. The transfected cells were incubated with either the control buffer or the indicated chemotherapeutic agent for 24 h. Subsequently, the cells were fixed for DNA content analysis with BD FACSCalibur flow cytometry system. (C) Protein lysate from the treated cells were harvested for phosphor‐CHK1 (p‐CHK1) expression analysis. (D) The suppression of BRCA1 sensitized HK1 cells to CDDP and DOX treatment. HK1 cells were transfected with BRCA1‐specific siRNAs (si‐BRCA1) or siRNA control (si‐NEG) and the protein lysates were collected for BRCA1 expression analysis 24 h after transfection (left panel). The transfected HK1 cells were incubated with different concentrations of CDDP or DOX for 48 h before CCK‐8 analysis. The IC50 value was determined by fitting a sigmoidal dose‐response curve to the data using GraphPad Prism 5 program. Sum‐of‐squares F‐test was used as the comparison method (right panel). (E) Clonogenic survival assays. Approximately 500 or 1000 transfected cells were seeded into the 6‐well plate and treated with CDDP or DOX for 24 h. The cells were cultured for 14‐18 d in normal medium before staining, and colonies containing more than 30 cells were counted. The number of colonies generated from the mock treatment was compared (set as 100%). All the experiments were performed in triplicate and the Student's t ‐test was conducted, compared with the control transfected cells. * P < 0.05; ** P < 0.01

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Western Blot, Transfection, Incubation, Control, Flow Cytometry, Expressing, CCK-8 Assay, Comparison, Cell Culture, Staining, Generated

The EBV‐miRNAs impair cisplatin‐ and doxorubicin‐induced DNA damage response in nasopharyngeal epithelial cells. The representative images of the RAD51 foci staining in HK1 cells (upper left panel) and NP69 cells (upper right panel) are shown. The cells transfected with either siRNA control (si‐NEG), BRCA1‐specific siRNA (si‐BRCA1) or miR‐BARTs mimics were treated with cisplatin (CDDP) and doxorubicin (DOX), followed by immunostaining with the RAD51 antibody. At least 100 nuclei were randomly selected for counting, and the cells containing more than five apparent RAD51 foci in the nucleus were considered positive. The percentage of the RAD51‐positive cells with mean + SD from three independent experiments are shown in the lower panel. Student's t ‐test was used to compare them with the control transfected cells (miR‐NEG) in each set of experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The EBV‐miRNAs impair cisplatin‐ and doxorubicin‐induced DNA damage response in nasopharyngeal epithelial cells. The representative images of the RAD51 foci staining in HK1 cells (upper left panel) and NP69 cells (upper right panel) are shown. The cells transfected with either siRNA control (si‐NEG), BRCA1‐specific siRNA (si‐BRCA1) or miR‐BARTs mimics were treated with cisplatin (CDDP) and doxorubicin (DOX), followed by immunostaining with the RAD51 antibody. At least 100 nuclei were randomly selected for counting, and the cells containing more than five apparent RAD51 foci in the nucleus were considered positive. The percentage of the RAD51‐positive cells with mean + SD from three independent experiments are shown in the lower panel. Student's t ‐test was used to compare them with the control transfected cells (miR‐NEG) in each set of experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Staining, Transfection, Control, Immunostaining

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