gene exp kcnj6 hs00158423 m1  (Thermo Fisher)


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    Thermo Fisher gene exp kcnj6 hs00158423 m1
    General characterization of neurons derived from PARK2 KO and healthy isogenic induced pluripotent stem cells (iPSCs). ( A ) Graphical overview of the differentiation of iPSCs to neural stem cells (NSCs) and fully committed dopaminergic neurons. ( B ) Immunofluorescence analysis of MAP2 (mature neurons, red) and TH (dopaminergic neurons, green) expression in PARK2 KO iPSC and control iPSC lines at day 25 of differentiation. Cell nuclei are visualized using DAPI (blue). Representative pictures are shown. Scale bar: 50 µm. ( C , D ) Quantitative assessment of ( C ) MAP2 + mature neurons and (D) TH + dopaminergic neurons in PARK2 KO and control iPSC lines. Data are presented as mean ± SEM, n = 15, 5 independent differentiations. ( E , F ) Western blotting and densitometry analysis of ( E ) MAP2 and ( F ) TH protein expression levels in PARK2 KO and control iPSC lines. Protein expression levels were normalized to α-actin. Full-length blots are presented in Supplementary Fig. . Data are presented as mean ± SEM, n = 3, 3 independent experiments. ( G ) qRT-PCR analysis for midbrain/dopaminergic markers (EN1, NURR1, and <t>GIRK2).</t> GAPDH, 18S, HPRT were used as endogenous references. Data were normalized to control levels and presented as mean ± SEM, n = 4, 2 independent experiments.
    Gene Exp Kcnj6 Hs00158423 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Lysosomal perturbations in human dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation"

    Article Title: Lysosomal perturbations in human dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-67091-6

    General characterization of neurons derived from PARK2 KO and healthy isogenic induced pluripotent stem cells (iPSCs). ( A ) Graphical overview of the differentiation of iPSCs to neural stem cells (NSCs) and fully committed dopaminergic neurons. ( B ) Immunofluorescence analysis of MAP2 (mature neurons, red) and TH (dopaminergic neurons, green) expression in PARK2 KO iPSC and control iPSC lines at day 25 of differentiation. Cell nuclei are visualized using DAPI (blue). Representative pictures are shown. Scale bar: 50 µm. ( C , D ) Quantitative assessment of ( C ) MAP2 + mature neurons and (D) TH + dopaminergic neurons in PARK2 KO and control iPSC lines. Data are presented as mean ± SEM, n = 15, 5 independent differentiations. ( E , F ) Western blotting and densitometry analysis of ( E ) MAP2 and ( F ) TH protein expression levels in PARK2 KO and control iPSC lines. Protein expression levels were normalized to α-actin. Full-length blots are presented in Supplementary Fig. . Data are presented as mean ± SEM, n = 3, 3 independent experiments. ( G ) qRT-PCR analysis for midbrain/dopaminergic markers (EN1, NURR1, and GIRK2). GAPDH, 18S, HPRT were used as endogenous references. Data were normalized to control levels and presented as mean ± SEM, n = 4, 2 independent experiments.
    Figure Legend Snippet: General characterization of neurons derived from PARK2 KO and healthy isogenic induced pluripotent stem cells (iPSCs). ( A ) Graphical overview of the differentiation of iPSCs to neural stem cells (NSCs) and fully committed dopaminergic neurons. ( B ) Immunofluorescence analysis of MAP2 (mature neurons, red) and TH (dopaminergic neurons, green) expression in PARK2 KO iPSC and control iPSC lines at day 25 of differentiation. Cell nuclei are visualized using DAPI (blue). Representative pictures are shown. Scale bar: 50 µm. ( C , D ) Quantitative assessment of ( C ) MAP2 + mature neurons and (D) TH + dopaminergic neurons in PARK2 KO and control iPSC lines. Data are presented as mean ± SEM, n = 15, 5 independent differentiations. ( E , F ) Western blotting and densitometry analysis of ( E ) MAP2 and ( F ) TH protein expression levels in PARK2 KO and control iPSC lines. Protein expression levels were normalized to α-actin. Full-length blots are presented in Supplementary Fig. . Data are presented as mean ± SEM, n = 3, 3 independent experiments. ( G ) qRT-PCR analysis for midbrain/dopaminergic markers (EN1, NURR1, and GIRK2). GAPDH, 18S, HPRT were used as endogenous references. Data were normalized to control levels and presented as mean ± SEM, n = 4, 2 independent experiments.

    Techniques Used: Derivative Assay, Immunofluorescence, Expressing, Western Blot, Quantitative RT-PCR

    gene exp kcnj6 hs00158423 m1  (Thermo Fisher)


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    Thermo Fisher gene exp kcnj6 hs00158423 m1
    General characterization of neurons derived from PARK2 KO and healthy isogenic induced pluripotent stem cells (iPSCs). ( A ) Graphical overview of the differentiation of iPSCs to neural stem cells (NSCs) and fully committed dopaminergic neurons. ( B ) Immunofluorescence analysis of MAP2 (mature neurons, red) and TH (dopaminergic neurons, green) expression in PARK2 KO iPSC and control iPSC lines at day 25 of differentiation. Cell nuclei are visualized using DAPI (blue). Representative pictures are shown. Scale bar: 50 µm. ( C , D ) Quantitative assessment of ( C ) MAP2 + mature neurons and (D) TH + dopaminergic neurons in PARK2 KO and control iPSC lines. Data are presented as mean ± SEM, n = 15, 5 independent differentiations. ( E , F ) Western blotting and densitometry analysis of ( E ) MAP2 and ( F ) TH protein expression levels in PARK2 KO and control iPSC lines. Protein expression levels were normalized to α-actin. Full-length blots are presented in Supplementary Fig. . Data are presented as mean ± SEM, n = 3, 3 independent experiments. ( G ) qRT-PCR analysis for midbrain/dopaminergic markers (EN1, NURR1, and <t>GIRK2).</t> GAPDH, 18S, HPRT were used as endogenous references. Data were normalized to control levels and presented as mean ± SEM, n = 4, 2 independent experiments.
    Gene Exp Kcnj6 Hs00158423 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Lysosomal perturbations in human dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation"

    Article Title: Lysosomal perturbations in human dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-67091-6

    General characterization of neurons derived from PARK2 KO and healthy isogenic induced pluripotent stem cells (iPSCs). ( A ) Graphical overview of the differentiation of iPSCs to neural stem cells (NSCs) and fully committed dopaminergic neurons. ( B ) Immunofluorescence analysis of MAP2 (mature neurons, red) and TH (dopaminergic neurons, green) expression in PARK2 KO iPSC and control iPSC lines at day 25 of differentiation. Cell nuclei are visualized using DAPI (blue). Representative pictures are shown. Scale bar: 50 µm. ( C , D ) Quantitative assessment of ( C ) MAP2 + mature neurons and (D) TH + dopaminergic neurons in PARK2 KO and control iPSC lines. Data are presented as mean ± SEM, n = 15, 5 independent differentiations. ( E , F ) Western blotting and densitometry analysis of ( E ) MAP2 and ( F ) TH protein expression levels in PARK2 KO and control iPSC lines. Protein expression levels were normalized to α-actin. Full-length blots are presented in Supplementary Fig. . Data are presented as mean ± SEM, n = 3, 3 independent experiments. ( G ) qRT-PCR analysis for midbrain/dopaminergic markers (EN1, NURR1, and GIRK2). GAPDH, 18S, HPRT were used as endogenous references. Data were normalized to control levels and presented as mean ± SEM, n = 4, 2 independent experiments.
    Figure Legend Snippet: General characterization of neurons derived from PARK2 KO and healthy isogenic induced pluripotent stem cells (iPSCs). ( A ) Graphical overview of the differentiation of iPSCs to neural stem cells (NSCs) and fully committed dopaminergic neurons. ( B ) Immunofluorescence analysis of MAP2 (mature neurons, red) and TH (dopaminergic neurons, green) expression in PARK2 KO iPSC and control iPSC lines at day 25 of differentiation. Cell nuclei are visualized using DAPI (blue). Representative pictures are shown. Scale bar: 50 µm. ( C , D ) Quantitative assessment of ( C ) MAP2 + mature neurons and (D) TH + dopaminergic neurons in PARK2 KO and control iPSC lines. Data are presented as mean ± SEM, n = 15, 5 independent differentiations. ( E , F ) Western blotting and densitometry analysis of ( E ) MAP2 and ( F ) TH protein expression levels in PARK2 KO and control iPSC lines. Protein expression levels were normalized to α-actin. Full-length blots are presented in Supplementary Fig. . Data are presented as mean ± SEM, n = 3, 3 independent experiments. ( G ) qRT-PCR analysis for midbrain/dopaminergic markers (EN1, NURR1, and GIRK2). GAPDH, 18S, HPRT were used as endogenous references. Data were normalized to control levels and presented as mean ± SEM, n = 4, 2 independent experiments.

    Techniques Used: Derivative Assay, Immunofluorescence, Expressing, Western Blot, Quantitative RT-PCR

    gene exp kcnj6 hs00158423 m1  (Thermo Fisher)


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    Thermo Fisher gene exp kcnj6 hs00158423 m1
    A) Graphical overview of the differentiation of iPSCs to neural stem cells (NSCs) and fully committed dopaminergic neurons. B) Immunofluorescence analysis of MAP2 (mature neurons, red), and TH (dopaminergic neurons, green) expression in PARK2 KO iPSC and control iPSC lines at day 25 of differentiation. Cell nuclei are visualized using DAPI (blue). Representative pictures are shown. Scale bar: 50 μm. C-D) Quantitative assessment of C) MAP2+ mature neurons and D) TH+ dopaminergic neurons in PARK2 KO and control iPSC lines. All data are presented as mean ± SEM, n=15; 5 independent differentiations. E-F) Western blotting and densitometry analysis of E) MAP2 and F) TH protein expression levels in PARK2 KO and control iPSC lines. Protein expression levels were normalized to α-actin. Data are presented as mean ± SEM; 3 independent experiments. G) qPCR analysis for midbrain/dopaminergic markers (EN1, NURR1 and <t>GIRK2).</t> GAPDH, 18S, HPRT were used as endogenous references. Data were normalized to control levels and presented as mean ± SEM, 2 independent experiments.
    Gene Exp Kcnj6 Hs00158423 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Lysosomal perturbations in dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation"

    Article Title: Lysosomal perturbations in dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation

    Journal: bioRxiv

    doi: 10.1101/734244

    A) Graphical overview of the differentiation of iPSCs to neural stem cells (NSCs) and fully committed dopaminergic neurons. B) Immunofluorescence analysis of MAP2 (mature neurons, red), and TH (dopaminergic neurons, green) expression in PARK2 KO iPSC and control iPSC lines at day 25 of differentiation. Cell nuclei are visualized using DAPI (blue). Representative pictures are shown. Scale bar: 50 μm. C-D) Quantitative assessment of C) MAP2+ mature neurons and D) TH+ dopaminergic neurons in PARK2 KO and control iPSC lines. All data are presented as mean ± SEM, n=15; 5 independent differentiations. E-F) Western blotting and densitometry analysis of E) MAP2 and F) TH protein expression levels in PARK2 KO and control iPSC lines. Protein expression levels were normalized to α-actin. Data are presented as mean ± SEM; 3 independent experiments. G) qPCR analysis for midbrain/dopaminergic markers (EN1, NURR1 and GIRK2). GAPDH, 18S, HPRT were used as endogenous references. Data were normalized to control levels and presented as mean ± SEM, 2 independent experiments.
    Figure Legend Snippet: A) Graphical overview of the differentiation of iPSCs to neural stem cells (NSCs) and fully committed dopaminergic neurons. B) Immunofluorescence analysis of MAP2 (mature neurons, red), and TH (dopaminergic neurons, green) expression in PARK2 KO iPSC and control iPSC lines at day 25 of differentiation. Cell nuclei are visualized using DAPI (blue). Representative pictures are shown. Scale bar: 50 μm. C-D) Quantitative assessment of C) MAP2+ mature neurons and D) TH+ dopaminergic neurons in PARK2 KO and control iPSC lines. All data are presented as mean ± SEM, n=15; 5 independent differentiations. E-F) Western blotting and densitometry analysis of E) MAP2 and F) TH protein expression levels in PARK2 KO and control iPSC lines. Protein expression levels were normalized to α-actin. Data are presented as mean ± SEM; 3 independent experiments. G) qPCR analysis for midbrain/dopaminergic markers (EN1, NURR1 and GIRK2). GAPDH, 18S, HPRT were used as endogenous references. Data were normalized to control levels and presented as mean ± SEM, 2 independent experiments.

    Techniques Used: Immunofluorescence, Expressing, Western Blot

    gene exp kcnj6 hs00158423 m1  (Thermo Fisher)


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    Thermo Fisher gene exp kcnj6 hs00158423 m1
    Bcl-XL effects on dopaminergic maturation. A, percentage of TH+ cells after 7 days of differentiation of control or Bcl-XL-overexpressing hVM1 cells. hVM1-ø cells were treated with 50 μm Z-VAD-fmk (p < 0.05, Kruskal Wallis ANOVA, post hoc Mann-Whitney U test; *, versus hVM-ø cells; $, versus hVM1-ø + Z-VAD-fmk). B, pictures illustrate ICC staining for TH and Hoechst. Scale bar, 20 μm. C–E, Q-RT-PCR for VMAT2, DAT, and <t>GIRK2</t> in dividing and 7-day differentiated control or Bcl-XL-overexpressing cells. Data represent mean ± S.E. (n = 3) and were normalized to GAPDH gene expression. RQs were referred to hVM1-ø cells under division conditions (p < 0.05, ANOVA, post hoc Fisher test; *, versus hVM1-ø cells; †, versus Div; $, versus hVM1-low Bcl-XL). F and G, GIRK2 and DAT Q-RT-PCR data from 7-day differentiated cells were relative to TH expression levels, respectively (n = 3, p < 0.05, ANOVA, post hoc Fisher test; *, versus hVM1-ø cells). H and I, DA detection by HPLC in 7-day differentiated Bcl-XL-overexpressing cell lines. The histograms represent intracellular DA content and released DA (in basal or KCl-induced depolarization conditions) (n = 3; *, p < 0.05, Mann-Whitney U test). DA could not be reliably detected in control cells, possibly due to the high rate of cell death by day 7 of differentiation. J, DA/TH double ICC in 7-day differentiated hVM1-low Bcl-XL and hVM1-high Bcl-XL cells. Scale bar, 10 μm. K and L, double ICC for TH and GIRK2 and TH and Pitx3 in 7-day differentiated cells (scale bar, 10 μm).
    Gene Exp Kcnj6 Hs00158423 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "In Vitro and in Vivo Enhanced Generation of Human A9 Dopamine Neurons from Neural Stem Cells by Bcl-X L"

    Article Title: In Vitro and in Vivo Enhanced Generation of Human A9 Dopamine Neurons from Neural Stem Cells by Bcl-X L

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.054312

    Bcl-XL effects on dopaminergic maturation. A, percentage of TH+ cells after 7 days of differentiation of control or Bcl-XL-overexpressing hVM1 cells. hVM1-ø cells were treated with 50 μm Z-VAD-fmk (p < 0.05, Kruskal Wallis ANOVA, post hoc Mann-Whitney U test; *, versus hVM-ø cells; $, versus hVM1-ø + Z-VAD-fmk). B, pictures illustrate ICC staining for TH and Hoechst. Scale bar, 20 μm. C–E, Q-RT-PCR for VMAT2, DAT, and GIRK2 in dividing and 7-day differentiated control or Bcl-XL-overexpressing cells. Data represent mean ± S.E. (n = 3) and were normalized to GAPDH gene expression. RQs were referred to hVM1-ø cells under division conditions (p < 0.05, ANOVA, post hoc Fisher test; *, versus hVM1-ø cells; †, versus Div; $, versus hVM1-low Bcl-XL). F and G, GIRK2 and DAT Q-RT-PCR data from 7-day differentiated cells were relative to TH expression levels, respectively (n = 3, p < 0.05, ANOVA, post hoc Fisher test; *, versus hVM1-ø cells). H and I, DA detection by HPLC in 7-day differentiated Bcl-XL-overexpressing cell lines. The histograms represent intracellular DA content and released DA (in basal or KCl-induced depolarization conditions) (n = 3; *, p < 0.05, Mann-Whitney U test). DA could not be reliably detected in control cells, possibly due to the high rate of cell death by day 7 of differentiation. J, DA/TH double ICC in 7-day differentiated hVM1-low Bcl-XL and hVM1-high Bcl-XL cells. Scale bar, 10 μm. K and L, double ICC for TH and GIRK2 and TH and Pitx3 in 7-day differentiated cells (scale bar, 10 μm).
    Figure Legend Snippet: Bcl-XL effects on dopaminergic maturation. A, percentage of TH+ cells after 7 days of differentiation of control or Bcl-XL-overexpressing hVM1 cells. hVM1-ø cells were treated with 50 μm Z-VAD-fmk (p < 0.05, Kruskal Wallis ANOVA, post hoc Mann-Whitney U test; *, versus hVM-ø cells; $, versus hVM1-ø + Z-VAD-fmk). B, pictures illustrate ICC staining for TH and Hoechst. Scale bar, 20 μm. C–E, Q-RT-PCR for VMAT2, DAT, and GIRK2 in dividing and 7-day differentiated control or Bcl-XL-overexpressing cells. Data represent mean ± S.E. (n = 3) and were normalized to GAPDH gene expression. RQs were referred to hVM1-ø cells under division conditions (p < 0.05, ANOVA, post hoc Fisher test; *, versus hVM1-ø cells; †, versus Div; $, versus hVM1-low Bcl-XL). F and G, GIRK2 and DAT Q-RT-PCR data from 7-day differentiated cells were relative to TH expression levels, respectively (n = 3, p < 0.05, ANOVA, post hoc Fisher test; *, versus hVM1-ø cells). H and I, DA detection by HPLC in 7-day differentiated Bcl-XL-overexpressing cell lines. The histograms represent intracellular DA content and released DA (in basal or KCl-induced depolarization conditions) (n = 3; *, p < 0.05, Mann-Whitney U test). DA could not be reliably detected in control cells, possibly due to the high rate of cell death by day 7 of differentiation. J, DA/TH double ICC in 7-day differentiated hVM1-low Bcl-XL and hVM1-high Bcl-XL cells. Scale bar, 10 μm. K and L, double ICC for TH and GIRK2 and TH and Pitx3 in 7-day differentiated cells (scale bar, 10 μm).

    Techniques Used: MANN-WHITNEY, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing

    Bcl-XL effects on proneural and dopaminergic gene expression. Cell lines were analyzed under division (Div) or after 3 and 7 days (d3 and d7) of differentiation. A–C, proneural gene expression was determined from division to day 7 of differentiation by analyzing NGN2 (A), NEUROD1 (B), and MASH1 (C). D and E, expression of genes involved in DAn patterning. EN1 and LMX1B were analyzed in proliferating cells. In relation to EN1 gene expression, mRNA levels remained elevated in Bcl-XL overexpressing cells as compared with hVM1-ø cells during the whole differentiation period (not shown). The Western blot shows En1 protein levels in proliferating cells. β-actin was used as loading control. F and G, expression assay of genes involved in DAn maturation; quantitative expression of NURR1 and PITX3 genes in proliferating cells (Div) and 3-day (d3) or 7-day (d7) differentiated cells (results for VMAT2, DAT, and GIRK2 were shown in Fig. 5). NURR1 and PITX3 were analyzed and reported in proliferating and in early differentiated cells (d3) because these genes were also essential for postmitotic DAn. Data represent mean ± S.E. (n = 3 or 5) and were normalized to GAPDH gene expression. RQ was calibrated by referring all data to hVM1-ø cell under division conditions (p < 0.05, ANOVA, post hoc Fisher test; *, versus Div; †, versus hVM1-ø; $, versus hVM1-low Bcl-XL).
    Figure Legend Snippet: Bcl-XL effects on proneural and dopaminergic gene expression. Cell lines were analyzed under division (Div) or after 3 and 7 days (d3 and d7) of differentiation. A–C, proneural gene expression was determined from division to day 7 of differentiation by analyzing NGN2 (A), NEUROD1 (B), and MASH1 (C). D and E, expression of genes involved in DAn patterning. EN1 and LMX1B were analyzed in proliferating cells. In relation to EN1 gene expression, mRNA levels remained elevated in Bcl-XL overexpressing cells as compared with hVM1-ø cells during the whole differentiation period (not shown). The Western blot shows En1 protein levels in proliferating cells. β-actin was used as loading control. F and G, expression assay of genes involved in DAn maturation; quantitative expression of NURR1 and PITX3 genes in proliferating cells (Div) and 3-day (d3) or 7-day (d7) differentiated cells (results for VMAT2, DAT, and GIRK2 were shown in Fig. 5). NURR1 and PITX3 were analyzed and reported in proliferating and in early differentiated cells (d3) because these genes were also essential for postmitotic DAn. Data represent mean ± S.E. (n = 3 or 5) and were normalized to GAPDH gene expression. RQ was calibrated by referring all data to hVM1-ø cell under division conditions (p < 0.05, ANOVA, post hoc Fisher test; *, versus Div; †, versus hVM1-ø; $, versus hVM1-low Bcl-XL).

    Techniques Used: Expressing, Western Blot

    Q-RT-PCR in vivo of human genes and behavioral results. A–E, gene expression. Rats were transplanted as described under “Experimental Procedures.” After 2 months, brains were processed for RNA extraction and Q-RT-PCR detection of β-III tubulin (neurons) (A), DAn (TH) (B), SNpc origin (GIRK2) (D), and maturation (DAT) of DAn (E). Data show higher levels of neuronal, dopaminergic, and SNpc genes in hVM1-Bcl-XL grafts compared with control grafts (hVM1-ø), suggestive of more neurons and mature DA neurons (n = 5 and 6, respectively, mean ± S.E.; *, p < 0.05, Mann-Whitney U test). C, the ratio TH/β-III-tubulin represents the dopaminergic field of the generated neurons after 2 months of transplantation. Determination of gene expression was made with human-specific probes, not cross-reacting with rat genes. RNA integrity was confirmed by 18 S rRNA expression. RQ was calibrated by referring all data to the hVM1-ø cells transplanted group. F and G, behavioral results. F, contralateral apomorphine-induced rotation (for hVM1-ø and hVM1-high Bcl-XL groups, n = 12 and 11, respectively). G, intrastriatal grafts of hVM1-high Bcl-XL cells induce partial compensation of the lesion-induced rotational asymmetry in the d-amphetamine-induced rotation test (for hVM1-ø and hVM1-high Bcl-XL groups n = 13 and 9, respectively). In both cases, data are expressed as turns/min. Rotation tests were run at pregrafting (PG) and 2 months post-transplantation (2M). Data represent mean ± S.E. Data were analyzed by two-way ANOVA, with least square difference post hoc test (*, p < 0.05 versus hVM1-ø).
    Figure Legend Snippet: Q-RT-PCR in vivo of human genes and behavioral results. A–E, gene expression. Rats were transplanted as described under “Experimental Procedures.” After 2 months, brains were processed for RNA extraction and Q-RT-PCR detection of β-III tubulin (neurons) (A), DAn (TH) (B), SNpc origin (GIRK2) (D), and maturation (DAT) of DAn (E). Data show higher levels of neuronal, dopaminergic, and SNpc genes in hVM1-Bcl-XL grafts compared with control grafts (hVM1-ø), suggestive of more neurons and mature DA neurons (n = 5 and 6, respectively, mean ± S.E.; *, p < 0.05, Mann-Whitney U test). C, the ratio TH/β-III-tubulin represents the dopaminergic field of the generated neurons after 2 months of transplantation. Determination of gene expression was made with human-specific probes, not cross-reacting with rat genes. RNA integrity was confirmed by 18 S rRNA expression. RQ was calibrated by referring all data to the hVM1-ø cells transplanted group. F and G, behavioral results. F, contralateral apomorphine-induced rotation (for hVM1-ø and hVM1-high Bcl-XL groups, n = 12 and 11, respectively). G, intrastriatal grafts of hVM1-high Bcl-XL cells induce partial compensation of the lesion-induced rotational asymmetry in the d-amphetamine-induced rotation test (for hVM1-ø and hVM1-high Bcl-XL groups n = 13 and 9, respectively). In both cases, data are expressed as turns/min. Rotation tests were run at pregrafting (PG) and 2 months post-transplantation (2M). Data represent mean ± S.E. Data were analyzed by two-way ANOVA, with least square difference post hoc test (*, p < 0.05 versus hVM1-ø).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vivo, Expressing, RNA Extraction, MANN-WHITNEY, Generated, Transplantation Assay

    Proposed model of Bcl-XL effects on human VM NSC differentiation. In a control situation, hVM1-ø cells first differentiate to neuronal (NPC) and glial progenitors and then generate neurons (β-III-tubulin+) and glial cells (GFAP+). Additional signals are required to generate DAn (TH+) and mature DAn (also expressing markers like VMAT-2, DAT, and GIRK2). Cell death occurs during the whole of the differentiation process. Bcl-XL exerts three actions. First, it counteracts cell death (at multiple levels). Second, Bcl-XL promotes neuron generation by inducing proneural factors (Ngn2 and NeuroD1) at early times during differentiation. Third, Bcl-XL potentiates dopaminergic differentiation, first enhancing DA-ergic patterning factors (Lm1xb, En1, Nurr1, and Pitx3) and later inducing factors involved in maturation of the DAn (GIRK2, VMAT2, and DAT). Phenotypic markers in boldface type are increased in a Bcl-XL dose-dependent way.
    Figure Legend Snippet: Proposed model of Bcl-XL effects on human VM NSC differentiation. In a control situation, hVM1-ø cells first differentiate to neuronal (NPC) and glial progenitors and then generate neurons (β-III-tubulin+) and glial cells (GFAP+). Additional signals are required to generate DAn (TH+) and mature DAn (also expressing markers like VMAT-2, DAT, and GIRK2). Cell death occurs during the whole of the differentiation process. Bcl-XL exerts three actions. First, it counteracts cell death (at multiple levels). Second, Bcl-XL promotes neuron generation by inducing proneural factors (Ngn2 and NeuroD1) at early times during differentiation. Third, Bcl-XL potentiates dopaminergic differentiation, first enhancing DA-ergic patterning factors (Lm1xb, En1, Nurr1, and Pitx3) and later inducing factors involved in maturation of the DAn (GIRK2, VMAT2, and DAT). Phenotypic markers in boldface type are increased in a Bcl-XL dose-dependent way.

    Techniques Used: Expressing

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    Thermo Fisher gene exp kcnj6 hs00158423 m1
    Gene Exp Kcnj6 Hs00158423 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp kcnj6 hs00158423 m1
    General characterization of neurons derived from PARK2 KO and healthy isogenic induced pluripotent stem cells (iPSCs). ( A ) Graphical overview of the differentiation of iPSCs to neural stem cells (NSCs) and fully committed dopaminergic neurons. ( B ) Immunofluorescence analysis of MAP2 (mature neurons, red) and TH (dopaminergic neurons, green) expression in PARK2 KO iPSC and control iPSC lines at day 25 of differentiation. Cell nuclei are visualized using DAPI (blue). Representative pictures are shown. Scale bar: 50 µm. ( C , D ) Quantitative assessment of ( C ) MAP2 + mature neurons and (D) TH + dopaminergic neurons in PARK2 KO and control iPSC lines. Data are presented as mean ± SEM, n = 15, 5 independent differentiations. ( E , F ) Western blotting and densitometry analysis of ( E ) MAP2 and ( F ) TH protein expression levels in PARK2 KO and control iPSC lines. Protein expression levels were normalized to α-actin. Full-length blots are presented in Supplementary Fig. . Data are presented as mean ± SEM, n = 3, 3 independent experiments. ( G ) qRT-PCR analysis for midbrain/dopaminergic markers (EN1, NURR1, and <t>GIRK2).</t> GAPDH, 18S, HPRT were used as endogenous references. Data were normalized to control levels and presented as mean ± SEM, n = 4, 2 independent experiments.
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    General characterization of neurons derived from PARK2 KO and healthy isogenic induced pluripotent stem cells (iPSCs). ( A ) Graphical overview of the differentiation of iPSCs to neural stem cells (NSCs) and fully committed dopaminergic neurons. ( B ) Immunofluorescence analysis of MAP2 (mature neurons, red) and TH (dopaminergic neurons, green) expression in PARK2 KO iPSC and control iPSC lines at day 25 of differentiation. Cell nuclei are visualized using DAPI (blue). Representative pictures are shown. Scale bar: 50 µm. ( C , D ) Quantitative assessment of ( C ) MAP2 + mature neurons and (D) TH + dopaminergic neurons in PARK2 KO and control iPSC lines. Data are presented as mean ± SEM, n = 15, 5 independent differentiations. ( E , F ) Western blotting and densitometry analysis of ( E ) MAP2 and ( F ) TH protein expression levels in PARK2 KO and control iPSC lines. Protein expression levels were normalized to α-actin. Full-length blots are presented in Supplementary Fig. . Data are presented as mean ± SEM, n = 3, 3 independent experiments. ( G ) qRT-PCR analysis for midbrain/dopaminergic markers (EN1, NURR1, and GIRK2). GAPDH, 18S, HPRT were used as endogenous references. Data were normalized to control levels and presented as mean ± SEM, n = 4, 2 independent experiments.

    Journal: Scientific Reports

    Article Title: Lysosomal perturbations in human dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation

    doi: 10.1038/s41598-020-67091-6

    Figure Lengend Snippet: General characterization of neurons derived from PARK2 KO and healthy isogenic induced pluripotent stem cells (iPSCs). ( A ) Graphical overview of the differentiation of iPSCs to neural stem cells (NSCs) and fully committed dopaminergic neurons. ( B ) Immunofluorescence analysis of MAP2 (mature neurons, red) and TH (dopaminergic neurons, green) expression in PARK2 KO iPSC and control iPSC lines at day 25 of differentiation. Cell nuclei are visualized using DAPI (blue). Representative pictures are shown. Scale bar: 50 µm. ( C , D ) Quantitative assessment of ( C ) MAP2 + mature neurons and (D) TH + dopaminergic neurons in PARK2 KO and control iPSC lines. Data are presented as mean ± SEM, n = 15, 5 independent differentiations. ( E , F ) Western blotting and densitometry analysis of ( E ) MAP2 and ( F ) TH protein expression levels in PARK2 KO and control iPSC lines. Protein expression levels were normalized to α-actin. Full-length blots are presented in Supplementary Fig. . Data are presented as mean ± SEM, n = 3, 3 independent experiments. ( G ) qRT-PCR analysis for midbrain/dopaminergic markers (EN1, NURR1, and GIRK2). GAPDH, 18S, HPRT were used as endogenous references. Data were normalized to control levels and presented as mean ± SEM, n = 4, 2 independent experiments.

    Article Snippet: Relative gene expression was assessed using the following TagMan assays: GAPDH , Hs02758991_g1; 18 S , Hs03003631_g1; HPRT , Hs02800695_m1; EN1 , Hs00154977_m1; NURR1 , Hs00428691_m1; GIRK2 , Hs00158423_m1.

    Techniques: Derivative Assay, Immunofluorescence, Expressing, Western Blot, Quantitative RT-PCR