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Addgene inc pcdna d3cpv
Measurement of peroxisomal Ca 2+ in cardiomyocytes. (A) Experimental paradigm of Ca 2+ measurement in excitable cells. The peak after thapsigargin (Tg) addition represents Ca 2+ increase due to the SERCA inhibition and Ca 2+ retention in the cytosol. (B) Cytosolic Ca 2+ measurement in NRCMs following the experimental design in (A), n = 25 (Tg), 22 (control) from three experiments. Addition of Tg is compared to the addition of Tg-free buffer (control). (C) Basal levels are not different before the treatment in (B). (D) After Tg addition in (B) cytosolic Ca 2+ increases. (E) Peroxisomal Ca 2+ measurement in NRCMs following the experimental design in (A). Addition of Tg is compared to the addition of Tg-free buffer (control), n = 20 (Tg), 31 (control) from three experiments. F) Basal levels of Ca 2+ are not different before the treatment in (E). (G) Peroxisomal Ca 2+ increases after Tg addition in (E). (H) HiPSC-CMs generation. Donor skin fibroblasts were reprogrammed to hiPSCs, which were then differentiated to CMs. (I) hiPSC-CMs were stained for cardiac troponin (cTnT) and analyzed by flow cytometry. Negative control without primary antibody. 94.8% of iPSC-CMs are cTnT-positive (cTNT + ). (J) Immunofluorescence staining visualized α-actinin protein expression and regular sarcomeric organization. Scale bar: 20 μm. (K) Cytosolic Ca 2+ measurement in hiPSC-CMs with <t>D3cpV</t> following the experimental paradigm for excitable cells in (A). Addition of Tg is compared to the addition of Tg-free buffer (control) to avoid artefacts and false results due to mechanical effect on the cells due to the addition itself. n = 24 (Tg), 27 (control) from three differentiation experiments. (L) No difference is found between two groups before the treatment in (K). (M) Tg addition in (K) results in cytosolic Ca 2+ increase. (N) Peroxisomal Ca 2+ measurement in hiPSC-CMs with D3cpV-px following the experimental design in for excitable cells depicted in (A). Addition of Tg is compared to the addition of Tg-free buffer (control). n = 26 (Tg), 33 (control) from three differentiation experimnets. (O) Basal levels of Ca 2+ are not different before the treatment in (N). (P) Peroxisomal Ca 2+ increases after Tg addition in (M). (B, E, K, N) Data presented as means from three independent experiments. (C, D, F, G, L, M, O, P) Unpaired Student’s t-test was used for the statistical analysis. ****p
Pcdna D3cpv, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Peroxisomes contribute to intracellular calcium dynamics"

Article Title: Peroxisomes contribute to intracellular calcium dynamics

Journal: bioRxiv

doi: 10.1101/2020.09.02.279174

Measurement of peroxisomal Ca 2+ in cardiomyocytes. (A) Experimental paradigm of Ca 2+ measurement in excitable cells. The peak after thapsigargin (Tg) addition represents Ca 2+ increase due to the SERCA inhibition and Ca 2+ retention in the cytosol. (B) Cytosolic Ca 2+ measurement in NRCMs following the experimental design in (A), n = 25 (Tg), 22 (control) from three experiments. Addition of Tg is compared to the addition of Tg-free buffer (control). (C) Basal levels are not different before the treatment in (B). (D) After Tg addition in (B) cytosolic Ca 2+ increases. (E) Peroxisomal Ca 2+ measurement in NRCMs following the experimental design in (A). Addition of Tg is compared to the addition of Tg-free buffer (control), n = 20 (Tg), 31 (control) from three experiments. F) Basal levels of Ca 2+ are not different before the treatment in (E). (G) Peroxisomal Ca 2+ increases after Tg addition in (E). (H) HiPSC-CMs generation. Donor skin fibroblasts were reprogrammed to hiPSCs, which were then differentiated to CMs. (I) hiPSC-CMs were stained for cardiac troponin (cTnT) and analyzed by flow cytometry. Negative control without primary antibody. 94.8% of iPSC-CMs are cTnT-positive (cTNT + ). (J) Immunofluorescence staining visualized α-actinin protein expression and regular sarcomeric organization. Scale bar: 20 μm. (K) Cytosolic Ca 2+ measurement in hiPSC-CMs with D3cpV following the experimental paradigm for excitable cells in (A). Addition of Tg is compared to the addition of Tg-free buffer (control) to avoid artefacts and false results due to mechanical effect on the cells due to the addition itself. n = 24 (Tg), 27 (control) from three differentiation experiments. (L) No difference is found between two groups before the treatment in (K). (M) Tg addition in (K) results in cytosolic Ca 2+ increase. (N) Peroxisomal Ca 2+ measurement in hiPSC-CMs with D3cpV-px following the experimental design in for excitable cells depicted in (A). Addition of Tg is compared to the addition of Tg-free buffer (control). n = 26 (Tg), 33 (control) from three differentiation experimnets. (O) Basal levels of Ca 2+ are not different before the treatment in (N). (P) Peroxisomal Ca 2+ increases after Tg addition in (M). (B, E, K, N) Data presented as means from three independent experiments. (C, D, F, G, L, M, O, P) Unpaired Student’s t-test was used for the statistical analysis. ****p
Figure Legend Snippet: Measurement of peroxisomal Ca 2+ in cardiomyocytes. (A) Experimental paradigm of Ca 2+ measurement in excitable cells. The peak after thapsigargin (Tg) addition represents Ca 2+ increase due to the SERCA inhibition and Ca 2+ retention in the cytosol. (B) Cytosolic Ca 2+ measurement in NRCMs following the experimental design in (A), n = 25 (Tg), 22 (control) from three experiments. Addition of Tg is compared to the addition of Tg-free buffer (control). (C) Basal levels are not different before the treatment in (B). (D) After Tg addition in (B) cytosolic Ca 2+ increases. (E) Peroxisomal Ca 2+ measurement in NRCMs following the experimental design in (A). Addition of Tg is compared to the addition of Tg-free buffer (control), n = 20 (Tg), 31 (control) from three experiments. F) Basal levels of Ca 2+ are not different before the treatment in (E). (G) Peroxisomal Ca 2+ increases after Tg addition in (E). (H) HiPSC-CMs generation. Donor skin fibroblasts were reprogrammed to hiPSCs, which were then differentiated to CMs. (I) hiPSC-CMs were stained for cardiac troponin (cTnT) and analyzed by flow cytometry. Negative control without primary antibody. 94.8% of iPSC-CMs are cTnT-positive (cTNT + ). (J) Immunofluorescence staining visualized α-actinin protein expression and regular sarcomeric organization. Scale bar: 20 μm. (K) Cytosolic Ca 2+ measurement in hiPSC-CMs with D3cpV following the experimental paradigm for excitable cells in (A). Addition of Tg is compared to the addition of Tg-free buffer (control) to avoid artefacts and false results due to mechanical effect on the cells due to the addition itself. n = 24 (Tg), 27 (control) from three differentiation experiments. (L) No difference is found between two groups before the treatment in (K). (M) Tg addition in (K) results in cytosolic Ca 2+ increase. (N) Peroxisomal Ca 2+ measurement in hiPSC-CMs with D3cpV-px following the experimental design in for excitable cells depicted in (A). Addition of Tg is compared to the addition of Tg-free buffer (control). n = 26 (Tg), 33 (control) from three differentiation experimnets. (O) Basal levels of Ca 2+ are not different before the treatment in (N). (P) Peroxisomal Ca 2+ increases after Tg addition in (M). (B, E, K, N) Data presented as means from three independent experiments. (C, D, F, G, L, M, O, P) Unpaired Student’s t-test was used for the statistical analysis. ****p

Techniques Used: Inhibition, Staining, Flow Cytometry, Negative Control, Immunofluorescence, Expressing

Measurement of peroxisomal Ca 2+ in paced cardiomyocytes. (A ) D3cpV transfected NRCMs are stimulated with 1 Hz. Images are taken every 50 ms. Oscillations of FRET ratio are seen, n = 3. (B) FFT from the data in (A). Signal increases are rhythmic and correspond to the pacing frequency. (C) D3cpV-px transfected NRCMs are stimulated with 1 Hz. Images are taken every 100 ms. FRET ratio oscillations are see. n = 3. (D) FFT from the data in (C). Signal increases are rhythmic and correspond to the pacing frequency.
Figure Legend Snippet: Measurement of peroxisomal Ca 2+ in paced cardiomyocytes. (A ) D3cpV transfected NRCMs are stimulated with 1 Hz. Images are taken every 50 ms. Oscillations of FRET ratio are seen, n = 3. (B) FFT from the data in (A). Signal increases are rhythmic and correspond to the pacing frequency. (C) D3cpV-px transfected NRCMs are stimulated with 1 Hz. Images are taken every 100 ms. FRET ratio oscillations are see. n = 3. (D) FFT from the data in (C). Signal increases are rhythmic and correspond to the pacing frequency.

Techniques Used: Transfection

New sensors for peroxisomal Ca 2+ (A) Genetically encoded calcium indicators (GECIs) targeted to peroxisomes. D3cpv-px and D1cpV-px are FRET sensors with modified CaM sites. Pericam-px is a single-fluorophore based GECI that has M13 and CaM as Ca 2+ binding sites. In the absence of Ca 2+ , the emission measured when the sensor is excited with 420 nm is higher than when excited with 505 nm. The ratio 505/420 is a measure for the Ca 2+ concentration. (B) Subcellular localization of GECIs used in this study. (C) Peroxisomal GECIs colocalize with the peroxisomal membrane marker PEX14. HeLa cells were transfected with the GECIs and stained with anti-PEX14 antibodies. The images in the left part of the panel show one cell each (scale bar 10 µm). The cropped areas are marked and magnified in the right part of the panel (scale bar 2 µm). (D-F) D3cpv-px, D1cpv-px, and pericam-px are Ca 2+ sensitive. Images false-colored with LUT show representative cells before (left) and after (right) Ca 2+ addition. Curves presented as mean ± SEM. Scale bar: 10 µm. (D) Addition of 1 mM Ca 2+ to D3cpV-px expressing cells results in 1.5-fold FRET ratio increase, n = 60 cells from three independent experiments. (E) FRET ratio increases 1.08 times when 1 mM Ca 2+ is added to D1cpv-px expressing cells, n = 33 cells from three experiments. (F) Ca 2+ addition leads to 1.5-fold increase in 505/420 ratio with Pericam-px, n = 75 cells from three experiments. (G) Measurement of D3cpV-px during cytosol whashout. No change in signal is detected. (H) Measurement of pericam-px during cytosol whashout. No difference of signal before and after cytosol washout is detected, n = 43 cells for D3cpV-px in (G) and n = 45 cells for pericam-px in (H).
Figure Legend Snippet: New sensors for peroxisomal Ca 2+ (A) Genetically encoded calcium indicators (GECIs) targeted to peroxisomes. D3cpv-px and D1cpV-px are FRET sensors with modified CaM sites. Pericam-px is a single-fluorophore based GECI that has M13 and CaM as Ca 2+ binding sites. In the absence of Ca 2+ , the emission measured when the sensor is excited with 420 nm is higher than when excited with 505 nm. The ratio 505/420 is a measure for the Ca 2+ concentration. (B) Subcellular localization of GECIs used in this study. (C) Peroxisomal GECIs colocalize with the peroxisomal membrane marker PEX14. HeLa cells were transfected with the GECIs and stained with anti-PEX14 antibodies. The images in the left part of the panel show one cell each (scale bar 10 µm). The cropped areas are marked and magnified in the right part of the panel (scale bar 2 µm). (D-F) D3cpv-px, D1cpv-px, and pericam-px are Ca 2+ sensitive. Images false-colored with LUT show representative cells before (left) and after (right) Ca 2+ addition. Curves presented as mean ± SEM. Scale bar: 10 µm. (D) Addition of 1 mM Ca 2+ to D3cpV-px expressing cells results in 1.5-fold FRET ratio increase, n = 60 cells from three independent experiments. (E) FRET ratio increases 1.08 times when 1 mM Ca 2+ is added to D1cpv-px expressing cells, n = 33 cells from three experiments. (F) Ca 2+ addition leads to 1.5-fold increase in 505/420 ratio with Pericam-px, n = 75 cells from three experiments. (G) Measurement of D3cpV-px during cytosol whashout. No change in signal is detected. (H) Measurement of pericam-px during cytosol whashout. No difference of signal before and after cytosol washout is detected, n = 43 cells for D3cpV-px in (G) and n = 45 cells for pericam-px in (H).

Techniques Used: Modification, Chick Chorioallantoic Membrane Assay, Binding Assay, Concentration Assay, Marker, Transfection, Staining, Expressing

Measurement of peroxisomal Ca 2+ in HeLa cells. (A) Comparison of cytosolic and peroxisomal responces to ionomycin (Iono). In comparison to cytosol, peroxisomal signal increases gradually, n = 16 cells for D3cpV and n = 9 cells for D3cpV-px. (B) Experimental paradigm of a two-step Ca 2+ measurement in non-excitable cells. 1 st peak after histamine (His) addition: ER-store depletion. 2 nd peak, after addition of extracellular Ca 2+ : PM-based uptake. (C) Measurement with D3cpV-px according to the paradigm in (B). Two Ca 2+ peaks of the experimental paradigm are detectable with D3cpV-px, n ≥ 50 cells from three experiments. (D) Absolute Ca 2+ concentration dynamics calculated from the data in (C). (E) Basal and maximum (max) Ca 2+ concentrations in peroxisomes based on (C). (F) Measurement with pericam-px according to the paradigm in (B), n = 27 cells from three experiments. (G) Simultaneous measurement of cytosolic (blue) and peroxisomal (green) Ca 2+ . No delay of signal increase after histamine addition, but a delayed drop of the signal in peroxisomes. Left y axis: of D3cpV-px (peroxisomal sensor). Right y axis: F n /F 0 ratio of R-GECO1 (cytosolic sensor), n = 35 cells from three experiments. (H) Decline of F n /F 0 ratio per millisecond (ms) in the linear part of the curves in (G) (from second 65 to 115, Student’s t-test). Kinetic delay in decrease in peroxisomal signal is seen. (I) Comparison of cytosolic, mitochondrial and peroxisomal Ca 2+ response measured following the paradigm in (B). Characteristic two peaks present in all three compartments. (J) Basal levels of Ca 2+ in peroxisomes are similar to mitochondria. Analysis performed based on the data from (I). (K) Peroxisomal Ca 2+ increase upon ER-store depletion is smaller than that of cytosol or mitochondria. Analysis performed based on the data from (I). (L) Peroxisomal Ca 2+ increase upon PM-based cellular uptake of Ca 2+ is comparable to mitochondria. Analysis performed based on the data from (I). (A, C, D, F) Data presented as mean ± SEM. (J-L) One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. ***p
Figure Legend Snippet: Measurement of peroxisomal Ca 2+ in HeLa cells. (A) Comparison of cytosolic and peroxisomal responces to ionomycin (Iono). In comparison to cytosol, peroxisomal signal increases gradually, n = 16 cells for D3cpV and n = 9 cells for D3cpV-px. (B) Experimental paradigm of a two-step Ca 2+ measurement in non-excitable cells. 1 st peak after histamine (His) addition: ER-store depletion. 2 nd peak, after addition of extracellular Ca 2+ : PM-based uptake. (C) Measurement with D3cpV-px according to the paradigm in (B). Two Ca 2+ peaks of the experimental paradigm are detectable with D3cpV-px, n ≥ 50 cells from three experiments. (D) Absolute Ca 2+ concentration dynamics calculated from the data in (C). (E) Basal and maximum (max) Ca 2+ concentrations in peroxisomes based on (C). (F) Measurement with pericam-px according to the paradigm in (B), n = 27 cells from three experiments. (G) Simultaneous measurement of cytosolic (blue) and peroxisomal (green) Ca 2+ . No delay of signal increase after histamine addition, but a delayed drop of the signal in peroxisomes. Left y axis: of D3cpV-px (peroxisomal sensor). Right y axis: F n /F 0 ratio of R-GECO1 (cytosolic sensor), n = 35 cells from three experiments. (H) Decline of F n /F 0 ratio per millisecond (ms) in the linear part of the curves in (G) (from second 65 to 115, Student’s t-test). Kinetic delay in decrease in peroxisomal signal is seen. (I) Comparison of cytosolic, mitochondrial and peroxisomal Ca 2+ response measured following the paradigm in (B). Characteristic two peaks present in all three compartments. (J) Basal levels of Ca 2+ in peroxisomes are similar to mitochondria. Analysis performed based on the data from (I). (K) Peroxisomal Ca 2+ increase upon ER-store depletion is smaller than that of cytosol or mitochondria. Analysis performed based on the data from (I). (L) Peroxisomal Ca 2+ increase upon PM-based cellular uptake of Ca 2+ is comparable to mitochondria. Analysis performed based on the data from (I). (A, C, D, F) Data presented as mean ± SEM. (J-L) One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. ***p

Techniques Used: Concentration Assay

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Article Title: Peroxisomes contribute to intracellular calcium dynamics
Article Snippet: .. DNA constructsD3cpV-px (PST 1738) was generated from (pcDNA-)D3cpV (kind gift from A. Palmer and R. Tsien ( ) (Addgene #36323)) by amplifying an insert with OST 1599 (GCGCATCGAT GGTGATGGCC AAGTAAACTA TGAAGAG) and OST 1600 (GCGCGAATTC TTAGAGCTTC GATTTCAGAC TTCCCTCGA) primers. .. The product was then reinserted into D3cpV using ClaI and EcoRI restriction sites. (pcDNA-)4mtD3cpV was a kind gift from A. Palmer and R. Tsien ( ) (Addgene #36324).

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    Addgene inc pcdna d3cpv
    Measurement of peroxisomal Ca 2+ in cardiomyocytes. (A) Experimental paradigm of Ca 2+ measurement in excitable cells. The peak after thapsigargin (Tg) addition represents Ca 2+ increase due to the SERCA inhibition and Ca 2+ retention in the cytosol. (B) Cytosolic Ca 2+ measurement in NRCMs following the experimental design in (A), n = 25 (Tg), 22 (control) from three experiments. Addition of Tg is compared to the addition of Tg-free buffer (control). (C) Basal levels are not different before the treatment in (B). (D) After Tg addition in (B) cytosolic Ca 2+ increases. (E) Peroxisomal Ca 2+ measurement in NRCMs following the experimental design in (A). Addition of Tg is compared to the addition of Tg-free buffer (control), n = 20 (Tg), 31 (control) from three experiments. F) Basal levels of Ca 2+ are not different before the treatment in (E). (G) Peroxisomal Ca 2+ increases after Tg addition in (E). (H) HiPSC-CMs generation. Donor skin fibroblasts were reprogrammed to hiPSCs, which were then differentiated to CMs. (I) hiPSC-CMs were stained for cardiac troponin (cTnT) and analyzed by flow cytometry. Negative control without primary antibody. 94.8% of iPSC-CMs are cTnT-positive (cTNT + ). (J) Immunofluorescence staining visualized α-actinin protein expression and regular sarcomeric organization. Scale bar: 20 μm. (K) Cytosolic Ca 2+ measurement in hiPSC-CMs with <t>D3cpV</t> following the experimental paradigm for excitable cells in (A). Addition of Tg is compared to the addition of Tg-free buffer (control) to avoid artefacts and false results due to mechanical effect on the cells due to the addition itself. n = 24 (Tg), 27 (control) from three differentiation experiments. (L) No difference is found between two groups before the treatment in (K). (M) Tg addition in (K) results in cytosolic Ca 2+ increase. (N) Peroxisomal Ca 2+ measurement in hiPSC-CMs with D3cpV-px following the experimental design in for excitable cells depicted in (A). Addition of Tg is compared to the addition of Tg-free buffer (control). n = 26 (Tg), 33 (control) from three differentiation experimnets. (O) Basal levels of Ca 2+ are not different before the treatment in (N). (P) Peroxisomal Ca 2+ increases after Tg addition in (M). (B, E, K, N) Data presented as means from three independent experiments. (C, D, F, G, L, M, O, P) Unpaired Student’s t-test was used for the statistical analysis. ****p
    Pcdna D3cpv, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna d3cpv/product/Addgene inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcdna d3cpv - by Bioz Stars, 2020-09
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    Measurement of peroxisomal Ca 2+ in cardiomyocytes. (A) Experimental paradigm of Ca 2+ measurement in excitable cells. The peak after thapsigargin (Tg) addition represents Ca 2+ increase due to the SERCA inhibition and Ca 2+ retention in the cytosol. (B) Cytosolic Ca 2+ measurement in NRCMs following the experimental design in (A), n = 25 (Tg), 22 (control) from three experiments. Addition of Tg is compared to the addition of Tg-free buffer (control). (C) Basal levels are not different before the treatment in (B). (D) After Tg addition in (B) cytosolic Ca 2+ increases. (E) Peroxisomal Ca 2+ measurement in NRCMs following the experimental design in (A). Addition of Tg is compared to the addition of Tg-free buffer (control), n = 20 (Tg), 31 (control) from three experiments. F) Basal levels of Ca 2+ are not different before the treatment in (E). (G) Peroxisomal Ca 2+ increases after Tg addition in (E). (H) HiPSC-CMs generation. Donor skin fibroblasts were reprogrammed to hiPSCs, which were then differentiated to CMs. (I) hiPSC-CMs were stained for cardiac troponin (cTnT) and analyzed by flow cytometry. Negative control without primary antibody. 94.8% of iPSC-CMs are cTnT-positive (cTNT + ). (J) Immunofluorescence staining visualized α-actinin protein expression and regular sarcomeric organization. Scale bar: 20 μm. (K) Cytosolic Ca 2+ measurement in hiPSC-CMs with D3cpV following the experimental paradigm for excitable cells in (A). Addition of Tg is compared to the addition of Tg-free buffer (control) to avoid artefacts and false results due to mechanical effect on the cells due to the addition itself. n = 24 (Tg), 27 (control) from three differentiation experiments. (L) No difference is found between two groups before the treatment in (K). (M) Tg addition in (K) results in cytosolic Ca 2+ increase. (N) Peroxisomal Ca 2+ measurement in hiPSC-CMs with D3cpV-px following the experimental design in for excitable cells depicted in (A). Addition of Tg is compared to the addition of Tg-free buffer (control). n = 26 (Tg), 33 (control) from three differentiation experimnets. (O) Basal levels of Ca 2+ are not different before the treatment in (N). (P) Peroxisomal Ca 2+ increases after Tg addition in (M). (B, E, K, N) Data presented as means from three independent experiments. (C, D, F, G, L, M, O, P) Unpaired Student’s t-test was used for the statistical analysis. ****p

    Journal: bioRxiv

    Article Title: Peroxisomes contribute to intracellular calcium dynamics

    doi: 10.1101/2020.09.02.279174

    Figure Lengend Snippet: Measurement of peroxisomal Ca 2+ in cardiomyocytes. (A) Experimental paradigm of Ca 2+ measurement in excitable cells. The peak after thapsigargin (Tg) addition represents Ca 2+ increase due to the SERCA inhibition and Ca 2+ retention in the cytosol. (B) Cytosolic Ca 2+ measurement in NRCMs following the experimental design in (A), n = 25 (Tg), 22 (control) from three experiments. Addition of Tg is compared to the addition of Tg-free buffer (control). (C) Basal levels are not different before the treatment in (B). (D) After Tg addition in (B) cytosolic Ca 2+ increases. (E) Peroxisomal Ca 2+ measurement in NRCMs following the experimental design in (A). Addition of Tg is compared to the addition of Tg-free buffer (control), n = 20 (Tg), 31 (control) from three experiments. F) Basal levels of Ca 2+ are not different before the treatment in (E). (G) Peroxisomal Ca 2+ increases after Tg addition in (E). (H) HiPSC-CMs generation. Donor skin fibroblasts were reprogrammed to hiPSCs, which were then differentiated to CMs. (I) hiPSC-CMs were stained for cardiac troponin (cTnT) and analyzed by flow cytometry. Negative control without primary antibody. 94.8% of iPSC-CMs are cTnT-positive (cTNT + ). (J) Immunofluorescence staining visualized α-actinin protein expression and regular sarcomeric organization. Scale bar: 20 μm. (K) Cytosolic Ca 2+ measurement in hiPSC-CMs with D3cpV following the experimental paradigm for excitable cells in (A). Addition of Tg is compared to the addition of Tg-free buffer (control) to avoid artefacts and false results due to mechanical effect on the cells due to the addition itself. n = 24 (Tg), 27 (control) from three differentiation experiments. (L) No difference is found between two groups before the treatment in (K). (M) Tg addition in (K) results in cytosolic Ca 2+ increase. (N) Peroxisomal Ca 2+ measurement in hiPSC-CMs with D3cpV-px following the experimental design in for excitable cells depicted in (A). Addition of Tg is compared to the addition of Tg-free buffer (control). n = 26 (Tg), 33 (control) from three differentiation experimnets. (O) Basal levels of Ca 2+ are not different before the treatment in (N). (P) Peroxisomal Ca 2+ increases after Tg addition in (M). (B, E, K, N) Data presented as means from three independent experiments. (C, D, F, G, L, M, O, P) Unpaired Student’s t-test was used for the statistical analysis. ****p

    Article Snippet: DNA constructsD3cpV-px (PST 1738) was generated from (pcDNA-)D3cpV (kind gift from A. Palmer and R. Tsien ( ) (Addgene #36323)) by amplifying an insert with OST 1599 (GCGCATCGAT GGTGATGGCC AAGTAAACTA TGAAGAG) and OST 1600 (GCGCGAATTC TTAGAGCTTC GATTTCAGAC TTCCCTCGA) primers.

    Techniques: Inhibition, Staining, Flow Cytometry, Negative Control, Immunofluorescence, Expressing

    Measurement of peroxisomal Ca 2+ in paced cardiomyocytes. (A ) D3cpV transfected NRCMs are stimulated with 1 Hz. Images are taken every 50 ms. Oscillations of FRET ratio are seen, n = 3. (B) FFT from the data in (A). Signal increases are rhythmic and correspond to the pacing frequency. (C) D3cpV-px transfected NRCMs are stimulated with 1 Hz. Images are taken every 100 ms. FRET ratio oscillations are see. n = 3. (D) FFT from the data in (C). Signal increases are rhythmic and correspond to the pacing frequency.

    Journal: bioRxiv

    Article Title: Peroxisomes contribute to intracellular calcium dynamics

    doi: 10.1101/2020.09.02.279174

    Figure Lengend Snippet: Measurement of peroxisomal Ca 2+ in paced cardiomyocytes. (A ) D3cpV transfected NRCMs are stimulated with 1 Hz. Images are taken every 50 ms. Oscillations of FRET ratio are seen, n = 3. (B) FFT from the data in (A). Signal increases are rhythmic and correspond to the pacing frequency. (C) D3cpV-px transfected NRCMs are stimulated with 1 Hz. Images are taken every 100 ms. FRET ratio oscillations are see. n = 3. (D) FFT from the data in (C). Signal increases are rhythmic and correspond to the pacing frequency.

    Article Snippet: DNA constructsD3cpV-px (PST 1738) was generated from (pcDNA-)D3cpV (kind gift from A. Palmer and R. Tsien ( ) (Addgene #36323)) by amplifying an insert with OST 1599 (GCGCATCGAT GGTGATGGCC AAGTAAACTA TGAAGAG) and OST 1600 (GCGCGAATTC TTAGAGCTTC GATTTCAGAC TTCCCTCGA) primers.

    Techniques: Transfection

    New sensors for peroxisomal Ca 2+ (A) Genetically encoded calcium indicators (GECIs) targeted to peroxisomes. D3cpv-px and D1cpV-px are FRET sensors with modified CaM sites. Pericam-px is a single-fluorophore based GECI that has M13 and CaM as Ca 2+ binding sites. In the absence of Ca 2+ , the emission measured when the sensor is excited with 420 nm is higher than when excited with 505 nm. The ratio 505/420 is a measure for the Ca 2+ concentration. (B) Subcellular localization of GECIs used in this study. (C) Peroxisomal GECIs colocalize with the peroxisomal membrane marker PEX14. HeLa cells were transfected with the GECIs and stained with anti-PEX14 antibodies. The images in the left part of the panel show one cell each (scale bar 10 µm). The cropped areas are marked and magnified in the right part of the panel (scale bar 2 µm). (D-F) D3cpv-px, D1cpv-px, and pericam-px are Ca 2+ sensitive. Images false-colored with LUT show representative cells before (left) and after (right) Ca 2+ addition. Curves presented as mean ± SEM. Scale bar: 10 µm. (D) Addition of 1 mM Ca 2+ to D3cpV-px expressing cells results in 1.5-fold FRET ratio increase, n = 60 cells from three independent experiments. (E) FRET ratio increases 1.08 times when 1 mM Ca 2+ is added to D1cpv-px expressing cells, n = 33 cells from three experiments. (F) Ca 2+ addition leads to 1.5-fold increase in 505/420 ratio with Pericam-px, n = 75 cells from three experiments. (G) Measurement of D3cpV-px during cytosol whashout. No change in signal is detected. (H) Measurement of pericam-px during cytosol whashout. No difference of signal before and after cytosol washout is detected, n = 43 cells for D3cpV-px in (G) and n = 45 cells for pericam-px in (H).

    Journal: bioRxiv

    Article Title: Peroxisomes contribute to intracellular calcium dynamics

    doi: 10.1101/2020.09.02.279174

    Figure Lengend Snippet: New sensors for peroxisomal Ca 2+ (A) Genetically encoded calcium indicators (GECIs) targeted to peroxisomes. D3cpv-px and D1cpV-px are FRET sensors with modified CaM sites. Pericam-px is a single-fluorophore based GECI that has M13 and CaM as Ca 2+ binding sites. In the absence of Ca 2+ , the emission measured when the sensor is excited with 420 nm is higher than when excited with 505 nm. The ratio 505/420 is a measure for the Ca 2+ concentration. (B) Subcellular localization of GECIs used in this study. (C) Peroxisomal GECIs colocalize with the peroxisomal membrane marker PEX14. HeLa cells were transfected with the GECIs and stained with anti-PEX14 antibodies. The images in the left part of the panel show one cell each (scale bar 10 µm). The cropped areas are marked and magnified in the right part of the panel (scale bar 2 µm). (D-F) D3cpv-px, D1cpv-px, and pericam-px are Ca 2+ sensitive. Images false-colored with LUT show representative cells before (left) and after (right) Ca 2+ addition. Curves presented as mean ± SEM. Scale bar: 10 µm. (D) Addition of 1 mM Ca 2+ to D3cpV-px expressing cells results in 1.5-fold FRET ratio increase, n = 60 cells from three independent experiments. (E) FRET ratio increases 1.08 times when 1 mM Ca 2+ is added to D1cpv-px expressing cells, n = 33 cells from three experiments. (F) Ca 2+ addition leads to 1.5-fold increase in 505/420 ratio with Pericam-px, n = 75 cells from three experiments. (G) Measurement of D3cpV-px during cytosol whashout. No change in signal is detected. (H) Measurement of pericam-px during cytosol whashout. No difference of signal before and after cytosol washout is detected, n = 43 cells for D3cpV-px in (G) and n = 45 cells for pericam-px in (H).

    Article Snippet: DNA constructsD3cpV-px (PST 1738) was generated from (pcDNA-)D3cpV (kind gift from A. Palmer and R. Tsien ( ) (Addgene #36323)) by amplifying an insert with OST 1599 (GCGCATCGAT GGTGATGGCC AAGTAAACTA TGAAGAG) and OST 1600 (GCGCGAATTC TTAGAGCTTC GATTTCAGAC TTCCCTCGA) primers.

    Techniques: Modification, Chick Chorioallantoic Membrane Assay, Binding Assay, Concentration Assay, Marker, Transfection, Staining, Expressing

    Measurement of peroxisomal Ca 2+ in HeLa cells. (A) Comparison of cytosolic and peroxisomal responces to ionomycin (Iono). In comparison to cytosol, peroxisomal signal increases gradually, n = 16 cells for D3cpV and n = 9 cells for D3cpV-px. (B) Experimental paradigm of a two-step Ca 2+ measurement in non-excitable cells. 1 st peak after histamine (His) addition: ER-store depletion. 2 nd peak, after addition of extracellular Ca 2+ : PM-based uptake. (C) Measurement with D3cpV-px according to the paradigm in (B). Two Ca 2+ peaks of the experimental paradigm are detectable with D3cpV-px, n ≥ 50 cells from three experiments. (D) Absolute Ca 2+ concentration dynamics calculated from the data in (C). (E) Basal and maximum (max) Ca 2+ concentrations in peroxisomes based on (C). (F) Measurement with pericam-px according to the paradigm in (B), n = 27 cells from three experiments. (G) Simultaneous measurement of cytosolic (blue) and peroxisomal (green) Ca 2+ . No delay of signal increase after histamine addition, but a delayed drop of the signal in peroxisomes. Left y axis: of D3cpV-px (peroxisomal sensor). Right y axis: F n /F 0 ratio of R-GECO1 (cytosolic sensor), n = 35 cells from three experiments. (H) Decline of F n /F 0 ratio per millisecond (ms) in the linear part of the curves in (G) (from second 65 to 115, Student’s t-test). Kinetic delay in decrease in peroxisomal signal is seen. (I) Comparison of cytosolic, mitochondrial and peroxisomal Ca 2+ response measured following the paradigm in (B). Characteristic two peaks present in all three compartments. (J) Basal levels of Ca 2+ in peroxisomes are similar to mitochondria. Analysis performed based on the data from (I). (K) Peroxisomal Ca 2+ increase upon ER-store depletion is smaller than that of cytosol or mitochondria. Analysis performed based on the data from (I). (L) Peroxisomal Ca 2+ increase upon PM-based cellular uptake of Ca 2+ is comparable to mitochondria. Analysis performed based on the data from (I). (A, C, D, F) Data presented as mean ± SEM. (J-L) One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. ***p

    Journal: bioRxiv

    Article Title: Peroxisomes contribute to intracellular calcium dynamics

    doi: 10.1101/2020.09.02.279174

    Figure Lengend Snippet: Measurement of peroxisomal Ca 2+ in HeLa cells. (A) Comparison of cytosolic and peroxisomal responces to ionomycin (Iono). In comparison to cytosol, peroxisomal signal increases gradually, n = 16 cells for D3cpV and n = 9 cells for D3cpV-px. (B) Experimental paradigm of a two-step Ca 2+ measurement in non-excitable cells. 1 st peak after histamine (His) addition: ER-store depletion. 2 nd peak, after addition of extracellular Ca 2+ : PM-based uptake. (C) Measurement with D3cpV-px according to the paradigm in (B). Two Ca 2+ peaks of the experimental paradigm are detectable with D3cpV-px, n ≥ 50 cells from three experiments. (D) Absolute Ca 2+ concentration dynamics calculated from the data in (C). (E) Basal and maximum (max) Ca 2+ concentrations in peroxisomes based on (C). (F) Measurement with pericam-px according to the paradigm in (B), n = 27 cells from three experiments. (G) Simultaneous measurement of cytosolic (blue) and peroxisomal (green) Ca 2+ . No delay of signal increase after histamine addition, but a delayed drop of the signal in peroxisomes. Left y axis: of D3cpV-px (peroxisomal sensor). Right y axis: F n /F 0 ratio of R-GECO1 (cytosolic sensor), n = 35 cells from three experiments. (H) Decline of F n /F 0 ratio per millisecond (ms) in the linear part of the curves in (G) (from second 65 to 115, Student’s t-test). Kinetic delay in decrease in peroxisomal signal is seen. (I) Comparison of cytosolic, mitochondrial and peroxisomal Ca 2+ response measured following the paradigm in (B). Characteristic two peaks present in all three compartments. (J) Basal levels of Ca 2+ in peroxisomes are similar to mitochondria. Analysis performed based on the data from (I). (K) Peroxisomal Ca 2+ increase upon ER-store depletion is smaller than that of cytosol or mitochondria. Analysis performed based on the data from (I). (L) Peroxisomal Ca 2+ increase upon PM-based cellular uptake of Ca 2+ is comparable to mitochondria. Analysis performed based on the data from (I). (A, C, D, F) Data presented as mean ± SEM. (J-L) One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. ***p

    Article Snippet: DNA constructsD3cpV-px (PST 1738) was generated from (pcDNA-)D3cpV (kind gift from A. Palmer and R. Tsien ( ) (Addgene #36323)) by amplifying an insert with OST 1599 (GCGCATCGAT GGTGATGGCC AAGTAAACTA TGAAGAG) and OST 1600 (GCGCGAATTC TTAGAGCTTC GATTTCAGAC TTCCCTCGA) primers.

    Techniques: Concentration Assay