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( A ) Representative LysoSensor Yellow/Blue DND-160 fluorescence images showing lysosomal pH in Ctrl and cKO <t>primary</t> <t>RTECs</t> treated with HG + PA, with quantification. ( B ) Calculated lysosomal pH values in indicated group, derived from the LysoSensor Yellow/Blue fluorescence ratio using a calibration curve. ( C ) Measurement of lysosomal phospholipase A 2 (PLA 2 ), phospholipase C (PLC), and phospholipase D (PLD) activities in indicated group. ( D ) Immunoblot analysis of EGFR degradation following <t>EGF</t> stimulation in Ctrl and cKO primary RTECs under HG + PA conditions, with quantification. ( E and F ) Immunoblotting of cathepsin B (CTSB) maturation (E) and LC3 and p62 (F) in HG + PA-treated Ctrl and cKO RTECs, with quantification. ( G ) Cell viability assay of HG + PA-treated Ctrl and cKO RTECs. ( H ) Calculated lysosomal pH measurement in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( I and J ) Measurement of lysosomal phospholipase A2 activities (I) and cathepsin activities (J) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( K and L ) Immunofluorescence staining of Nile Red (K) or LipidTOX (L) (red) together with LAMP1 (green) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1, with corresponding quantitative analyses. ( M ) Cell viability analysis of HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. Data are presented as mean ± SEM. P values were determined by unpaired two-tailed Student’s t test for (A) to (G) and by one-way ANOVA followed by Tukey’s multiple-comparisons test for (H) to (M). Scale bar: 10 μm in (A), (K), and (L). n = 6 for (A), (K), and (L), n = 4 for (B) and (H), n = 3 for (C) to (F), (I), (J), and (M), n = 5 for (G). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.
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( A ) Representative LysoSensor Yellow/Blue DND-160 fluorescence images showing lysosomal pH in Ctrl and cKO <t>primary</t> <t>RTECs</t> treated with HG + PA, with quantification. ( B ) Calculated lysosomal pH values in indicated group, derived from the LysoSensor Yellow/Blue fluorescence ratio using a calibration curve. ( C ) Measurement of lysosomal phospholipase A 2 (PLA 2 ), phospholipase C (PLC), and phospholipase D (PLD) activities in indicated group. ( D ) Immunoblot analysis of EGFR degradation following <t>EGF</t> stimulation in Ctrl and cKO primary RTECs under HG + PA conditions, with quantification. ( E and F ) Immunoblotting of cathepsin B (CTSB) maturation (E) and LC3 and p62 (F) in HG + PA-treated Ctrl and cKO RTECs, with quantification. ( G ) Cell viability assay of HG + PA-treated Ctrl and cKO RTECs. ( H ) Calculated lysosomal pH measurement in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( I and J ) Measurement of lysosomal phospholipase A2 activities (I) and cathepsin activities (J) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( K and L ) Immunofluorescence staining of Nile Red (K) or LipidTOX (L) (red) together with LAMP1 (green) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1, with corresponding quantitative analyses. ( M ) Cell viability analysis of HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. Data are presented as mean ± SEM. P values were determined by unpaired two-tailed Student’s t test for (A) to (G) and by one-way ANOVA followed by Tukey’s multiple-comparisons test for (H) to (M). Scale bar: 10 μm in (A), (K), and (L). n = 6 for (A), (K), and (L), n = 4 for (B) and (H), n = 3 for (C) to (F), (I), (J), and (M), n = 5 for (G). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.
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( A ) Representative LysoSensor Yellow/Blue DND-160 fluorescence images showing lysosomal pH in Ctrl and cKO <t>primary</t> <t>RTECs</t> treated with HG + PA, with quantification. ( B ) Calculated lysosomal pH values in indicated group, derived from the LysoSensor Yellow/Blue fluorescence ratio using a calibration curve. ( C ) Measurement of lysosomal phospholipase A 2 (PLA 2 ), phospholipase C (PLC), and phospholipase D (PLD) activities in indicated group. ( D ) Immunoblot analysis of EGFR degradation following <t>EGF</t> stimulation in Ctrl and cKO primary RTECs under HG + PA conditions, with quantification. ( E and F ) Immunoblotting of cathepsin B (CTSB) maturation (E) and LC3 and p62 (F) in HG + PA-treated Ctrl and cKO RTECs, with quantification. ( G ) Cell viability assay of HG + PA-treated Ctrl and cKO RTECs. ( H ) Calculated lysosomal pH measurement in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( I and J ) Measurement of lysosomal phospholipase A2 activities (I) and cathepsin activities (J) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( K and L ) Immunofluorescence staining of Nile Red (K) or LipidTOX (L) (red) together with LAMP1 (green) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1, with corresponding quantitative analyses. ( M ) Cell viability analysis of HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. Data are presented as mean ± SEM. P values were determined by unpaired two-tailed Student’s t test for (A) to (G) and by one-way ANOVA followed by Tukey’s multiple-comparisons test for (H) to (M). Scale bar: 10 μm in (A), (K), and (L). n = 6 for (A), (K), and (L), n = 4 for (B) and (H), n = 3 for (C) to (F), (I), (J), and (M), n = 5 for (G). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.
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( A ) Representative LysoSensor Yellow/Blue DND-160 fluorescence images showing lysosomal pH in Ctrl and cKO <t>primary</t> <t>RTECs</t> treated with HG + PA, with quantification. ( B ) Calculated lysosomal pH values in indicated group, derived from the LysoSensor Yellow/Blue fluorescence ratio using a calibration curve. ( C ) Measurement of lysosomal phospholipase A 2 (PLA 2 ), phospholipase C (PLC), and phospholipase D (PLD) activities in indicated group. ( D ) Immunoblot analysis of EGFR degradation following <t>EGF</t> stimulation in Ctrl and cKO primary RTECs under HG + PA conditions, with quantification. ( E and F ) Immunoblotting of cathepsin B (CTSB) maturation (E) and LC3 and p62 (F) in HG + PA-treated Ctrl and cKO RTECs, with quantification. ( G ) Cell viability assay of HG + PA-treated Ctrl and cKO RTECs. ( H ) Calculated lysosomal pH measurement in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( I and J ) Measurement of lysosomal phospholipase A2 activities (I) and cathepsin activities (J) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( K and L ) Immunofluorescence staining of Nile Red (K) or LipidTOX (L) (red) together with LAMP1 (green) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1, with corresponding quantitative analyses. ( M ) Cell viability analysis of HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. Data are presented as mean ± SEM. P values were determined by unpaired two-tailed Student’s t test for (A) to (G) and by one-way ANOVA followed by Tukey’s multiple-comparisons test for (H) to (M). Scale bar: 10 μm in (A), (K), and (L). n = 6 for (A), (K), and (L), n = 4 for (B) and (H), n = 3 for (C) to (F), (I), (J), and (M), n = 5 for (G). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.
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( A ) Representative LysoSensor Yellow/Blue DND-160 fluorescence images showing lysosomal pH in Ctrl and cKO <t>primary</t> <t>RTECs</t> treated with HG + PA, with quantification. ( B ) Calculated lysosomal pH values in indicated group, derived from the LysoSensor Yellow/Blue fluorescence ratio using a calibration curve. ( C ) Measurement of lysosomal phospholipase A 2 (PLA 2 ), phospholipase C (PLC), and phospholipase D (PLD) activities in indicated group. ( D ) Immunoblot analysis of EGFR degradation following <t>EGF</t> stimulation in Ctrl and cKO primary RTECs under HG + PA conditions, with quantification. ( E and F ) Immunoblotting of cathepsin B (CTSB) maturation (E) and LC3 and p62 (F) in HG + PA-treated Ctrl and cKO RTECs, with quantification. ( G ) Cell viability assay of HG + PA-treated Ctrl and cKO RTECs. ( H ) Calculated lysosomal pH measurement in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( I and J ) Measurement of lysosomal phospholipase A2 activities (I) and cathepsin activities (J) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( K and L ) Immunofluorescence staining of Nile Red (K) or LipidTOX (L) (red) together with LAMP1 (green) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1, with corresponding quantitative analyses. ( M ) Cell viability analysis of HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. Data are presented as mean ± SEM. P values were determined by unpaired two-tailed Student’s t test for (A) to (G) and by one-way ANOVA followed by Tukey’s multiple-comparisons test for (H) to (M). Scale bar: 10 μm in (A), (K), and (L). n = 6 for (A), (K), and (L), n = 4 for (B) and (H), n = 3 for (C) to (F), (I), (J), and (M), n = 5 for (G). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.
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( A ) Representative LysoSensor Yellow/Blue DND-160 fluorescence images showing lysosomal pH in Ctrl and cKO primary RTECs treated with HG + PA, with quantification. ( B ) Calculated lysosomal pH values in indicated group, derived from the LysoSensor Yellow/Blue fluorescence ratio using a calibration curve. ( C ) Measurement of lysosomal phospholipase A 2 (PLA 2 ), phospholipase C (PLC), and phospholipase D (PLD) activities in indicated group. ( D ) Immunoblot analysis of EGFR degradation following EGF stimulation in Ctrl and cKO primary RTECs under HG + PA conditions, with quantification. ( E and F ) Immunoblotting of cathepsin B (CTSB) maturation (E) and LC3 and p62 (F) in HG + PA-treated Ctrl and cKO RTECs, with quantification. ( G ) Cell viability assay of HG + PA-treated Ctrl and cKO RTECs. ( H ) Calculated lysosomal pH measurement in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( I and J ) Measurement of lysosomal phospholipase A2 activities (I) and cathepsin activities (J) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( K and L ) Immunofluorescence staining of Nile Red (K) or LipidTOX (L) (red) together with LAMP1 (green) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1, with corresponding quantitative analyses. ( M ) Cell viability analysis of HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. Data are presented as mean ± SEM. P values were determined by unpaired two-tailed Student’s t test for (A) to (G) and by one-way ANOVA followed by Tukey’s multiple-comparisons test for (H) to (M). Scale bar: 10 μm in (A), (K), and (L). n = 6 for (A), (K), and (L), n = 4 for (B) and (H), n = 3 for (C) to (F), (I), (J), and (M), n = 5 for (G). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Journal: Science Advances

Article Title: Renoprotective effects of tubular glucagon receptor activation mediated by V-ATPase

doi: 10.1126/sciadv.aeg2534

Figure Lengend Snippet: ( A ) Representative LysoSensor Yellow/Blue DND-160 fluorescence images showing lysosomal pH in Ctrl and cKO primary RTECs treated with HG + PA, with quantification. ( B ) Calculated lysosomal pH values in indicated group, derived from the LysoSensor Yellow/Blue fluorescence ratio using a calibration curve. ( C ) Measurement of lysosomal phospholipase A 2 (PLA 2 ), phospholipase C (PLC), and phospholipase D (PLD) activities in indicated group. ( D ) Immunoblot analysis of EGFR degradation following EGF stimulation in Ctrl and cKO primary RTECs under HG + PA conditions, with quantification. ( E and F ) Immunoblotting of cathepsin B (CTSB) maturation (E) and LC3 and p62 (F) in HG + PA-treated Ctrl and cKO RTECs, with quantification. ( G ) Cell viability assay of HG + PA-treated Ctrl and cKO RTECs. ( H ) Calculated lysosomal pH measurement in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( I and J ) Measurement of lysosomal phospholipase A2 activities (I) and cathepsin activities (J) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. ( K and L ) Immunofluorescence staining of Nile Red (K) or LipidTOX (L) (red) together with LAMP1 (green) in HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1, with corresponding quantitative analyses. ( M ) Cell viability analysis of HG + PA-treated Ctrl and cKO RTECs following Forskolin treatment with or without BafA1. Data are presented as mean ± SEM. P values were determined by unpaired two-tailed Student’s t test for (A) to (G) and by one-way ANOVA followed by Tukey’s multiple-comparisons test for (H) to (M). Scale bar: 10 μm in (A), (K), and (L). n = 6 for (A), (K), and (L), n = 4 for (B) and (H), n = 3 for (C) to (F), (I), (J), and (M), n = 5 for (G). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: RTECs were serum-starved for 4 hours and stimulated with recombinant human EGF (100 ng/ml, MCE HY-P7109) for 0–120 min at 37°C.

Techniques: Fluorescence, Derivative Assay, Western Blot, Viability Assay, Immunofluorescence, Staining, Two Tailed Test