Journal: Biology Open

Article Title: BAG6 contributes to glucose uptake by supporting the cell surface translocation of the glucose transporter GLUT4

doi: 10.1242/bio.047324

Figure Lengend Snippet: Transgenic cell lines with Flag-tag inserted in the extracellular domain of GLUT4. (A) Schematic of the Flag-inserted GLUT4-mCherry (left panel) and T7-tagged IR (right panel) proteins used in this study. Both of these gene products were stably co-expressed in CHO-K1 cells. A triple repeat of the Flag epitope tag (3×Flag) was inserted into the first extracellular loop of GLUT4 between Pro 66 and Gly 67 . With this inserted epitope, cell surface GLUT4 can be detected on non-permeabilized cells using an anti-Flag M2 antibody. The C-terminus of GLUT4 was fused with mCherry to quantify the total level of GLUT4 expression. The C-terminus of the IR β-chain was fused with a 3×T7 epitope tag (IR-T7). (B) Stable expression of Flag-inserted GLUT4-mCherry protein and IR-T7 protein in a transgenic CHO-K1 cell line (clone 8-20) was verified by western blot analysis. α-Tubulin was used as a loading control. The non-transfected parental CHO-K1 cell line was used as a negative control (wild-type). (C) Insulin stimulates the cell surface expression of Flag GLUT4-mCherry. Plasma membrane exposed (shown as green) or total (shown as red) GLUT4 protein levels were examined with (Insulin) or without (Vehicle) insulin treatment (1 μg/ml). In order to detect cell surface GLUT4 exclusively, the plasma membrane was intentionally left non-permeabilized. Transgenic cell line clone 8-20 was used in this experiment. Scale bar: 20 µm. A Keyence BZ-X700 fluorescence microscope was used for observations. (D,E) Cell surface Flag-positive cells were counted with (+) or without (−) insulin. n =962 cells for control, and n =647 cells for insulin treatment. Statistical significance in E was determined by a chi-square test. ** P <0.01 compared with control. (F) Anti-mCherry immunoblot analysis showed that insulin treatment did not influence the total expression level of Flag GLUT4-mCherry fusion protein. α-Tubulin was used as a loading control.

Article Snippet: Subsequently, the T7-positive cells were transfected with the pCI-puro- Flag GLUT4-mCherry expression vector, and were selected with 7.5 μg/ml puromycin (Wako).

Techniques: Transgenic Assay, FLAG-tag, Stable Transfection, Expressing, Western Blot, Transfection, Negative Control, Fluorescence, Microscopy

Journal: Biology Open

Article Title: BAG6 contributes to glucose uptake by supporting the cell surface translocation of the glucose transporter GLUT4

doi: 10.1242/bio.047324

Figure Lengend Snippet: BAG6 deficiency induces defects in the cell surface expression of GLUT4. (A) Efficacy of BAG6 knockdown and the expression levels of Flag GLUT4-mCherry protein in CHO-K1 cells. Note that BAG6 depletion did not influence the total expression level of GLUT4. (B) At 72 h after transfection with siRNA duplexes (5 nM each) for Bag6 siRNA#2 (lower panels) or control (upper panels) into a transgenic CHO-K1 cell line (clone 8-9), plasma membrane-exposed (Flag-signals on non-permeabilized cells are shown as green in the left panels) or total (mCherry signals are shown as red in the right panels) GLUT4 protein levels were observed with insulin treatment. Keyence BZ-X700 fluorescence microscope was used for observations. Scale bar: 20 µm. See also Fig. S1 . (C) Cell surface Flag-positive cells were counted under the respective conditions and the positive rates are plotted as a bar graph. Transgenic cell line clone 8-20 was used in this experiment. The quantified data represent positive rates. n =1159 cells for control siRNA, n =1445 cells for Bag6 siRNA#2. Statistical significance was determined by a chi-square test. ** P <0.01 compared with control. (D) Cellular distribution of IR-T7 (green) in control or BAG6 knockdown cells. T7-immunosignals were detected under a cell membrane-permeabilized condition. Nuclear DNA was stained with Hoechst 33342 (shown as blue). Scale bar: 20 µm. (E) The incorporation of 2-NBDG into a transgenic CHO-K1 cell line (clone 8-20) was quantified as in Fig. 1 . Insulin (1 μg/ml) was included in the medium. BAY-876 (150 nM) was included as a glucose transporter inhibitor as indicated. The value from control cells was defined as 1.0. The graph represents the mean±s.d. calculated from three independent biological replicates. Statistical significance was determined by Student's t -test. * P <0.05; n.s., not significant.

Article Snippet: Subsequently, the T7-positive cells were transfected with the pCI-puro- Flag GLUT4-mCherry expression vector, and were selected with 7.5 μg/ml puromycin (Wako).

Techniques: Expressing, Transfection, Transgenic Assay, Fluorescence, Microscopy, Staining

Journal: Biology Open

Article Title: BAG6 contributes to glucose uptake by supporting the cell surface translocation of the glucose transporter GLUT4

doi: 10.1242/bio.047324

Figure Lengend Snippet: Live-cell flow cytometry analysis indicates that BAG6 is necessary for the cell surface expression of GLUT4. (A) BAG6 depletion downregulates the cell surface expression of GLUT4. Live-cell flow cytometry analysis of a non-permeabilized CHO-K1 cell line (clone 8-20) with an anti-Flag M2 antibody. The flow cytometry patterns of negative control and Bag6 siRNA#2 are indicated as black and red lines, respectively. BAG6 knockdown was performed with three independent siRNA duplexes as described in the Materials and Methods, which all gave similar results (see Fig. S2A,B ). Representative results for Bag6 siRNA#2 are shown. Insulin (1 μg/ml) was included in the culture medium. The data were obtained by logarithmic scale analysis. (B) Quantitative evaluations of the flow cytometric fluorescence intensity of cell surface GLUT4 (determined by anti-Flag immunosignals) of transgenic CHO-K1 cells (clone 8-20). The data shown are the calculated cell surface GLUT4 ratio normalized by the intensity of control siRNA cells without insulin. See also Fig. S2C ,D . (C) Quantitative evaluations of the fluorescence intensity of cell surface GLUT4 of a different transgenic CHO-K1 cell line (clone 8-9) that was isolated independently to clone 8-20. (D) Akt phosphorylation at Ser 473 was examined using an anti-phospho Akt antibody. The results suggest that insulin stimulates Akt phosphorylation in a PI3K-dependent manner, and BAG6 depletion did not affect Akt phosphorylation. (E,F) Live cell flow cytometry analysis as in A. To block PI3K/Akt-dependent GLUT4 translocation, 100 nM wortmannin was added to the culture medium at 10 min before insulin stimulation (clone 8-20). The flow cytometry patterns are indicated as negative control (E,F, black lines), wortmannin treatment (E, red line), and Rab8a siRNA (F, red line). All the data were confirmed by at least three independent biological replicates. Statistical significance was determined by Student's t -test. * P <0.05, ** P <0.01.

Article Snippet: Subsequently, the T7-positive cells were transfected with the pCI-puro- Flag GLUT4-mCherry expression vector, and were selected with 7.5 μg/ml puromycin (Wako).

Techniques: Flow Cytometry, Expressing, Negative Control, Fluorescence, Transgenic Assay, Isolation, Blocking Assay, Translocation Assay

Journal: Biology Open

Article Title: BAG6 contributes to glucose uptake by supporting the cell surface translocation of the glucose transporter GLUT4

doi: 10.1242/bio.047324

Figure Lengend Snippet: Defective intracellular distribution of insulin-stimulated GLUT4 in BAG6-suppressed cells. (A–C) Intracellular localization of Flag GLUT4-mCherry (red) in the presence (Insulin) or absence (Vehicle) of insulin stimulation with or without Rab8a siRNA (A), Bag6 siRNA#2 (B) and Bag6 siRNA#3 (C). Fluorescent mCherry-derived signals were detected using a laser scanning confocal microscopy system (LSM710). Note that this experiment used a different transgenic cell line (clone 51-25) because we noticed that the endogenous expression of IR in CHO-K1 cells is sufficient for insulin responsiveness and that IR transfection is dispensable for insulin-dependent Akt phosphorylation. We observed similar results with clone 8-20. Nuclei were stained with Hoechst 33342 (shown as blue). Peri-nuclear-localized GLUT4 signals are indicated by white arrowheads. Scale bars: 10 µm. (D) Quantification of the number of cells with the peri-nuclear localization of GLUT4 with or without Bag6 siRNA#2. Statistical significance was determined by chi-square test. * P <0.05, ** P <0.01.

Article Snippet: Subsequently, the T7-positive cells were transfected with the pCI-puro- Flag GLUT4-mCherry expression vector, and were selected with 7.5 μg/ml puromycin (Wako).

Techniques: Derivative Assay, Confocal Microscopy, Transgenic Assay, Expressing, Transfection, Staining

Journal: Biology Open

Article Title: BAG6 contributes to glucose uptake by supporting the cell surface translocation of the glucose transporter GLUT4

doi: 10.1242/bio.047324

Figure Lengend Snippet: Cell surface expression of GLUT4 is downregulated by BAG6, Rab8a and Stx6 depletion. (A) Flow cytometry analysis of GLUT4 with BAG6, Rab8a and Stx6 knockdown. The intensity of cell surface GLUT4 signals is indicated as relative values to the signal of the basal (no insulin) condition. (B) Insulin-stimulated Akt phosphorylation at Ser 473 was not perturbed by BAG6, Rab8a and Stx6 knockdown. (C) Flow cytometry quantification of cell surface GLUT4 expression with BAG6 knockdown in the presence of insulin (clone 8-20). To block PI3K/Akt-dependent GLUT4 translocation, 100 nM wortmannin (Wort.) was added to the culture medium at 10 min before insulin stimulation. All data were confirmed by at least three independent biological replicates. Statistical significance was determined by Student's t -test. * P <0.05, ** P <0.01.

Article Snippet: Subsequently, the T7-positive cells were transfected with the pCI-puro- Flag GLUT4-mCherry expression vector, and were selected with 7.5 μg/ml puromycin (Wako).

Techniques: Expressing, Flow Cytometry, Blocking Assay, Translocation Assay

Journal: Biology Open

Article Title: BAG6 contributes to glucose uptake by supporting the cell surface translocation of the glucose transporter GLUT4

doi: 10.1242/bio.047324

Figure Lengend Snippet: BAG6 has a partial role in Stx6 biogenesis. (A) CHO-K1 cells transfected with 5 nM Bag6 siRNA#2 duplex or control siRNA were fractionated into cytosolic and membrane-associated/insoluble fractions and were probed with an anti-Stx6 antibody to detect the cytoplasmic defective form of endogenous Stx6. The BAG6 blot indicates the depletion of BAG6 protein by its siRNA. α-Tubulin was used as a cytoplasmic marker and calnexin was used as a membrane fraction marker. (B) Schematic of the C-terminal OPG-tagged and N-terminal T7- (or Flag-) tagged Stx6 used in this study. The amino acid sequences of the OPG tag are indicated with the N-glycosylation site at Asn 300 . TMD indicates the transmembrane domain. (C) C-terminally OPG-tagged Stx6 was glycosylated. Flag-Stx6-OPG protein was expressed in CHO-K1 cells and immunoprecipitated with an anti-Flag antibody. The precipitates were incubated with or without five units of the de-glycosylation enzyme PNGase F and subjected to western blot analysis with an anti-Flag antibody. Glycosylated (indicated as G) and non-glycosylated (indicated as N) signals are indicated. (D) Glycosylation of OPG-tagged Stx6 was reduced modestly by BAG6 knockdown. T7-tagged Stx6-OPG was expressed in CHO-K1 cells with or without Bag6 siRNA (duplexes #2 and #3), and probed with anti-Stx6 and an anti-T7 antibodies. Low-mobility glycosylated (G) and high-mobility non-glycosylated (N) signals of Stx6-OPG are indicated. α-Tubulin was used as a loading control. Endo. indicates a specific signal for endogenous Stx6 protein. (E) Abnormal distribution of endogenous Stx6 (Endo. Stx6, shown as green) in BAG6-depleted CHO-K1 cells (with Bag6 siRNA#2). Fluorescent signals were detected using a laser scanning confocal microscopy system (LSM710). Nuclei were stained with Hoechst 33342 (shown as blue). Scale bar: 10 µm. (F) Intracellular localization of Flag GLUT4-mCherry (red) in the presence (Insulin, b,d) or absence (Vehicle, a,c) of insulin stimulation with (c,d) or without (a,b) Stx6 siRNA. Fluorescent mCherry-derived signals were detected as in Fig. 5 . Scale bar: 10 µm.

Article Snippet: Subsequently, the T7-positive cells were transfected with the pCI-puro- Flag GLUT4-mCherry expression vector, and were selected with 7.5 μg/ml puromycin (Wako).

Techniques: Transfection, Marker, Immunoprecipitation, Incubation, Western Blot, Confocal Microscopy, Staining, Derivative Assay

Journal: Biology Open

Article Title: BAG6 contributes to glucose uptake by supporting the cell surface translocation of the glucose transporter GLUT4

doi: 10.1242/bio.047324

Figure Lengend Snippet: Schematic of the possible roles of BAG6 in insulin-stimulated GLUT4 translocation. Insulin (and downstream, PI3K-/Akt-/AS160-) -dependent translocation of GLUT4 from the peri-nuclear compartment to the cell surface depends on Rab8a small GTPase. BAG6 is critical for the degradation of the GDP-bound inactive form of Rab8a , whose accumulation impairs the function of GTP-bound Rab8a. BAG6 also plays a partly redundant role in the assembly of newly synthesized Stx6 into the ER membrane. Collectively, dysfunction of BAG6 results in accumulation of GDP-bound Rab8a, as well as cytosolically mislocalized Stx6 whose accumulation impairs the function of membrane-anchored Stx6. Therefore, defects in BAG6 lead to defective cell surface expression of GLUT4 in response to insulin, which in turn leads to reduced glucose incorporation into the cells.

Article Snippet: Subsequently, the T7-positive cells were transfected with the pCI-puro- Flag GLUT4-mCherry expression vector, and were selected with 7.5 μg/ml puromycin (Wako).

Techniques: Translocation Assay, Synthesized, Expressing

Journal: Neuroscience

Article Title: Morphological Changes in Different Populations of Bladder Afferent Neurons Detected by Herpes Simplex Virus (HSV) Vectors with Cell Type-Specific Promoters in Mice with Spinal Cord Injury

doi: 10.1016/j.neuroscience.2017.09.024

Figure Lengend Snippet: HSV expression vector schematic. A: The vector backbone vLG was generated from the HSV KOS strain in which BAC sequences were inserted into the TK locus (UL23). Deletions were introduced in the internal repeat region and the genes encoding immediate early proteins ICP4 and ICP27, rendering the vector replication-defective. The Gateway destination cassette was inserted into the remaining latency locus, replacing the latency promoter elements while maintaining a wild-type copy of ICP0 and the surrounding CTCF chromatin boundary elements. B: To generate PNS neuronal subtype targeted expression vectors, transgenes were recombined into the vector backbone via the Gateway cassette. Individual promoter sequences (1284 bp TRPV1 promoter [TRPV1p], 932 bp CGRP promoter [CGRPp], 553 bp CMV promoter [CMVp], and 970 bp NF200 promoter [NF200p]) incorporating a Kozak consensus translation initiation sequence were inserted into the pENTR1A transfer vector which contains attL sites for site-directed recombination with the attR-containing vector backbone. These Gateway plasmids were then recombined into the vLG vector backbone to generate the experimental vectors. The structure of the recombinant vector genomes were verified by FIGE analysis and the promoters and mCherry reporter gene were all sequenced verify the authenticity of the reagents.

Article Snippet: The 4 different (CMV, CGRP, TRPV1, NF200) promoter-mCherry reporter gene expression vectors were engineered by GateWay recombineering of the pENTR1A plasmids ( ) into the LAT locus ( Wolfe et al., 2010 ) with the 932 bp CGRP and 970 bp NF200 promoter sequences (SwitchGear Genomics, Carlsbad CA), the 553 bp CMV ( Thomsen et al., 1984 ; Stinski and Roehr, 1985 ), and 1284 bp TRPV1 ( Xue et al., 2007 ; personal communication with Mark Schumacher, UCSF) promoter sequences.

Techniques: Expressing, Plasmid Preparation, Generated, Sequencing, Recombinant

Journal: Neuroscience

Article Title: Morphological Changes in Different Populations of Bladder Afferent Neurons Detected by Herpes Simplex Virus (HSV) Vectors with Cell Type-Specific Promoters in Mice with Spinal Cord Injury

doi: 10.1016/j.neuroscience.2017.09.024

Figure Lengend Snippet: mCherry immunostaining. Positively stained neurons, in an L6 DRG section are indicated by arrows, 2 weeks after bladder wall injection of HSV virus encoding mCherry from_A: CMV promoter, B: CGRP promoter, C: TRPV1 promoter and D: NF200 promoter vectors in spinal intact (SI) mice. Scale bar = 50 µm.

Article Snippet: The 4 different (CMV, CGRP, TRPV1, NF200) promoter-mCherry reporter gene expression vectors were engineered by GateWay recombineering of the pENTR1A plasmids ( ) into the LAT locus ( Wolfe et al., 2010 ) with the 932 bp CGRP and 970 bp NF200 promoter sequences (SwitchGear Genomics, Carlsbad CA), the 553 bp CMV ( Thomsen et al., 1984 ; Stinski and Roehr, 1985 ), and 1284 bp TRPV1 ( Xue et al., 2007 ; personal communication with Mark Schumacher, UCSF) promoter sequences.

Techniques: Immunostaining, Staining, Injection, Virus

Journal: Neuroscience

Article Title: Morphological Changes in Different Populations of Bladder Afferent Neurons Detected by Herpes Simplex Virus (HSV) Vectors with Cell Type-Specific Promoters in Mice with Spinal Cord Injury

doi: 10.1016/j.neuroscience.2017.09.024

Figure Lengend Snippet: The average number of mCherry-positive neurons per DRG section at 2 weeks after bladder wall injection of HSV virus encoding mCherry from different subtype promoters in SI and SCI mice. A: The number of CMV promoter (CMVp)-mCherry HSV vector-labeled bladder afferent neurons (L1: U=3714, SI=83, SCI=107, p=0.053, L6: U=2887, SI=79, SCI=84, p=0.152). B: The numbers of CGRP promoter (CGRPp)-mCherry HSV vector-labeled bladder afferent neurons (L1: U=3311.5, SI=101, SCI=94, p=<0.001, L6: U=5222, SI=83, SCI=94, p=<0.001). C: The number of TRPV1 promoter (TRPV1p)-mCherry HSV vector-labeled bladder afferent neurons (L1: U=3978, SI=58, SCI=110, p=0.022, L6: U=1255, SI=88, SCI=70, p=<0.001). D: The number of NF200 promoter (NF200p)-mCherry HSV vector-labeled bladder afferent neurons (L1: U=3159.5, SI=83, SCI=94, p=0.767, L6: U=2819, SI=89, SCI=96, p=<0.001). Significance of p<0.001 determined by Mann-Whitney test indicated by a double asterisk. Data shown represent mean ± SEM. The number of DRG sections is described below each bar of the graph.

Article Snippet: The 4 different (CMV, CGRP, TRPV1, NF200) promoter-mCherry reporter gene expression vectors were engineered by GateWay recombineering of the pENTR1A plasmids ( ) into the LAT locus ( Wolfe et al., 2010 ) with the 932 bp CGRP and 970 bp NF200 promoter sequences (SwitchGear Genomics, Carlsbad CA), the 553 bp CMV ( Thomsen et al., 1984 ; Stinski and Roehr, 1985 ), and 1284 bp TRPV1 ( Xue et al., 2007 ; personal communication with Mark Schumacher, UCSF) promoter sequences.

Techniques: Injection, Virus, Plasmid Preparation, Labeling, MANN-WHITNEY

Journal: Neuroscience

Article Title: Morphological Changes in Different Populations of Bladder Afferent Neurons Detected by Herpes Simplex Virus (HSV) Vectors with Cell Type-Specific Promoters in Mice with Spinal Cord Injury

doi: 10.1016/j.neuroscience.2017.09.024

Figure Lengend Snippet: Cell size distribution of CGRP promoter (CGRPp) and NF200 promoter (NF200p) -mCherry HSV vector-labeled neurons. A: L6 DRG. B: L1 DRG. Filled and open columns represent cell-size distribution of CGRP promoter-mCherry and NF200 promoter-mCherry HSV vector-labeled bladder afferent neurons, respectively. Bin width = 50 µm2

Article Snippet: The 4 different (CMV, CGRP, TRPV1, NF200) promoter-mCherry reporter gene expression vectors were engineered by GateWay recombineering of the pENTR1A plasmids ( ) into the LAT locus ( Wolfe et al., 2010 ) with the 932 bp CGRP and 970 bp NF200 promoter sequences (SwitchGear Genomics, Carlsbad CA), the 553 bp CMV ( Thomsen et al., 1984 ; Stinski and Roehr, 1985 ), and 1284 bp TRPV1 ( Xue et al., 2007 ; personal communication with Mark Schumacher, UCSF) promoter sequences.

Techniques: Plasmid Preparation, Labeling

Journal: Neuroscience

Article Title: Morphological Changes in Different Populations of Bladder Afferent Neurons Detected by Herpes Simplex Virus (HSV) Vectors with Cell Type-Specific Promoters in Mice with Spinal Cord Injury

doi: 10.1016/j.neuroscience.2017.09.024

Figure Lengend Snippet: Cell size distribution of neuronal subtype vector-labeled bladder afferent neurons from L6 and L1 DRG. A: CMV promoter (CMVp)-mCherry HSV vector-labeled neurons (L6: U=700768, SI=1286, SCI=1154, p=0.018, L1: U=244049, SI=549, SCI=824, p=0.013). B: CGRP promoter (CGRPp)-mCherry HSV vector-labeled neurons (L6: U=260904, SI=558, SCI=872, p=0.021, L1: U=223004, SI=506, SCI=824, p=0.033). C: TRPV1 promoter (TRPV1p)-mCherry HSV vector-labeled neurons (L6: U=145547, SI=478, SCI=718, p=<0.001, L1: U=130034, SI=266, SCI=1104, p=0.004). D: NF200 promoter (NF200p)-mCherry HSV vector-labeled neurons (L6: U=117770, SI=556, SCI=441, p=0.285, L1: U=57829, SI=404, SCI=345, p=<0.001). Shaded and filled columns represent cell-size distribution of bladder afferent neurons from SI and SCI mice, respectively. Bin width = 50 µm2. Insets in A–D show the comparison of median cell size of bladder afferent neurons labeled by different promoter-mCherry HSV vectors. Data shown represent median ± IQR. Significance of p<0.05 indicated by a single asterisk, and significance of p< 0.01 indicated by a double asterisk determined using the Mann-Whitney test.

Article Snippet: The 4 different (CMV, CGRP, TRPV1, NF200) promoter-mCherry reporter gene expression vectors were engineered by GateWay recombineering of the pENTR1A plasmids ( ) into the LAT locus ( Wolfe et al., 2010 ) with the 932 bp CGRP and 970 bp NF200 promoter sequences (SwitchGear Genomics, Carlsbad CA), the 553 bp CMV ( Thomsen et al., 1984 ; Stinski and Roehr, 1985 ), and 1284 bp TRPV1 ( Xue et al., 2007 ; personal communication with Mark Schumacher, UCSF) promoter sequences.

Techniques: Plasmid Preparation, Labeling, Comparison, MANN-WHITNEY