Journal: Head & neck

Article Title: Novel mechanism of cisplatin resistance in head and neck squamous cell carcinoma involving extracellular vesicles and a copper transporter system.

doi: 10.1002/hed.27620

Figure Lengend Snippet: FIGURE 1 Expression of ATP7B and secretion of EVs by HNSCC cells. (A) Representative immunohistochemistry images of ATP7B expression in normal tissues and tumor tissues in the head and neck region. The upper row shows the low-magnification image and the lower row shows the high-magnification image. The box indicated by a dotted line shows the coincident area in each image. Scale bar: 200 μm. (B) Box and whisker plot of ATP7B-positive cells (%) in normal tissue (n = 5), tumor tissue from early-stage cases (stage I/II, n = 12) and tumor tissue from advanced cases (stage III/IV, n = 15) in the head and neck region. *p < 0.05, Kruskal–Wallis test. (C) Western blotting (WB) showing ATP7B protein expression in HNSCC cell lines. β-Actin was used as a loading control. (D) Representative TEM images of EVs derived from HNSCC cell lines. Scale bar: 100 nm. (E) Representative particle diameter distribution of EVs derived from HNSCC cell lines. Peak values were 100–200 nm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: SAS and HSC-3 cells were transfected with 5.0 μg of control short hairpin (sh)RNA plasmid (sc-108 060; Santa Cruz Biotechnology) or ATP7B shRNA plasmid (sc44 491-SH; Santa Cruz Biotechnology) with the use of 4D-NucleofectorTM (Lonza Group, Walkersville, MD).

Techniques: Expressing, Immunohistochemistry, Whisker Assay, Western Blot, Control, Derivative Assay

Journal: Head & neck

Article Title: Novel mechanism of cisplatin resistance in head and neck squamous cell carcinoma involving extracellular vesicles and a copper transporter system.

doi: 10.1002/hed.27620

Figure Lengend Snippet: FIGURE 2 Establishment of CDDP-resistant sublines of HNSCC cells. (A) The IC50 of CDDP in parental or resistant cells of HNSCC was evaluated after 24-h CDDP treatment (n = 6). **p < 0.01, Welch's t-test. (B) The CDDP concentration (μg/1 105 cells) in parental or resistant HNSCC cells was evaluated after 24-h CDDP treatment (n = 3). *p < 0.05, Welch's t-test. (C) WB showing cleaved caspase 3 and caspase 3 protein expression in parental or resistant cells of HNSCC treated with CDDP for 24 h. β-Actin = loading control. (D) WB showing ATP7B and CTR1 protein expression in parental or resistant cells of HNSCC. β-Actin = loading control. (E) Immunocytochemistry (ICC) showing ATP7B protein expression in parental or resistant cells of HNSCC. Top row: DAB staining; bottom row: Fluorescent staining. Scale bar: 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: SAS and HSC-3 cells were transfected with 5.0 μg of control short hairpin (sh)RNA plasmid (sc-108 060; Santa Cruz Biotechnology) or ATP7B shRNA plasmid (sc44 491-SH; Santa Cruz Biotechnology) with the use of 4D-NucleofectorTM (Lonza Group, Walkersville, MD).

Techniques: Concentration Assay, Expressing, Control, Immunocytochemistry, Staining

Journal: Head & neck

Article Title: Novel mechanism of cisplatin resistance in head and neck squamous cell carcinoma involving extracellular vesicles and a copper transporter system.

doi: 10.1002/hed.27620

Figure Lengend Snippet: FIGURE 3 The relationship between ATP7B knockdown and EV secretion. (A) WB showing ATP7B protein expression in shRNA- transfected HNSCC cells. β-Actin = loading control. (B) ICC showing ATP7B protein expression in shRNA-transfected HNSCC cells. Scale bar: 100 μm. (C) Cell viability at 24 h after CDDP addition. The survival rate was calculated relative to the control group (n = 3). *p < 0.05, Welch's t-test. (D): Representative TEM images of EVs derived from shRNA-transfected HNSCC cells. Scale bar: 100 nm. (E) Representative particle diameter distribution of EVs derived from shRNA-transfected HNSCC cells. Peak values were 100–200 nm. (F) EV protein per whole cell lysate (WCL) protein (%) of shRNA-transfected HNSCC cells (n = 9). *p < 0.05, Kruskal–Wallis test. (G) WB showing expressions of EpCAM, CD9, HSP90 and GAPD proteins as EV markers in shRNA-transfected HNSCC cell-derived EVs. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: SAS and HSC-3 cells were transfected with 5.0 μg of control short hairpin (sh)RNA plasmid (sc-108 060; Santa Cruz Biotechnology) or ATP7B shRNA plasmid (sc44 491-SH; Santa Cruz Biotechnology) with the use of 4D-NucleofectorTM (Lonza Group, Walkersville, MD).

Techniques: Knockdown, Expressing, shRNA, Transfection, Control, Derivative Assay

Journal: Head & neck

Article Title: Novel mechanism of cisplatin resistance in head and neck squamous cell carcinoma involving extracellular vesicles and a copper transporter system.

doi: 10.1002/hed.27620

Figure Lengend Snippet: FIGURE 4 The effect of GW4869 on EV secretion and ATP7B expression. (A) Amount of EV protein per WCL protein (%) of HNSCC cells treated with or without GW4869 for 24 h (n = 6). *p < 0.05, Kruskal–Wallis test. (B) Representative images of HNSCC cells treated with or without GW4869 for 24 h. Scale bar: 100 μm. (C) ICC showing ATP7B protein expression in HNSCC cells treated with or without GW4869 for 24 h. Scale bar: 100 μm. (D) WB showing ATP7B protein expression in HNSCC cells treated with or without GW4869 for 24 h. β-Actin = loading control. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: SAS and HSC-3 cells were transfected with 5.0 μg of control short hairpin (sh)RNA plasmid (sc-108 060; Santa Cruz Biotechnology) or ATP7B shRNA plasmid (sc44 491-SH; Santa Cruz Biotechnology) with the use of 4D-NucleofectorTM (Lonza Group, Walkersville, MD).

Techniques: Expressing, Control

Journal: Head & neck

Article Title: Novel mechanism of cisplatin resistance in head and neck squamous cell carcinoma involving extracellular vesicles and a copper transporter system.

doi: 10.1002/hed.27620

Figure Lengend Snippet: FIGURE 5 Additive effect of GW4869 on the cell-killing of CDDP. (A) Representative images of HNSCC cells treated with GW4869 and/or CDDP for 24 h. Scale bar: 200 μm. (B) Number of surviving HNSCC cells treated with GW4869 and/or CDDP for 24 h (n = 9). *p < 0.05, Kruskal–Wallis test. (C) WB showing cleaved caspase 3 and caspase 3 protein expressions in HNSCC cells treated with GW4869 and/or CDDP for 24 h. β-Actin = loading control. (D) ICC showing ATP7B protein expression in HNSCC cells treated with GW4869 and/or CDDP for 24 h. Scale bar: 200 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: SAS and HSC-3 cells were transfected with 5.0 μg of control short hairpin (sh)RNA plasmid (sc-108 060; Santa Cruz Biotechnology) or ATP7B shRNA plasmid (sc44 491-SH; Santa Cruz Biotechnology) with the use of 4D-NucleofectorTM (Lonza Group, Walkersville, MD).

Techniques: Control, Expressing

Journal: Head & neck

Article Title: Novel mechanism of cisplatin resistance in head and neck squamous cell carcinoma involving extracellular vesicles and a copper transporter system.

doi: 10.1002/hed.27620

Figure Lengend Snippet: FIGURE 6 Graphical abstract. CDDP is normally incorporated into cells and then binds to nuclear chromosomal DNA, thereby inducing apoptosis and exerting a cytotoxic effect. ATP7B localizes to the late endosomes, where it encapsulates CDDPP and may export CDDP to the extracellular space using EVs. Our present findings also suggest that GW4869 may block the release of EVs from late endosomes by acting to inhibit ATP7B. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: SAS and HSC-3 cells were transfected with 5.0 μg of control short hairpin (sh)RNA plasmid (sc-108 060; Santa Cruz Biotechnology) or ATP7B shRNA plasmid (sc44 491-SH; Santa Cruz Biotechnology) with the use of 4D-NucleofectorTM (Lonza Group, Walkersville, MD).

Techniques: Blocking Assay

Journal: Scientific Reports

Article Title: Therapeutic potential of hepatocyte-like-cells converted from stem cells from human exfoliated deciduous teeth in fulminant Wilson’s disease

doi: 10.1038/s41598-018-38275-y

Figure Lengend Snippet: Hepatic functions and ATP7B expression of SHED-Heps. ( a – e ) In vitro hepatic function assays of SHED-Heps. Culture of SHED-Heps and SHED and measuring of human albumin (hALB), glucose, triglyceride (TG), and urea in the conditioned medium are performed according to the Methods. ( a ) Xenobiotic activity of SHED-Heps and SHED via CYP3A4 is analyzed under dexamethasone stimulation (50 μM). ( b ) Low density lipoprotein (LDL) uptake and bile acid transport are analyzed by DiI-Ac-LDL ( c ) and cholyl-lysyl-fluorescein (CLF) ( d ) staining, respectively. ( e – g ) QRT-PCR shows the expression of ATPase copper transporting beta gene ( ATP7B ) in SHED and SHED-Heps. The results are shown as the ratio to the expression in SHED- Heps ( e ). Immunofluorescent staining demonstrates the expression of ATP7B in SHED and SHED- Heps. Control IgG, isotype-matched IgG staining. ( f ) QRT-PCR shows the effect of ATP7B siRNA treatment in SHED and SHED-Heps. The results are shown as the ratio to the expression of 18S ribosomal RNA ( 18S ). siATP7B, ATP7B siRNA pre-treatment; siCTRL, scrambled control siRNA pre-treatment ( g ) Immunofluorescent staining shows the effect of ATP7B. siRNA treatment in SHED and SHED-Heps. ( h ) ( a , b , e , g ) n = 3 for all groups. **P < 0.01, ***P < 0.005. Graph bars show the means ± SEM. ( c , d , f , h ) Nuclei are stained with DAPI. Bars = 30 μm ( c , d ), 20 μm ( f , h ).

Article Snippet: SHED and SHED-Heps were treated for 48 h with Lipofectamine RNAiMAX (Thermo Fisher Scientific) mixed with human ATP7B siRNA (20 nM; Santa Cruz Biotechnology, Santa Cruz, CA), human stanniocalcin 1 (STC1) siRNA (20 nM; Santa Cruz Biotechnology, Santa Cruz), and the corresponding control scrambled siRNA (Santa Cruz Biotechnology).

Techniques: Expressing, In Vitro, Activity Assay, Staining, Quantitative RT-PCR, Control

Journal: Scientific Reports

Article Title: Therapeutic potential of hepatocyte-like-cells converted from stem cells from human exfoliated deciduous teeth in fulminant Wilson’s disease

doi: 10.1038/s41598-018-38275-y

Figure Lengend Snippet: Suppressive effects and integration of transplanted SHED-Heps on copper accumulated hepatic failure in fulminant LEC rats. SHED and SHED-Heps are transplanted in copper-overloaded LEC rats at 6 weeks of the age. ( a , b ) Histological assays of liver tissues at 10 weeks of the age. Representative images of liver tissues are analyzed by hematoxylin and eosin staining (HE). CV , central vein. ( a ) Copper accumulation is analyzed by Rhodanine staining. Yellow arrows, copper deposition. ( b , c ) Biochemical assay shows the copper contents in the liver tissues at 10 weeks of the age. ( d , e ) Post-transplant kinetics of DiR- labeled SHED and SHED-Hep cells in fulminant LEC rats after 2 weeks (2w) of SHED- and SHED- Hep-transplantation. In vivo tracing shows that DiR labeling is detected in the part of liver of rats. ( d ) Ex vivo tracing shows that DiR labeling is detected in liver and spleen, but not in lung and kidney, of rats. ( e , f , g ) Integration of transplanted SHED- and SHED-Heps in the liver tissues of fulminant LEC rats after 4 weeks of the transplantation. Immunohistochemial assay demonstrates the localization of human albumin (hALB) positive cells in the parenchyma of recipient liver tissues at 10 weeks of the age. Nuclei are stained with hematoxylin. ( f ) Double immunofluorescence shows that localization of human albumin (hALB, red) and human ATP7B (hATP7B, green) in the parenchymal cells of recipient liver tissues of SHED- and SHED-Hep-transplanted fulminant LEC rats. Nuclei are stained with DAPI. ( g ) ( a – g ) LEA, control LEA rats; LEC, non-transplanted fulminant LEC rats; SHED-T, SHED-transplanted fulminant LEC rats; SHED-Hep-T, SHED-Hep-transplanted fulminant LEC rats. ( a , b , f , g ) Bars = 50 μm ( a ), 100 μm ( b , f ), 30 μm ( g ). ( c ) n = 3 for all groups. Graph bars show the means ± SD. *P < 0.05 and ***P < 0.005.

Article Snippet: SHED and SHED-Heps were treated for 48 h with Lipofectamine RNAiMAX (Thermo Fisher Scientific) mixed with human ATP7B siRNA (20 nM; Santa Cruz Biotechnology, Santa Cruz, CA), human stanniocalcin 1 (STC1) siRNA (20 nM; Santa Cruz Biotechnology, Santa Cruz), and the corresponding control scrambled siRNA (Santa Cruz Biotechnology).

Techniques: Staining, Labeling, Transplantation Assay, In Vivo, Ex Vivo, Immunofluorescence, Control

Journal: Scientific Reports

Article Title: Therapeutic potential of hepatocyte-like-cells converted from stem cells from human exfoliated deciduous teeth in fulminant Wilson’s disease

doi: 10.1038/s41598-018-38275-y

Figure Lengend Snippet: In vitro copper metabolism of SHED-Heps. ( a – c ) In vitro copper metabolism assay in SHED-Heps and SHED via ATP7B. SHED, SHED-Heps, and ATP7B-knock-downed SHED-Heps are cultured for 6 h under CuSO 4 (600 μM) treatment and are subsequently cultured without CuSO 4 . Intracellular accumulated copper is measured at indicated time. Biochemical assay shows the intracellular copper contents in SHED and SHED-Heps ( a ), ATP7B- knock-downed SHED ( b ), and ATP7B-knock-downed SHED-Heps ( c ). ( d – f ) I n vitro survival assay in SHED and SHED-Heps via ATP7B under copper stimulation. SHED, SHED-Heps, and ATP7B-knock-downed SHED-Heps are cultured under the different stimulation of CuSO 4 (0, 125, 250, and 500 μM). The cell viability of the cells is measured after 3 days of the stimulation. Biochemical assay shows the viability of SHED and SHED-Heps ( d ), ATP7B-knock-downed SHED ( e ), and ATP7B- knock-downed SHED-Heps ( f ). ( g , h ) ATP7B-siRNA-treated SHED and ATP7B-siRNA-treated SHED-Heps are transplanted into fulminant LEC rats at 6 weeks of the age. The survival of LEC rats transplanted with ATP7B-siRNA-treated SHED (si ATP7B -SHED-T; ( g ), n = 5) and ATP7B-siRNA- treated SHED-Heps (si ATP7B -SHED-Hep-T; ( h ), n = 5) is assay by Kaplan-Meier curve. ( a – f) The results are shown as the ratio to the copper concentration at 0 h in each group. n = 3 for all groups. *P < 0.05 and ***P < 0.005. ns: no significance. Graph bars show the means ± SEM. ( a ) ††† P < 0.005 (vs. SHED at each tipe point). NS: no significance (vs. SHED at each tipe point). ( b , c , e , f ) siATP7B, ATP7B-siRNA treatment; siCTRL, control scrambled siRNA treatment. ( d ) ### P < 0.005 (vs. 0 h of each group). NS: no significance (vs. 0 h of each group). ( g , h ) CTRL-siRNA-SHED-Hep-T, fulminant LEC rats transplanted with control-siRNA-treated SHED (n = 5).

Article Snippet: SHED and SHED-Heps were treated for 48 h with Lipofectamine RNAiMAX (Thermo Fisher Scientific) mixed with human ATP7B siRNA (20 nM; Santa Cruz Biotechnology, Santa Cruz, CA), human stanniocalcin 1 (STC1) siRNA (20 nM; Santa Cruz Biotechnology, Santa Cruz), and the corresponding control scrambled siRNA (Santa Cruz Biotechnology).

Techniques: In Vitro, Cell Culture, Clonogenic Cell Survival Assay, Concentration Assay, Control