Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: A , B Basal expression levels of DUSP16 in NPC cell lines were determined by quantitative real-time PCR (qPCR). Data were expressed as means ± standard error of the mean (mean ± SEM, n = 3 biologically independent samples). Two-tail unpaired t -test showed significantly higher DUSP16 expression in C666-1 and HK-1 cells compared to that in HONE-1 cells. * P < 0.05, ** P < 0.001 ( A ). DUSP16 protein expression in these cells was determined by western blot analysis. The data are representative of two experiments with similar results ( B ). C Expression of DUSP16 in HK-1 and C666-1 in response to cisplatin treatment was determined by qPCR and western blot analysis. Induction of DUSP16 mRNA expression at each time point was expressed as means ± SEM ( n = 3 biologically independent samples). Statistical analysis was performed using two-tailed unpaired t -test. * P < 0.05, ** P < 0.001. D Flow cytometric analysis measuring apoptosis in HK-1 and C666-1 cell lines at 48 h after cisplatin treatment. Bar charts show the percentage of apoptotic (AnnexinV + 7AAD + ) and surviving (AnnexinV − 7AAD − ) cells, respectively, in both untreated and cisplatin-treated groups with mean ± SEM ( n = 3 biologically independent samples). Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. The data are representa t ive of three experiments with similar results. E , F DUSP16 protein expression in colorectal cancer cell DLD-1 and HCT116 ( E ) and in gastric cancer cell Ags and Nugc3 was evaluated by western blot analysis. The data are representative of 3 experiments with similar results. G , H Phospho-JNK (pJNK), phospho-P38 (pP38), phospho-ERK (pERK) and their total protein expression (tJNK, tP38, and tERK) in HK-1 ( G ), and C666-1 ( H ) cells upon cisplatin treatment was examined by western blot analysis. The data are representative of 3 experiments with similar results. Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: A mRNA and protein levels of DUSP16 in HK-1 cells transfected with vector or human DUSP16 cDNA were quantified by qPCR and western blot analysis, respectively. Bar chart shows mRNA expression of vector ( n = 2) and DUSP16 ( n = 4) expressing clones. B , C Comparison of apoptosis induction between vector- and DUSP16 -transfected cells after cisplatin treatment was carried out by staining the cells with AnnexinV and 7AAD followed by flow cytometry analysis ( B ). Bar charts show the percentage of apoptotic (both AnnexinV + 7AAD + and AnnexinV + 7AAD − cells) and surviving cells, respectively, in both untreated and cisplatin-treated groups with mean ± SEM, n = 3 biologically independent samples ( C ). Statistical analysis was performed using two-tailed unpaired t -test. The data are representative of 3 experiments with similar results. D DUSP16 mRNA expression in DLD-1 cells transfected with vector ( n = 6) or human DUSP16 cDNA ( n = 6) quantified by qPCR. Statistical analysis was performed using two-tailed unpaired t -test. DUSP16 protein expression in these clones was assessed by Western blot. E , F Apoptosis induction between the vector- and DUSP16 -transfected DLD-1 cells after cisplatin treatment was analyzed ( E ) and bar charts show the percentage of apoptotic and surviving cells in both untreated and cisplatin-treated groups with mean ± SEM, n = 3 biologically independent samples ( F ). Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. G DUSP16 mRNA and protein expression in Nugc3 cells transfected with vector or human DUSP16 cDNA was determined by qPCR and immunoblotting, respectively. Dot plot shows DUSP16 mRNA expression in vector ( n = 3) and DUSP16 ( n = 5) expressing clones. Statistical analysis was performed using two-tailed unpaired t -test. H , I Apoptosis induction between vector- and DUSP16 -transfected Nugc3 cells after cisplatin treatment was analyzed and bar charts ( I ) show the percentage of apoptotic and surviving cells in untreated and cisplatin-treated cells, respectively, with mean ± SEM, n = 3 biologically independent samples. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. The data are representative of 3 experiments with similar results. Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Clone Assay, Comparison, Staining, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: A Comparison of apoptosis induction between HK-1 and C666-1 cells in response to carboplatin treatment by flow cytometry analysis. Bar chart shows the percentage of apoptotic cells with mean values ± standard error of the mean (SEM), n = 3 biologically independent samples. The data are representative of three experiments with similar results. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. B Comparison of apoptosis induction between vector- and DUSP16 -transfected HK-1 cells after oxaliplatin or carboplatin treatment by flow cytometry analysis. Bar chart shows the percentage of apoptotic cells (including both AnnexinV + 7AAD + and AnnexinV + 7AAD − cells) with mean values ± SEM, n = 3 biologically independent samples. The data are representative of three experiments with similar results. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. C Comparison of apoptosis induction between HCT116 and DLD-1 cells in response to oxaliplatin treatment by flow cytometry analysis. Bar chart shows the percentage of apoptotic cells with mean values ± SEM, n = biologically independent samples. The data are representative of three experiments with similar results. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. D Comparison of apoptosis induction between vector- and DUSP16 -transfected HCT116 cells after oxaliplatin or carboplatin treatment by flow cytometry analysis. Bar chart shows the percentage of apoptotic cells with mean values ± SEM, n = 3 biologically independent samples. The data are representative of three experiments with similar results. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Comparison, Flow Cytometry, Two Tailed Test, Plasmid Preparation, Transfection

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: A – C Western blot analysis of p-MAPKs, total MAPKs, c-Myc, cleaved caspase 9 (C-C9), and cleaved caspase 3 (C-C3) was carried out on vector- and DUSP16 -transfected HK-1 ( A ), DLD-1 ( B ), and Nugc3 ( C ) cells after cisplatin treatment. The data is representative of at least two experiments with similar results. D Western blot analysis of cisplatin-induced cytochrome c (Cyt c) release in cytosolic fractions of vector- and DUSP16 -transfected HK-1 and DLD-1 cells. The data are representative of three experiments with similar results. E Scatterplots depicting changes in the mitochondrial membrane potential in vector- and DUSP16 -transfected HK-1 cells upon cisplatin treatment. F Western blot analysis of BAX expression in mitochondrial and cytosolic fractions of vector- and DUSP16 -transfected HK-1 and DLD-1 cells. The data are representative of three experiments with similar results. G Western blot analysis of BAX wild-type (WT) and knockout (KO) DLD-1 cell clones with or without DUSP16 overexpression. The data are representative of two experiments with similar results. H BAX WT and KO DLD-1 cells with or without DUSP16 overexpression were treated with cisplatin for 48 h to examine cell apoptosis by staining with AnnexinV + and 7AAD + followed by flow cytometry analysis. Bar chart shows the percentage of apoptotic cells with mean values ± SEM, n = 3 biologically independent samples. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. The data are representative of three experiments with similar results. Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Western Blot, Plasmid Preparation, Transfection, Membrane, Expressing, Knock-Out, Clone Assay, Over Expression, Staining, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: A Vector- or DUSP16 -transfected HK-1 cells were inoculated into NSG mice. Cisplatin was injected at 3 mg/kg body weight every 3 days. After 20 days, tumors were harvested to measure the sizes and weights. The data are expressed as dots representing the weight of each tumor; the scale bars are the mean ± standard error of the mean (SEM) of tumor weights from six mice in each treatment group of the representative experiment (mean ± SEM, n = 6). Statistical analysis was performed using two-tailed unpaired t -test. B Growth of untreated and cisplatin-treated xenografts of vector- and DUSP16 -transfected DLD-1 cells. Box-and-whisker plots show means ± SEM of tumor weights from four mice from each treatment group per experiment (mean ± SEM, n = 4). Boxes correspond to the 25th, 50th/median and 75th percentiles; whiskers denote maximum and minimum. Statistical analysis was performed using two-tailed unpaired t -test. C , D IHC analysis for cleaved caspase 3 of HK-1 tumor sections; representative images of cleaved caspase 3 staining ( n = 6) were shown ( C ). Boxed regions were magnified and presented under the respective panels. Black arrows highlight the caspase 3 staining. Box-and-whisker plots show means ± SEM of the means of cleaved caspase 3 staining in each group of mice ( D ). Boxes correspond to the 25th, 50th/median and 75th percentiles; whiskers denote maximum and minimum. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001 (mean ± SEM, n = 3). E , F IHC analysis for Ki67 of tumor sections. Representative images of Ki67 staining were shown ( E ). Boxed regions were magnified and presented under the respective panels. Black arrows highlighted the Ki67 staining. Box-and-whisker plots show means ± SEM of Ki67 staining ( F ) from each group of mice *** P < 0.0001 (mean ± SEM, n = 3). The data are representative of three experiments with similar results. Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Transfection, Injection, Two Tailed Test, Whisker Assay, Staining

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: C666-1 cells were transfected with DUSP16 CRISPR/Cas9 constructs to knockdown DUSP16 expression. A Bar chart shows the mRNA expression in vector and DUSP16 construct transfected cells with mean values ± SEM, n = 3 biologically independent samples. Statistical analysis was performed using two-tailed unpaired t -test. Protein levels of DUSP16 were quantified by immunoblotting. *** P < 0.0001. B Apoptosis of control and DUSP16 knockdown (KD) cells after cisplatin treatment was measured by AnnexinV/7AAD staining and flow cytometry. C Bar charts show the percentage of apoptotic and surviving (unstained) cells with mean values ± SEM, n = 3 biologically independent samples. Statistical analysis was performed using two-tailed unpaired t -test. The data are representative of three experiments with similar results. D Western blot analysis of the pJNK, pP38, pERK, their total protein levels, and c-Myc in control or DUSP16-KD C666-1 cells was carried out after cisplatin treatment. The data are representative of three experiments with similar results. Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Transfection, CRISPR, Construct, Knockdown, Expressing, Plasmid Preparation, Two Tailed Test, Western Blot, Control, Staining, Flow Cytometry

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: A Mice were inoculated with control or DUSP16-KD cells. Cisplatin was injected at 3 mg/kg body weight every 3 days. After 20 days, tumors were harvested, and the sizes and weights were measured. The data are expressed as dots representing the weight of each tumor; the scale bars are the mean ± SEM of tumor weights from 4 (the vector control group) or 5 mice (the other groups) of the representative experiment. Statistical analysis was performed using two-tailed unpaired t -test. B Representative images of immunohistochemistry analysis for cleaved caspase 3 and Ki67 staining of tumor sections were shown. Boxed regions were magnified and presented under the respective panels. Black arrows highlight the caspase 3 and Ki67 staining, respectively. Box-and-whisker plots show mean values ± SEM of cleaved caspase 3 and Ki67 staining from each group of mice ( n = 4). Boxes correspond to the 25th, 50th/median and 75th percentiles; whiskers denote maximum and minimum. Black scale bars = 200 μm, white scale bars = 50 μm. Statistical analysis was performed using two-tailed unpaired t -test. * P < 0.05. The data are representative of three experiments with similar results. C Representative images of HNSCC specimens ( n = 50) showing negative (0 +), low (1+), intermediate (2+), and high (3+) staining of DUSP16 in tumor cells. Black scale bars = 200 μm. D Patients with higher DUSP16 expression showed lower disease-free survival (DFS) compared to patients with low DUSP16 expression (log-rank test, two-sided, P = 0.042). E Representative images of breast cancer specimens ( n = 113) showing negative (0 +), low (1 +), and high (3 +) staining of DUSP16 in tumor cells. Black scale bars = 200 μm. F Among the 113 breast cancer patients (age 28–64) treated with cisplatin, carboplatin, or lobaplatin after surgery, high DUSP16 protein expression in their tumors was associated with lower disease-free survival compared to patients with low DUSP16 expression (log-rank test, two-sided, P ˂ 0.0001). Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Control, Injection, Plasmid Preparation, Two Tailed Test, Immunohistochemistry, Staining, Whisker Assay, Expressing

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: A , B Basal expression levels of DUSP16 in NPC cell lines were determined by quantitative real-time PCR (qPCR). Data were expressed as means ± standard error of the mean (mean ± SEM, n = 3 biologically independent samples). Two-tail unpaired t -test showed significantly higher DUSP16 expression in C666-1 and HK-1 cells compared to that in HONE-1 cells. * P < 0.05, ** P < 0.001 ( A ). DUSP16 protein expression in these cells was determined by western blot analysis. The data are representative of two experiments with similar results ( B ). C Expression of DUSP16 in HK-1 and C666-1 in response to cisplatin treatment was determined by qPCR and western blot analysis. Induction of DUSP16 mRNA expression at each time point was expressed as means ± SEM ( n = 3 biologically independent samples). Statistical analysis was performed using two-tailed unpaired t -test. * P < 0.05, ** P < 0.001. D Flow cytometric analysis measuring apoptosis in HK-1 and C666-1 cell lines at 48 h after cisplatin treatment. Bar charts show the percentage of apoptotic (AnnexinV + 7AAD + ) and surviving (AnnexinV − 7AAD − ) cells, respectively, in both untreated and cisplatin-treated groups with mean ± SEM ( n = 3 biologically independent samples). Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. The data are representa t ive of three experiments with similar results. E , F DUSP16 protein expression in colorectal cancer cell DLD-1 and HCT116 ( E ) and in gastric cancer cell Ags and Nugc3 was evaluated by western blot analysis. The data are representative of 3 experiments with similar results. G , H Phospho-JNK (pJNK), phospho-P38 (pP38), phospho-ERK (pERK) and their total protein expression (tJNK, tP38, and tERK) in HK-1 ( G ), and C666-1 ( H ) cells upon cisplatin treatment was examined by western blot analysis. The data are representative of 3 experiments with similar results. Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: A mRNA and protein levels of DUSP16 in HK-1 cells transfected with vector or human DUSP16 cDNA were quantified by qPCR and western blot analysis, respectively. Bar chart shows mRNA expression of vector ( n = 2) and DUSP16 ( n = 4) expressing clones. B , C Comparison of apoptosis induction between vector- and DUSP16 -transfected cells after cisplatin treatment was carried out by staining the cells with AnnexinV and 7AAD followed by flow cytometry analysis ( B ). Bar charts show the percentage of apoptotic (both AnnexinV + 7AAD + and AnnexinV + 7AAD − cells) and surviving cells, respectively, in both untreated and cisplatin-treated groups with mean ± SEM, n = 3 biologically independent samples ( C ). Statistical analysis was performed using two-tailed unpaired t -test. The data are representative of 3 experiments with similar results. D DUSP16 mRNA expression in DLD-1 cells transfected with vector ( n = 6) or human DUSP16 cDNA ( n = 6) quantified by qPCR. Statistical analysis was performed using two-tailed unpaired t -test. DUSP16 protein expression in these clones was assessed by Western blot. E , F Apoptosis induction between the vector- and DUSP16 -transfected DLD-1 cells after cisplatin treatment was analyzed ( E ) and bar charts show the percentage of apoptotic and surviving cells in both untreated and cisplatin-treated groups with mean ± SEM, n = 3 biologically independent samples ( F ). Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. G DUSP16 mRNA and protein expression in Nugc3 cells transfected with vector or human DUSP16 cDNA was determined by qPCR and immunoblotting, respectively. Dot plot shows DUSP16 mRNA expression in vector ( n = 3) and DUSP16 ( n = 5) expressing clones. Statistical analysis was performed using two-tailed unpaired t -test. H , I Apoptosis induction between vector- and DUSP16 -transfected Nugc3 cells after cisplatin treatment was analyzed and bar charts ( I ) show the percentage of apoptotic and surviving cells in untreated and cisplatin-treated cells, respectively, with mean ± SEM, n = 3 biologically independent samples. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. The data are representative of 3 experiments with similar results. Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Clone Assay, Comparison, Staining, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: A Comparison of apoptosis induction between HK-1 and C666-1 cells in response to carboplatin treatment by flow cytometry analysis. Bar chart shows the percentage of apoptotic cells with mean values ± standard error of the mean (SEM), n = 3 biologically independent samples. The data are representative of three experiments with similar results. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. B Comparison of apoptosis induction between vector- and DUSP16 -transfected HK-1 cells after oxaliplatin or carboplatin treatment by flow cytometry analysis. Bar chart shows the percentage of apoptotic cells (including both AnnexinV + 7AAD + and AnnexinV + 7AAD − cells) with mean values ± SEM, n = 3 biologically independent samples. The data are representative of three experiments with similar results. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. C Comparison of apoptosis induction between HCT116 and DLD-1 cells in response to oxaliplatin treatment by flow cytometry analysis. Bar chart shows the percentage of apoptotic cells with mean values ± SEM, n = biologically independent samples. The data are representative of three experiments with similar results. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. D Comparison of apoptosis induction between vector- and DUSP16 -transfected HCT116 cells after oxaliplatin or carboplatin treatment by flow cytometry analysis. Bar chart shows the percentage of apoptotic cells with mean values ± SEM, n = 3 biologically independent samples. The data are representative of three experiments with similar results. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Comparison, Flow Cytometry, Two Tailed Test, Plasmid Preparation, Transfection

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: A – C Western blot analysis of p-MAPKs, total MAPKs, c-Myc, cleaved caspase 9 (C-C9), and cleaved caspase 3 (C-C3) was carried out on vector- and DUSP16 -transfected HK-1 ( A ), DLD-1 ( B ), and Nugc3 ( C ) cells after cisplatin treatment. The data is representative of at least two experiments with similar results. D Western blot analysis of cisplatin-induced cytochrome c (Cyt c) release in cytosolic fractions of vector- and DUSP16 -transfected HK-1 and DLD-1 cells. The data are representative of three experiments with similar results. E Scatterplots depicting changes in the mitochondrial membrane potential in vector- and DUSP16 -transfected HK-1 cells upon cisplatin treatment. F Western blot analysis of BAX expression in mitochondrial and cytosolic fractions of vector- and DUSP16 -transfected HK-1 and DLD-1 cells. The data are representative of three experiments with similar results. G Western blot analysis of BAX wild-type (WT) and knockout (KO) DLD-1 cell clones with or without DUSP16 overexpression. The data are representative of two experiments with similar results. H BAX WT and KO DLD-1 cells with or without DUSP16 overexpression were treated with cisplatin for 48 h to examine cell apoptosis by staining with AnnexinV + and 7AAD + followed by flow cytometry analysis. Bar chart shows the percentage of apoptotic cells with mean values ± SEM, n = 3 biologically independent samples. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001. The data are representative of three experiments with similar results. Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Western Blot, Plasmid Preparation, Transfection, Membrane, Expressing, Knock-Out, Clone Assay, Over Expression, Staining, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: A Vector- or DUSP16 -transfected HK-1 cells were inoculated into NSG mice. Cisplatin was injected at 3 mg/kg body weight every 3 days. After 20 days, tumors were harvested to measure the sizes and weights. The data are expressed as dots representing the weight of each tumor; the scale bars are the mean ± standard error of the mean (SEM) of tumor weights from six mice in each treatment group of the representative experiment (mean ± SEM, n = 6). Statistical analysis was performed using two-tailed unpaired t -test. B Growth of untreated and cisplatin-treated xenografts of vector- and DUSP16 -transfected DLD-1 cells. Box-and-whisker plots show means ± SEM of tumor weights from four mice from each treatment group per experiment (mean ± SEM, n = 4). Boxes correspond to the 25th, 50th/median and 75th percentiles; whiskers denote maximum and minimum. Statistical analysis was performed using two-tailed unpaired t -test. C , D IHC analysis for cleaved caspase 3 of HK-1 tumor sections; representative images of cleaved caspase 3 staining ( n = 6) were shown ( C ). Boxed regions were magnified and presented under the respective panels. Black arrows highlight the caspase 3 staining. Box-and-whisker plots show means ± SEM of the means of cleaved caspase 3 staining in each group of mice ( D ). Boxes correspond to the 25th, 50th/median and 75th percentiles; whiskers denote maximum and minimum. Statistical analysis was performed using two-tailed unpaired t -test. *** P < 0.0001 (mean ± SEM, n = 3). E , F IHC analysis for Ki67 of tumor sections. Representative images of Ki67 staining were shown ( E ). Boxed regions were magnified and presented under the respective panels. Black arrows highlighted the Ki67 staining. Box-and-whisker plots show means ± SEM of Ki67 staining ( F ) from each group of mice *** P < 0.0001 (mean ± SEM, n = 3). The data are representative of three experiments with similar results. Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Transfection, Injection, Two Tailed Test, Whisker Assay, Staining

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: C666-1 cells were transfected with DUSP16 CRISPR/Cas9 constructs to knockdown DUSP16 expression. A Bar chart shows the mRNA expression in vector and DUSP16 construct transfected cells with mean values ± SEM, n = 3 biologically independent samples. Statistical analysis was performed using two-tailed unpaired t -test. Protein levels of DUSP16 were quantified by immunoblotting. *** P < 0.0001. B Apoptosis of control and DUSP16 knockdown (KD) cells after cisplatin treatment was measured by AnnexinV/7AAD staining and flow cytometry. C Bar charts show the percentage of apoptotic and surviving (unstained) cells with mean values ± SEM, n = 3 biologically independent samples. Statistical analysis was performed using two-tailed unpaired t -test. The data are representative of three experiments with similar results. D Western blot analysis of the pJNK, pP38, pERK, their total protein levels, and c-Myc in control or DUSP16-KD C666-1 cells was carried out after cisplatin treatment. The data are representative of three experiments with similar results. Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Transfection, CRISPR, Construct, Knockdown, Expressing, Plasmid Preparation, Two Tailed Test, Western Blot, Control, Staining, Flow Cytometry

Journal: Nature Communications

Article Title: DUSP16 promotes cancer chemoresistance through regulation of mitochondria-mediated cell death

doi: 10.1038/s41467-021-22638-7

Figure Lengend Snippet: A Mice were inoculated with control or DUSP16-KD cells. Cisplatin was injected at 3 mg/kg body weight every 3 days. After 20 days, tumors were harvested, and the sizes and weights were measured. The data are expressed as dots representing the weight of each tumor; the scale bars are the mean ± SEM of tumor weights from 4 (the vector control group) or 5 mice (the other groups) of the representative experiment. Statistical analysis was performed using two-tailed unpaired t -test. B Representative images of immunohistochemistry analysis for cleaved caspase 3 and Ki67 staining of tumor sections were shown. Boxed regions were magnified and presented under the respective panels. Black arrows highlight the caspase 3 and Ki67 staining, respectively. Box-and-whisker plots show mean values ± SEM of cleaved caspase 3 and Ki67 staining from each group of mice ( n = 4). Boxes correspond to the 25th, 50th/median and 75th percentiles; whiskers denote maximum and minimum. Black scale bars = 200 μm, white scale bars = 50 μm. Statistical analysis was performed using two-tailed unpaired t -test. * P < 0.05. The data are representative of three experiments with similar results. C Representative images of HNSCC specimens ( n = 50) showing negative (0 +), low (1+), intermediate (2+), and high (3+) staining of DUSP16 in tumor cells. Black scale bars = 200 μm. D Patients with higher DUSP16 expression showed lower disease-free survival (DFS) compared to patients with low DUSP16 expression (log-rank test, two-sided, P = 0.042). E Representative images of breast cancer specimens ( n = 113) showing negative (0 +), low (1 +), and high (3 +) staining of DUSP16 in tumor cells. Black scale bars = 200 μm. F Among the 113 breast cancer patients (age 28–64) treated with cisplatin, carboplatin, or lobaplatin after surgery, high DUSP16 protein expression in their tumors was associated with lower disease-free survival compared to patients with low DUSP16 expression (log-rank test, two-sided, P ˂ 0.0001). Source data are provided as a Source data file.

Article Snippet: Knockdown of DUSP16 in C666-1 cells was performed by transfection with DUSP16 CRISPR/Cas9 KO Plasmid (sc-405727, Santa Cruz) and DUSP16 HDR plasmid (sc-405727-HDR, Santa Cruz) with UltraCruz® Transfection Reagent (sc-395739) according to the manufacturer’s instructions.

Techniques: Control, Injection, Plasmid Preparation, Two Tailed Test, Immunohistochemistry, Staining, Whisker Assay, Expressing