Journal: Cancer cell

Article Title: β2 adrenergic-neurotrophin feed-forward loop promotes pancreatic cancer

doi: 10.1016/j.ccell.2017.11.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: VECTASTAIN ABC HRP kit , Vector laboratories , Cat#PK-4000.

Techniques: Plasmid Preparation, Recombinant, Blocking Assay, Enzyme-linked Immunosorbent Assay, MTT Cell Proliferation, SYBR Green Assay, Western Blot, Knock-Out, Software

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: EGF induces translocation of EGFR to the Golgi. (a) HeLa cells were transfected with pDsRed-syntaxin 6. Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. EGFR was labeled with the indicated antibodies. The boxed areas are shown in detail in the insets. Insets 2–1 and 2–2 show representative colocalizations of EGFR and syntaxin 6. Scale bar, 10 μm. (b) Cells were serum starved overnight and then treated without or with EGF (50 ng/ml) for 20 min. Endogenous EGFR and syntaxin 6 were labeled with a primary antibodies and secondary fluorescein isothiocyanate (donor, green) and Texas-Red (acceptor; red) antibody. An Fc image was obtained using the Zeiss ZEN software. Scale bar, 20 μm. Quantitation of the FRET intensity is shown in the right. (c) Cell lysate was loaded onto the 0–30% OptiPrep density gradient medium and subjected to ultracentrifugation, and fractions were separated using the Gradient Station. The early endosome, the Golgi and ER markers were used to analyze fractions. S, short expose; L, long expose. (d) HeLa cells were treated with or without EGF (50 ng/ml) for 20 min after starvation overnight. The EGFR levels in the Golgi-enriched fraction (fraction 9) were analyzed using immunoblotting. (e) Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. One cell was used for z-stack scanning. Representative images were shown. The boxed areas are shown in detail in the insets. Scale bar, 10 μm. (f) Cells were transfected with GalNac T2 for 48 h or direct staining of endogenous marker, GM130. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for indicated time and analyzed using confocal microscope. Scale bar, 20 μm. Quantitation of colocalization of EGFR and endosomal markers is shown in the bottom. (g) HeLa cells were transfected with EGFP-GalNac T2. Cells were exposed to serum-free media overnight following treatment without or with EGF (50 ng/ml) for indicated time. Scale bar, 20 μm. The boxed areas are shown in the insets. Quantitation of colocalization of phospho-EGFR and total EGFR with the GalNac T2 is shown in the bottom. (h) HeLa cells were serum-starved overnight before EGF stimulation for indicated time. Total lysate and the Golgi-enriched fractions were performed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot to examine the phospho-1086 of EGFR and total EGFR levels.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Labeling, Software, Quantitation Assay, Western Blot, Staining, Marker, Microscopy, Polyacrylamide Gel Electrophoresis

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for the Golgi translocation of EGFR. (a) Cells were first transfected with syntaxin 6 or control (Ctrl) siRNAs for 24 h and then transfected with GalNac T2 for 48 h. Cells were then maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (b) Cells were transfected with syntaxin 6 or control siRNAs. After 72 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. The EGFR levels in the Golgi-enriched fraction were analyzed using immunoblotting. (c) Cells were transfected with CCD domain of syntaxin 6 or control vector. After 48 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. Cells were analyzed by confocal microscope. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (d) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and was restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated without or with EGF (50 ng/ml) for 20 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (e) Cells were transfected with syntaxin 6 or control siRNAs. After 24 h transfection, cells were transfected with GalNac T2 for 48 h. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and then analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Quantitation of colocalization of EGFR and endosomal markers is shown in the right. (f) HeLa cells were serum-starved overnight and stimulated without or with EGF (50 ng/ml) for 20 min. Cell lysates were immunoprecipitated with the indicated antibodies and subjected to immunoblot analysis as indicated. (g) In vitro transcribed and translated biotin-labeled syntaxin 6 was incubated with recombinant GST-fused EGFR fragments, pulled down using glutathione-Sepharose beads and visualized with horseradish peroxidase (HRP) conjugated streptavidin. CT, c-terminal domain; IB, immunoblot; KD, kimase domain fragment; TM, transmembrane domain fragment.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Confocal Microscopy, Quantitation Assay, Marker, Western Blot, Plasmid Preparation, Microscopy, shRNA, Immunoprecipitation, In Vitro, Labeling, Incubation, Recombinant

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Microtubules and dynein are required for EGF-induced Golgi transport of EGFR. (a) Serum-starved cells were treated with EGF. Double staining of EGFR and α-tubulin were subjected to confocal microscopy assay. Scale bars, 20 μm. (b) HeLa cells were transfected with GFP-GalNac T2, treated with microtubules or dynein inhibitors and then stimulated with EGF. The Golgi-enriched fractions were purified and subjected to immunoblot analysis with the indicated antibodies. (c) Serum-starved HeLa cells were treated as shown in (b) and then stimulated with EGF and analyzed by a confocal microscope. Scale bars, 20 μm. The boxed areas are shown in detail in the insets. Representative colocalization of EGFR and GalNac T2 is shown in inset 2–1. Quantitation of cells with Golgi-localized EGFR is shown in the lower panel. (d) HeLa cells were transfected with GFP-GalNac T2 expression plasmid and then transfected with control (ctrl) vector or CDK1 and cyclin B plasmids, respectively. Cells were then serum starved overnight, stimulated with EGF and further analyzed under a confocal microscope. Scale bar, 20 μm. Quantitative results are shown in the right. (e) Representative frames of time-lapse confocal microscopic image of cells treated with or without nocodazole. HeLa cells were transfected with EGFP–EGFR (green) and DsRed–syntaxin 6 (red) plasmids. After serum starvation overnight and EGF stimulation, images were collected at 30-s intervals as indicated. Scale bar, 5 μm. (f) Serum-starved HeLa cells were transfected with dynein shRNAs and then stimulated with EGF. Golgi-enriched fractions were purified and subjected to immunoblot analysis with indicated antibodies. DMSO, dimethyl sulfoxide; Noc, nocodazole; PT, paclitaxel; Van, vanadate.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Double Staining, Confocal Microscopy, Transfection, Purification, Western Blot, Microscopy, Quantitation Assay, Expressing, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for EGFR nuclear translocation. (a) HeLa cells were transfected with syntaxin 6 or control siRNAs and maintained in a serum-free media overnight and treated with EGF (50 ng/ml) for 30 min. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (b) Cells were transfected with syntaxin 6 or control siRNA and maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (c) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and vector control were restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (d) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (e) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Nuclear and non-nuclear fractions were subjected to immunoblot analysis with the indicated antibodies. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Quantitation Assay, Western Blot, shRNA, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Nuclear function of EGFR requires syntaxin 6 and microtubules. (a) After overnight serum starvation, cells were pretreated with the indicated inhibitors for 30-min treatment and then stimulated with EGF for 30 min, followed by chromatin-IP assay. For IgG control, lysate of cells without EGF stimulation was used. (b) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for 30 min, followed by chromatin-IP assy. For IgG control, lysate of cells without EGF stimulation was used. (c) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for indicated time. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) was used to analyze the mRNA level. (d) HeLa cells transfected with control siRNAs and siRNAs for syntaxin 6 were transfected with reporter plasmids containing CCND1 promoter. Then, after 24 h transfection, cells were maintained in serum-free media overnight and treated with EGF for indicated time. Total lysates were used for luciferase assay. Error bars were derived from three independent experiments. (e) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After transfection, 4 × 105 cells were seeded in a six-well plate, incubated for 72 h and then counted. (f) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 48 h transfection, cells were treated with BrdU (100 μm) for 1 h. Cells were assayed for BrdU incorporation by flow cytometry. (g) BT20 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (h) OVCAR3 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (i) A schematic model of syntaxin 6- and microtubule-mediated Golgi and nuclear transport of EGFR.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Chromatin Immunoprecipitation, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Derivative Assay, Incubation, BrdU Incorporation Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

doi: 10.1038/ncomms14405

Figure Lengend Snippet: ( a ) Diagram of the PC12-HttQ23 and Htt-Q74 proteins and experimental design. ( b ) Cells expressing either Htt-Q23 or Htt-Q74 were cultivated at 37 °C ( c ) in the presence of tetracycline (Dox) for 3 days to induce expression, exposed to heat shock 1 h at 42 °C, allowed to recover at 37 °C for 7 h (HS) and protein extracts immunoblotted with the indicated antibodies. HSF1 bands were quantitated using Quantity One image software (BioRad) and values normalized using GAPDH as loading control and referenced to control at 37 °C ( c ) in the absence of −Dox. ( c ) qRT-PCR analysis of the Hsp70 and Hsp25 genes in Htt-Q74-expressing cells as in B. HS (+Dox) group was compared with HS (−Dox) group. Error bars represent means±s.e.m., n =4. Unpaired t -test, *** P <0.001. ( d ) Analysis of Hsp70 promoter occupancy by HSF1. Error bars represent means±s.e.m., n =3. Unpaired t -test, ** P <0.01. ( e ) Mouse-derived striatal STHdh Q7 and STHdh Q111 cells were cultured at 33 °C ( c ), heat shocked at 42 °C 1 h with recovery at 33 °C for 7 h and immunoblotted with the indicated antibodies. HSF1 was quantified using Quantity One image software and normalized using GAPDH as loading control and referenced to control at 37 °C in the non-pathogenic STHdh Q7 . ( f ) Striatal samples from Wild type (WT) C57BL/6 or KIQ175 mice at 2, 6 and 12 months analysed by immunoblotting ( n =4). ( g ) Dorsal striatal sections from WT and KIQ175 mice ( n =3) at 6 months assayed by immunohistochemistry (IHC) for mHtt aggregation (1C2), HSF1 and HSF1-S303 phosphorylation using DAPI staining as control. Scale bar: 10 μm. ( h ) Gastrocnemius muscle extracts from WT and KI175 mice ( n =4) of the indicated age, immunoblotted for HSF1, Hsp70 and GAPDH. ( i ) Protein extracts from HD patient striatum and controls immunoblotted with indicated antibodies. ( j ) HSF1 phosphoproteomic analysis under non-pathogenic (−Dox) and pathogenic (+Dox) conditions in hsf1 −/− MEF inducible cell line expressing GFP-Htt-Q74 expressing HSF1. See for uncropped immunoblots. HSF1 represented by the Regulatory domain, DBD; DNA binding domain; LZ1-3 and LZ4; leucine zipper domains.

Article Snippet: HttQ74 cells were transfected with HA-Ub vector (Addgene #18712) by using Lipofectamine LTX Reagent (Invitrogen) following protocol instructions (Invitrogen protocols 25-0946 W). hsf1 −/− MEF-GFP-Htt-Q74 cells were transfected with pcDNA WT HSF1 or the HSF1 S303A mutant plasmid using Lipofectamine LTX reagent. hsf1 −/− MEFs were electroporated with FBXW7-FLAG tag and WT HSF1 or S303A plasmids using the SE Cell Line Nucleofector.

Techniques: Expressing, Software, Quantitative RT-PCR, Derivative Assay, Cell Culture, Western Blot, Immunohistochemistry, Staining, Binding Assay

Journal: Nature Communications

Article Title: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

doi: 10.1038/ncomms14405

Figure Lengend Snippet: ( a , b ) PC12 cells expressing Htt-Q74 for 1, 2 or 3 days followed by Heat Shock (HS) and recovery as indicated. Control and HS in the absence of Dox correspond to cells incubated during 3 days at 37 °C. ( c ) Htt-Q74 cells Dox-induced or not for 3 days and exposed to 2 μM 17-AAG and/or MG132 (5 μM) for 6 h and extracts immunoblotted with the indicated antibodies. ( d ) Diagram of the effects of 17-AAG and MG132 treatment and Htt-Q74 expression in HSF1. ( e ) Htt-Q74 cells transfected with human influenza haemagglutinin-ubiquitin (HA-Ub) plasmid Dox induced or not and treated with 5 μM MG132 treatment for 6 h. Whole-cell extract and HA immunoprecipitated (IP:HA) and HSF1 immunoprecipitated samples (IP:HSF1) were immunoblotted as indicated. ( f ) Transcript levels for indicated E3 ligases evaluated by qRT-PCR from striatum of WT and KIQ175 mice at 6 months. Error bars represent±s.e.m., ( n =3). Unpaired t -test, * P <0.05. ( g ) Human striatum samples from HD patients and controls and ( h ) Mouse striatum from 12 months old WT and KIQ175 mice were immunoblotted for HSF1 and Fbxw7. ( i ) Fbxw7 siRNA in STHdh Q7 and STHdh Q111 cells using scrambled RNA (Scr) as control. HSF1 was quantified using Quantity One image software and normalized using GAPDH as loading control and referenced to control at 37 °C in the non-pathogenic STHdh Q7 . ( j ) hsf1 −/− MEFs transfected with WT HSF1 or S303A mutant and Fbxw7-FLAG; samples immunoprecipitated with anti-FLAG and HSF1 detected. ( k ) hsf1 −/− MEFs expressing Dox-inducible Htt-Q74-GFP transfected with WT HSF1 or HSF1-S303A HS and 1 h recovery at 37 °C for 7 h. HSF1 was quantified using Quantity One image software and normalized using GAPDH as loading control and referenced to control (−Dox) expressing WT HSF1. See for uncropped immunoblots.

Article Snippet: HttQ74 cells were transfected with HA-Ub vector (Addgene #18712) by using Lipofectamine LTX Reagent (Invitrogen) following protocol instructions (Invitrogen protocols 25-0946 W). hsf1 −/− MEF-GFP-Htt-Q74 cells were transfected with pcDNA WT HSF1 or the HSF1 S303A mutant plasmid using Lipofectamine LTX reagent. hsf1 −/− MEFs were electroporated with FBXW7-FLAG tag and WT HSF1 or S303A plasmids using the SE Cell Line Nucleofector.

Techniques: Expressing, Incubation, Transfection, Plasmid Preparation, Immunoprecipitation, Quantitative RT-PCR, Software, Mutagenesis, Western Blot

Journal: Nature Communications

Article Title: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

doi: 10.1038/ncomms14405

Figure Lengend Snippet: ( a ) Mammalian CK2 holoenzyme subunit composition and function. ( b ) Htt-Q74 cells treated with CK2 kinase inhibitors TID43 or ( c ) Emodin 24 h before Htt-Q74 induction with Dox and heat shocked at 42 °C for 1 h followed by recovery at 37 °C for 7 h and extracts analysed by immunoblotting for Hsp70 and GAPDH. ( d ) Htt-Q74 cells treated with 5 μM TID43 and immunoblotted for HSF1 and P-HSF1-S303. ( e ) Fluorescent images for GFP-Htt-Q74 analysed microscopically in cells treated with DMSO or 1 μM TID43 as described in B. Scale bar: 200 μm. ( f ) Quantification of cells containing GFP-Htt-Q74 aggregates from E expressed as percentage of total number of cells evaluated. Error bars represent±s.e.m., ( n =500 cells). Unpaired t -test ** P <0.05. ( g ) hsf1 −/− MEFs expressing Dox-inducible Htt-Q74-GFP transfected with pcDNA or WT HSF1 and incubated with 1 μM TID43 24 h before Htt-Q74-GFP induction followed by heat shock at 42 °C during 1 and 7 h recovery at 37 °C. Cell viability expressed as % of viable cells under control conditions at 37 °C. Error bars represent±s.e.m., ( n =3). Unpaired t -test n.s., no significant, * P <0.05, ** P <0.01). ( h ) Htt-Q74 cells were transfected with siRNA against CK2β regulatory subunit or ( i ) CK2α and/or CK2α′ catalytic subunits using scrambled siRNA (Scr) as control 24 h before Htt-Q74 induction during 2 days followed by heat shock at 42 °C 1 h and recovery at 37 °C, 7 h. HSF1 was quantitated as in (F2H). All immunoblots shown for each panel contain the samples from the same membrane and were cropped to show only relevant data. See for uncropped immunoblots.

Article Snippet: HttQ74 cells were transfected with HA-Ub vector (Addgene #18712) by using Lipofectamine LTX Reagent (Invitrogen) following protocol instructions (Invitrogen protocols 25-0946 W). hsf1 −/− MEF-GFP-Htt-Q74 cells were transfected with pcDNA WT HSF1 or the HSF1 S303A mutant plasmid using Lipofectamine LTX reagent. hsf1 −/− MEFs were electroporated with FBXW7-FLAG tag and WT HSF1 or S303A plasmids using the SE Cell Line Nucleofector.

Techniques: Western Blot, Expressing, Transfection, Incubation

Journal: Nature Communications

Article Title: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

doi: 10.1038/ncomms14405

Figure Lengend Snippet: ( a ) CK2α, CK2α′ and CK2β protein levels in Htt-Q74 expressing cells under control ( c ) or heat shock conditions at 42 °C for 1 h (HS). CK2 subunit abundance was quantified using Quantity One image software normalized using GAPDH as loading control. CK2α′ ratio is shown and referenced to control (−Dox) cells. ( b ) CK2α, CK2α′ and CK2β striatal mRNA levels from WT and KIQ175 mice at 6 months of age. The value given for the amount of mRNA in the control group (WT) was set as 1. Error bars represent mean±s.e.m., ( n =4 animals). Values for the KIQ175 group were compared to the WT group. Statistical significance was measured by two-tailed unpaired t -test * P <0.05. ( c ) Protein levels for CK2α, CK2α′ and CK2β in the striatum and ( d ) gastrocnemius muscle of WT and KIQ175 mice at 6 months of age ( n =4). ( e ) Coronal section of the striatum of WT and KIQ175 at 6 months of age, showing co-localization of CK2α′ (red) with Ctip2 (green) and Fox1p (magenta) labelled MSNs in merged image. Scale bar: 10 μm. ( f ) CK2α, CK2α′ and CK2β qRT-PCR analysis and ( g ) protein levels in the striatum of HD patients and sex-age matched controls from 3 biospecimen banks . The value given for the amount of mRNA in the control group (C) was set as 1 for each gene. Error bars represent mean±s.e.m., ( n =7). One-tailed unpaired t -test * P <0.05, ** P <0.05, NS, no significant. Values for the Huntington's disease (HD) group were compared to the control (C) group. CK2α′ bands from immunoblots were quantified using Quantity One image software (BioRad) and the protein values were normalized using GAPDH as loading control and referenced to the corresponding age-sex-matched control patient. See for uncropped immunoblots.

Article Snippet: HttQ74 cells were transfected with HA-Ub vector (Addgene #18712) by using Lipofectamine LTX Reagent (Invitrogen) following protocol instructions (Invitrogen protocols 25-0946 W). hsf1 −/− MEF-GFP-Htt-Q74 cells were transfected with pcDNA WT HSF1 or the HSF1 S303A mutant plasmid using Lipofectamine LTX reagent. hsf1 −/− MEFs were electroporated with FBXW7-FLAG tag and WT HSF1 or S303A plasmids using the SE Cell Line Nucleofector.

Techniques: Expressing, Software, Two Tailed Test, Quantitative RT-PCR, One-tailed Test, Western Blot

Journal: bioRxiv

Article Title: PRMT7 ablation stimulates anti-tumor immunity and sensitizes melanoma to immune checkpoint blockade

doi: 10.1101/2021.07.28.454202

Figure Lengend Snippet: (A) RT-qPCR analysis of selected retrotransposons, IFNs and ISGs transcripts in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells. Bar graphs represent the mean fold-change ± SD. Data are representative of three independent experiments. Statistical significance was calculated by unpaired student t test (* p <0.1; ** p <0.01; *** p <0.001; **** p <0.0001). (B) The assessment of both sense and antisense transcripts of selected ERVs ( MuERV-L and IAP ) using strand-specific primers for RT-PCR (TASA-TD technique) in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells. β-actin was used as a negative control for antisense transcription. A representative experiment is shown of three independent experiments. (C) dsRNA enrichment of MuERV-L IAP, MuSD and Line-1 retrotransposons in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells by RT-qPCR analysis following RNase A treatment. (D) Total RNA extracted from sgCTL and sgPRMT7-1 B16 cells were treated with Mock, RNase III or RNase A (under high salt condition: 350 mM NaCl), dotted on Hybond N+ membrane and immunoblotted with the J2 antibody and visualized by methylene for loading control. Dots are denoted by numbers: 1, 3, 5 for sgCTL and 2, 4, 6 for sgPRMT7-1 cells nontreated (dots 1 and 2), treated with RNase III (dots 3 and 4) or RNase A (dots 5 and 6). (E) sgCTL and sgPRMT7-1 B16 cells were incubated with 0.5 mM sodium arsenite for 1h or 45°C (heat shock) treatment for 30 min. Cells were then fixed with 4% PFA and immunostained using anti-G3BP1 antibodies. A representative IF image is shown 60x magnification. DAPI, 4’,6-diamidino-2-phenylindole, was shown in blue as indicated. (F) The average number of SGs per cell of the staining done in (E) was quantified using image J software and presented as a bar plot ( n =60 to 70 cells per condition). Bar graphs show mean intensity ± SEM. Statistical significance was calculated by unpaired student t test (**** p <0.0001). (G) RT-qPCR analysis of DNMT mRNAs ( Dnmt1, Dnmt3a and Dnmt3b ) in sgCTL, sgPRMT7 B16 melanoma cells. Bar graphs represent the mean fold-change ± SD. Data are representative of three independent experiments. Statistical significance was calculated by unpaired student t test (**** p <0.0001). (H) Immunoblot of DNMT proteins (DNMT1, DNMT3a and DNMT3b) in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells. β-actin was used as the loading control. A representative experiment is shown out of three independent experiments. The molecular mass markers are indicated in the left in kDa. The DNMT bands are shown with arrowheads.

Article Snippet: 5 µg of total RNA extracted from B16.F10 cells was dissolved in 46 µl H2O and digested with 1U RNase A (Ambion #AM2270) under high salt condition: 3.5 µl NaCl (5 M stock) to a total volume of 50 µl and mixed well, followed by incubation for 30 min at 37°C.

Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Negative Control, Incubation, Staining, Software, Western Blot

Journal: Molecular cell

Article Title: Native Chromatin Proteomics Reveals Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance

doi: 10.1016/j.molcel.2019.10.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For each ChIP, approximately 2 μg of antibody was pre-incubated with 30 μl of Invitrogen Dynabeads Protein A, Protein G or IgG (for TAP tagged strains): anti-H3K9me2 (Abcam 1220) with protein A, anti-H3K14ac (Millipore 07–353) with protein-A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-GFP (Abcam 290) with protein A, and anti-Myc (Covance 9E10, MMS-150P) with protein G. For H3K9me3 ChIP, 1 μg of anti-H3K9me3 antibody (Diagenode, C15500003) was first incubated with 30 μl Dynabeads M-280 Streptavidin beads (Invitrogen) at 4 °C for 1 hr, followed by blocking with 5 μM biotin, according to the manufacturer’s instructions, prior to using for each immunoprecipitation.

Techniques: Purification, Recombinant, Silver Staining, Protease Inhibitor, Hybridization, Western Blot, Staining, Microscopy, Mass Spectrometry, Software, Real-time Polymerase Chain Reaction

Journal: Cell

Article Title: The Parkinson’s disease protein alpha-synuclein is a modulator of Processing-bodies and mRNA stability

doi: 10.1016/j.cell.2022.05.008

Figure Lengend Snippet: Key resources table

Article Snippet: BCA Protein Assay Kit , Pierce , 23225.

Techniques: Western Blot, Virus, Recombinant, Protease Inhibitor, Lysis, Magnetic Beads, Membrane, Transfection, Expressing, Bicinchoninic Acid Protein Assay, Silver Staining, In Situ, Sample Prep, Luciferase, Reporter Assay, Multiplex sample analysis, Biomarker Discovery, Marker, Generated, Software, Mass Spectrometry, Imaging

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Location of missense and nonsense mutations in SLC9A6/NHE6 variants of patients with Christianson syndrome. A, schematic planar drawing of the predicted membrane topology of the longest splice-variant of mammalian NHE6 and location of the mutations (yellow circles). Two consensus N-linked glycosylation sites (128NVT and 145NVS) within extracellular loop 2 have been verified experimentally (data not shown) and are illustrated in the drawing. B, phylogenetic comparison of the primary sequence of segments containing the various mutations in NHE6. The affected residues are shaded in black.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Membrane, Variant Assay, Glycoproteomics, Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Structure homology modeling of transmembrane NHE6 variants associated with Christianson syndrome. A, front (membrane aspect, left panel) and top (extracellular aspect, right panel) views of a 3D structure homology model of dimeric human NHE6 based on the crystal structure of the bacterial T. thermophilus Na+/H+ antiporter NapA (TtNapA) (Protein Data Bank accession code 5bz3; 2.30 Å, 15% identity, 27% similarity), which provided the broadest coverage, highest resolution, and best spatial fit compared with other crystallized bacterial Na+/H+ antiporters. The proposed structure includes only the membrane-spanning helices (M2–M12; amino acids 74–540) that aligned with homologous segments of TtNapA. The top view includes the locations of the transmembrane-localized residues Leu-188 and Gly-383 mutated in CS. B, molecular dynamics simulation of structural changes predicted to occur in TM4 (highlighted in cyan) upon substitution of Leu-188 with Pro (L188P). The monomeric forms of NHE6 WT and L188P are illustrated. C and D, structural perturbations predicted to occur in the intramembranous re-entrant (R) loop between helices M8 and M9 upon substitution of Gly-383 with Asp (G383D). C, upper and lower panels show front and top views, respectively, of Gly-383, which is packed tightly against amino acids Phe-373, Ala-376, and Glu-377 (top left). Mutation of Gly-383 to Asp would result in steric clashes between these residues and interfere with the packing between these segments of the R-loop. D, G383D substitution would also disrupt interactions of R-loop residues Trp-379, Phe-381, and especially Thr-382 with residues Asp-92 and Ile-296 in helix M7 and residues Ile-330, Phe-331, and Ser-334 in helix M8.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Membrane, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the biosynthetic maturation of NHE6 variants. A, AP-1 cells transiently expressing NHE6GFP WT or CS-linked variants were lysed at the indicated time points (6–48 h) post-transfection. Equal amounts of proteins (20 μg) were subjected to SDS-PAGE and immunoblotting with a polyclonal anti-GFP antibody. NHE6 migrates as multiple bands: higher molecular weight bands represent the fully-glycosylated (fg) and core-glycosylated (cg) dimeric (d) forms of the exchanger that do not fully dissociate under SDS-PAGE conditions, whereas lower molecular weight bands represent fully-glycosylated and core-glycosylated forms of the dissociated monomeric (m) protein. The blots were stripped and reprobed with a mouse monoclonal anti-GAPDH antibody to control for protein loading. B, ratios of fully-glycosylated protein (monomer and dimer)/total NHE6 protein (fg/total) were quantified by densitometry of X-ray films exposed in the linear range and analyzed using ImageJ software. Values represent the mean ± S.D. of three different experiments. Significance was determined by two-way ANOVA with a post hoc Tukey test. The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Population means of the NHE6 variants are significantly different (F value = 26.4, p value = 1.5 × 10−16). Population means as a function of time are significantly different (F value = 48.6, p value = 1.9 × 10−19). § indicates significance (p < 0.01) of the means of NHE6 variants within a cluster relative to the 12-h time point. Asterisks indicate significance (★, p < 0.05, and ★★, p < 0.01) of the means between clusters of NHE6 variants at the indicated time points.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Expressing, Transfection, SDS Page, Western Blot, Molecular Weight, Control, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the protein stability of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT of CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide (CHX) for the indicated time points and lysed, and equal amounts of protein (20 μg) were analyzed by Western blotting using a mouse monoclonal anti-HA antibody. Blots were reprobed with a mouse monoclonal anti-GAPDH antibody to control for loading. Blots are representative images from four separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Effect of proteasomal and lysosomal inhibitors on cellular clearance of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT or CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide (CHX) for the indicated time points in the presence of diluent (DMSO) and the proteasomal inhibitor MG-132 (40 μm) or the lysosomal inhibitor leupeptin/pepstatin (LeuP, 100 μg/ml). Total-cell lysates were analyzed by Western blotting with a mouse monoclonal HA antibody. Membranes were also probed for β-tubulin expression as a loading control. The immunoblots are representative of three separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Effect of the lysosomotropic agent chloroquine on cellular clearance of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT or CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide for the indicated time points in the presence of diluent (H2O) or chloroquine (CQ, 500 μm). Total-cell lysates were analyzed by Western blotting with a mouse monoclonal HA antibody. Membranes were also probed for β-tubulin expression as a loading control. The immunoblots are representative of four separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular detection of NHE6 variants in recycling endosomes in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were incubated with the recycling endosomal marker Alexa Fluor 488–conjugated transferrin (Tf-AF488, 10 μg/ml) for 45 min, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and Tf-AF488 channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with Tf-AF488 as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 6479.8, p value = 5.6 × 10−9), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Incubation, Marker, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular localization of certain CS variants in the endoplasmic reticulum in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were immunostained for endogenous CANX, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and CANX channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with CANX as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 3012.9, p value = 1.7 × 10−10), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the functional properties of NHE6 variants. A, biochemical determination of plasma membrane trafficking of NHE6GFP WT or CS-linked variants using a cell-surface biotinylation assay. Cell-surface proteins were labeled with N-hydroxysulfosuccinimidyl–SS–biotin in AP-1 cells expressing the NHE6GFP constructs after 48 h. Total-cell lysates (left panel; protein loading ranged from 10 to 50 μg of protein per sample as indicated below the blot) and biotinylated fractions (right panel; representing 20–100% of plasma membrane proteins extracted per sample) were examined by Western blotting with polyclonal anti-GFP and monoclonal anti-GAPDH antibodies. Representative blots from three experiments are shown. B and C, surface expression and endocytosis of external triple flag tag–labeled NHE6 (3FNHE6HA) constructs in transiently transfected (48 h) AP-1 cells using a cell-based ELISA. Mean intensity fluorescence (M.I.F.) units were determined as a function of the cellular protein concentration and then normalized as percentage (M.I.F. units for WT (100%): 25,100 ± 6,348, n = 4). The surface expression of each construct at time 0 min (before the start of internalization) is charted in B (n = 3–4 experiments). Significance from WT-expressing cells was determined using a one-way repeated measures ANOVA (F value = 463.3, p value = 0.0022), with a post hoc Dunnett's test; *, p < 0.05. Percentage internalization of NHE6 constructs normalized to the zero time point are presented in C and represent the mean ± S.D. (n = 3–4 experiments). The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q, and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Significance from WT cells at the 5- and 15-min time points was determined using a one-way ANOVA (F value = 9.43, p value = 4.48 × 10−5), with a post hoc Tukey test; ★, p < 0.05. D, transferrin uptake in HeLa cells transiently transfected (48 h) with GFP or NHE6GFP constructs. The initial uptake (5 min) of Alexa 633–conjugated transferrin (Tf-AF633) was measured in 1 × 104 GFP-positive HeLa cells per experiment by flow cytometry (M.I.F. units for GFP control: 10,204 ± 1554, n = 4). Data were normalized as a percentage and displayed as percent change from GFP control cells. Significance from control cells was determined using a one-way repeated measures ANOVA (F value = 320.7, p value = 3.8 × 10−4), with a post hoc Dunnett's test; ★★, p < 0.001. E, recycling endosomal pH (pHe) was measured in AP-1 cells in the absence or presence of transiently transfected (48 h) NHE6ChFP constructs by fluorescence ratio image analysis of the internalized pH-sensitive probe FITC-conjugated human transferrin (Tf–FITC). Data represent the average endosomal pHe per cell pooled from three separate experiments (8–12 cells per construct/experiment; n = 24–36). Significance was determined by one-way ANOVA (F value = 40.02, p value = 0), with a post-hoc Tukey test; ★★, p < 0.001. Data in B, D, and E are plotted as box charts, with the central white square indicating the mean; the box representing the S.E.; and the error bars showing the S.D. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Functional Assay, Clinical Proteomics, Membrane, Cell Surface Biotinylation Assay, Labeling, Expressing, Construct, Western Blot, FLAG-tag, Transfection, In-Cell ELISA, Fluorescence, Protein Concentration, Flow Cytometry, Control

Journal: Molecular Medicine Reports

Article Title: Dexmedetomidine mitigates oxidative stress in H9C2 cardiac myoblasts under a high-glucose environment via the PI3K/AKT signaling pathway

doi: 10.3892/mmr.2025.13616

Figure Lengend Snippet: Flow cytometry analysis of apoptosis. (A) HG group. (B) HG + H 2 O 2 group. (C) DP + HG + H 2 O 2 group. (D) LY294002 + HG + DP + H 2 O 2 group. (E) Quantification of H9C2 cell apoptosis. **P<0.01. Data are presented as the mean ± SEM, n=5 per group. HG, high-glucose; DP, dexmedetomidine preconditioning; H 2 O 2 , hydrogen peroxide.

Article Snippet: Following the treatments, apoptosis rates were assessed using the Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol, and analyzed by flow cytometry using a BD FACSCanto TM II (Becton, Dickinson and Company) and data were analyzed with FlowJo software (version 10.6.2; BD Biosciences).

Techniques: Flow Cytometry

Journal: Molecular Medicine Reports

Article Title: Dexmedetomidine mitigates oxidative stress in H9C2 cardiac myoblasts under a high-glucose environment via the PI3K/AKT signaling pathway

doi: 10.3892/mmr.2025.13616

Figure Lengend Snippet: Detection of the levels of apoptosis-related proteins by western blotting. (A) Protein bands of apoptosis-related proteins caspase-3, PI3K, AKT, BCL-2, BAX, p-PI3K and p-AKT in H9C2 cells from the four groups assessed by western blotting. GAPDH served as an internal control for sample loading. The differing band curvatures observed (‘frown’ effect for p-PI3K and ‘smile’ effect for t-PI3K) may result from minor membrane handling variations during blotting or localized gel inconsistencies during protein migration. These factors cause uneven stretching, folding or pressure, leading to the observed discrepancies. Semi-quantification of western blotting data of apoptosis-related proteins, including (B) caspase-3, (C) BCL-2, (D) BAX, (E) p/t-PI3K and (F) p/t-AKT, in H9C2 cells from the four groups. n=3 per group. *P<0.05, **P<0.01. HG, high-glucose; Dex, dexmedetomidine; DP, dexmedetomidine preconditioning; p, phosphorylated; t, total; H 2 O 2 , hydrogen peroxide.

Article Snippet: Following the treatments, apoptosis rates were assessed using the Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol, and analyzed by flow cytometry using a BD FACSCanto TM II (Becton, Dickinson and Company) and data were analyzed with FlowJo software (version 10.6.2; BD Biosciences).

Techniques: Western Blot, Control, Membrane, Migration

Journal: Oncology Letters

Article Title: Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines

doi: 10.3892/ol.2016.4520

Figure Lengend Snippet: CC-induced dose-dependent apoptosis of HEK-293 and human renal cancer SW-839 cells. (A) HEK-293 and (B) SW-839 cells were treated with various concentrations of CC (0, 5 and 10 µM) for 24 h, and stained with annexin V-fluorescein isothiocyanate/propidium iodide. The percentage of early-stage apoptotic cells is shown in the lower right quadrant, while the percentage of late-stage apoptotic cells is shown in the upper right quadrant. (C) Treatment of HEK-293 and SW-839 cells with 5 and 10 µM CC for 24 h induced cell apoptosis. CC, chelerythrine chloride; FL-H, fluorescence line height.

Article Snippet: One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Haimen, China) was used to stain the apoptotic tumor cells.

Techniques: Staining, Fluorescence

Journal: Oncology Letters

Article Title: Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines

doi: 10.3892/ol.2016.4520

Figure Lengend Snippet: Tumor growth suppression in a human renal cancer SW-839 xenograft nude mouse model. (A and B) Tumor volume of CC and control tumors. (C) Tumor weight of CC and control tumors. (D) No significant toxicity to mice was observed following treatment with CC (5 mg/kg/day), according to the body weight of the mice. (E) Representative TUNEL staining (red fluorescence) of SW-839 renal cancer xenografts. (F) Quantification of TUNEL + SW-839 cells in tumor xenografts was performed using cellSens Standard software. The data are presented as the mean ± standard deviation of three experiments. **P<0.01 vs. control cells. (G) IHC staining for Bcl-2 and Bcl-2-associated X protein on tumor sections, and (H) quantification of IHC staining using cellSens Standard software. CC, chelerythrine chloride; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; IHC, immunohistochemistry.

Article Snippet: One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Haimen, China) was used to stain the apoptotic tumor cells.

Techniques: Control, TUNEL Assay, Staining, Fluorescence, Software, Standard Deviation, Immunohistochemistry, End Labeling

Journal: Oncology Letters

Article Title: Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines

doi: 10.3892/ol.2016.4520

Figure Lengend Snippet: (Aa) Western blot analysis of the expression levels of apoptosis-associated proteins in HEK-293 and human renal cancer SW-839 cells following treatment with CC. Quantification of the expression of various proteins in HEK-293 and SW-839 cells, such as (Ab) p53, (Ac) Bax, (Ad) Bcl-2, (Ae) pro-caspase-3, (Af) cleaved caspase 3 and (Ag) PARP using GAPDH as a control. Multiple bands were observed in the cleaved caspase-3 lane due to non-specific binding of antibodies. (Ab-Ag) Quantification of western blotting (*P<0.05 and **P<0.01 vs. controls). (Ba) Western blot analysis of MAPK and Akt pathways after CC treatment in HEK-293 and SW-839 cells. The quantification of protein expression was performed for (Bb) pERK, (Bc) p-p38, (Bd) p-JNK and (Be) p-AKT, with GADPH as a control. The results are representative of ≥3 independent experiments. Multiple bands were observed in the p-ERK, p-JNK and JNK lanes due to non-specific binding of antibodies. *P<0.05 and **P<0.01 vs. controls. CC, chelerythrine chloride; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; PARP, poly (adenosine diphosphate-ribose) polymerase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p-, phospho-; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Article Snippet: One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Haimen, China) was used to stain the apoptotic tumor cells.

Techniques: Western Blot, Expressing, Control, Binding Assay