Table 1.
Primers used in qPCR.
Table 2.
siRNAs for silencing of gene expression in HK-2 cells.
Fig 1.
Identification of GADD45G interacting proteins using protein microarrays.
The microarray was treated with in the order of an Alexa-Fluor 635 nm (red) conjugated anti-GADD45G antibody, GADD45G protein, and an Alexa-Fluor 532 nm (green) conjugated anti-GADD45G antibody. Therefore, a protein spotted on the microarray that binds to GADD45G protein should be captured by an Alexa-Fluor 532 nm conjugated anti-GADD45G antibody and emits green color. The left panel shows a protein (duplicate) that emits green color which was identified as MAGEH1 (NP_054780). In contrast, GADD45G protein originally spotted in the microarray should be captured by both anti-GADD45G antibodies (Alexa-Fluor 635 nm and Alexa-Fluor 532 nm) and emits yellow color that combines red and green color (right panel).
Fig 2.
Co-immunoprecipitation to detect the interaction between GADD45G and MAGEH1.
(A) Detection of overexpressed GADD45G and HA-MAGEH1 protein by Western blot. HRE cells were infected with a serial amount of Ad-GADD45G and Ad-HA-MAGEH1 for 24 hours. GADD45G proteins were able to be detected at an MOI of 125 and above. HA-MAGEH1 proteins were able to be detected at an MOI of 63 and above by both anti-HA and anti-MAGEH1 antibodies. (B, C) HRE cells were infected with Ad-GADD45G and Ad-HA-MAGEH1 simultaneously at an MOI of 250 in the presence or absence of 25 μg/ml CsA to harvest cellular proteins 24 hours later. Co-immunoprecipitation (IP) studies were performed using lysates prepared from HRE cells overexpressing GADD45G and HA-MAGEH1 proteins, which were immunoprecipitated with a monoclonal anti-GADD45G antibody and then immunoblotted (IB) using an anti-HA antibody. For controls, cell lysates were subjected to IP using control IgG. (D, E) In a reciprocal fashion, proteins were immunoprecipitated with anti-HA antibody and then immunoblotted using an anti-GADD45G antibody. Ad-GADD45G represents adenoviral vectors expressing GADD45G; Ad-HA-MAGEH1, adenoviral vectors expressing hemagglutinin (HA)-tagged MAGEH1 adenoviral vectors; MOI, multiplicity of infection.
Fig 3.
Confocal laser scanning immunofluorescence analysis to detect the interaction between GADD45G and MAGEH1.
HRE cells were infected with Ad-GADD45G and Ad-HA-MAGEH1 simultaneously at an MOI of 250 for 24 hours. At the same time, HRE cells were not treated or treated with 25 μg/ml CsA for 24 hours and then subjected to immunofluorescent staining for the HA epitope (green) and GADD45G (red). In the merged image, yellow indicates costaining of the two proteins. DAPI staining (blue) was used to visualize the cell nucleus. The first row is showing cells not treated with CsA. The second row is showing cells treated with CsA. Scale bar: 10μm.
Fig 4.
Quantitative real-time PCR for mRNA expression in CsA-treated HRE cells.
(A) Time course of MAGEH1 gene expression in response to CsA treatment. HRE cells were incubated with 25 μg/ml CsA for the indicated time. n = 3 each. (B) Time course of GADD45G gene expression in response to CsA treatment. HRE cells were incubated with 25 μg/ml CsA for the indicated time. n = 3 each. (C) Silencing of MAGEH1 gene expression in sgMAGEH-HRE cells. n = 3 each. (D) Silencing of GADD45G gene expression in shGADD-HRE cells. n = 3 each. (E) Effect of silencing of MAGEH1 on GADD45G expression in sgMAGEH-HRE cells. n = 3 each. (F) Effect of silencing of GADD45G on MAGEH1 expression in shGADD-HRE cells. n = 3 each. Data were expressed as mean ± SD. One-way ANOVA followed by Bonferroni’s post-hoc comparisons tests (Panels A, B) or Student’s t-test (Panels C-F) was used to compare groups. A p value <0.05 was considered significant. #p<0.05 vs. all other groups; *p<0.05 between groups. GAPDH served as an internal control. sgMAGEH represents single guide RNA constructs to knockdown MAGEH1; sgCon, control single guide RNA constructs targeting no known genes; shGADD, short hairpin RNA constructs to knockdown GADD45G; shCon, control short hairpin RNA constructs targeting no known genes.
Fig 5.
Quantification of apoptosis and necrosis by flow cytometry in CsA-treated HRE cells: Effect of MAGEH1 knockdown.
sgMAGEH and sgCon stable HRE cells were incubated with 25 μg/ml CsA for the indicated time periods. (A) Representative scatter plots of flow cytometry. Quantification of (B) dead cells (Q1+Q2+Q4), (C) viable cells (PI negative and Annexin V negative, Q3), (D) necrotic/non-viable cells (PI positive and Annexin V negative, Q1) (E) late apoptotic/necrotic cells (PI positive and Annexin V positive, Q2), and (F) early apoptotic cells (PI negative and Annexin V positive, Q4). Each bar represents mean ± SD of 3 experiments. One-way ANOVA followed by Bonferroni’s post-hoc comparisons tests was used to compare groups. A p value <0.05 was considered significant. % p<0.05 vs. sgMAGEH; *p<0.05 vs. the corresponding sgCon group; #p<0.05 vs. all other corresponding groups. sgMAGEH represents single guide RNA constructs to knockdown MAGEH1; sgCon, control single guide RNA constructs targeting no known genes.
Fig 6.
Quantification of apoptosis and necrosis by flow cytometry in CsA-treated HRE cells: Effect of GADD45G knockdown.
shGADD and shCon stable HRE cells were treated with 25 μg/ml CsA for the indicated time periods. (A) Representative scatter plots of flow cytometry. Quantification of (B) dead cells (Q1+Q2+Q4), (C) viable cells (PI negative and Annexin V negative, Q3), (D) necrotic/non-viable cells (PI positive and Annexin V negative, Q1) (E) late apoptotic/necrotic cells (PI positive and Annexin V positive, Q2), and (F) early apoptotic cells (PI negative and Annexin V positive, Q4). Each bar represents mean ± SD of 3 experiments. One-way ANOVA followed by Bonferroni’s post-hoc comparisons tests was used to compare groups. A p value <0.05 was considered significant. #p<0.05 vs. all other corresponding groups; &p<0.05 vs. corresponding shCon/CsA groups; @p<0.05 vs. corresponding shGADD and shCon/CsA groups; $p<0.05 vs. corresponding shCon and shCon/CsA groups. shGADD represents shRNA constructs to knockdown GADD45G; shCon, control shRNA constructs targeting no known genes.
Fig 7.
siRNA induced gene silencing in HK-2 cells.
HK-2 cell line was transfected with siRNA for 24 h and then treated with 25 μg/ml CsA for 24 hours to measure mRNA expression using real-time PCR. (A) Transfection with MAGEH1 siRNA (siMAGEH) or control siRNA (siCon). n = 3 each. (B) Transfection with GADD45G siRNA (siGADD) or control siRNA (siCon). n = 3 each. GAPDH served as an internal control. Student’s t-test was used to compare groups. A p value <0.05 was considered significant. *p<0.05 between groups.
Fig 8.
Quantification of cell apoptosis and survival by flow cytometry in CsA-treated HK-2 cells: Effect of MAGEH1 knockdown.
HK-2 cell line was transfected with MAGEH1 siRNA or control siRNA. At 24 hours after transfection, cells were incubated with 12.5 μg/ml CsA for another 24 hours. (A) Representative scatter plots of flow cytometry. Quantification of (B) dead cells (Q1+Q2+Q4), (C) viable cells (PI negative and Annexin V negative, Q3), (D) necrotic/non-viable cells (PI positive and Annexin V negative, Q1) (E) late apoptotic/necrotic cells (PI positive and Annexin V positive, Q2), and (F) early apoptotic cells (PI negative and Annexin V positive, Q4). Each bar represents mean ± SD of 3 experiments. Statistical analysis was performed using One-way ANOVA followed by Bonferroni’s post-hoc comparisons tests. A p value <0.05 was considered significant. #p<0.05 vs. all other groups; &p<0.05 vs. siCon/CsA; ^p<0.05 vs. siMAGEH/CsA. siMAGEH stands for siRNA targeting MAGEH1; siCon, control siRNA targeting no known genes.
Fig 9.
Quantification of cell apoptosis and survival by flow cytometry in CsA-treated HK-2 cells: Effect of GADD45G knockdown.
HK-2 cell line was transfected with GADD45G siRNA or control siRNA. At 24 hours after transfection, cells were incubated with 12.5 μg/ml CsA for 24 another hours. (A) Representative scatter plots of flow cytometry. Quantification of (B) dead cells (Q1+Q2+Q4), (C) viable cells (PI negative and Annexin V negative, Q3), (D) necrotic/non-viable cells (PI positive and Annexin V negative, Q1) (E) late apoptotic/necrotic cells (PI positive and Annexin V positive, Q2), and (F) early apoptotic cells (PI negative and Annexin V positive, Q4). Each bar represents mean ± SD of 3 experiments. Statistical analysis was performed using One-way ANOVA followed by Bonferroni’s post-hoc comparisons tests. A p value <0.05 was considered significant. *p<0.05 vs. siCon; ^p<0.05 vs. siGADD/CsA; #p<0.05 vs. all other groups. siGADD stands for siRNA targeting GADD45G; siCon, control siRNA targeting no known genes.
Fig 10.
Western blot analysis of caspase activation in HRE cells: Effect of silencing of MAGEH1.
sgMAGEH and sgCon HRE stable cells were incubated with 25 μg/ml CSA for 24 and 48 hours to harvest protein for western blotting (A). Quantification of western blots (caspase 7, caspase 9, C-caspase 7, C-caspase 9) was performed using Image J at 24 hours (B), and 48 hours of incubation with CsA (C). C-caspases represent cleaved (active)-caspases; sgRNA MAGEH, single guide RNA constructs to knockdown MAGEH1; sgRNA Con, control single guide RNA constructs targeting no known genes.
Fig 11.
Western blot analysis of caspase activation in HRE cells: Effect of silencing of GADD45G.
shGADD and shCon HRE stable cells were incubated with 25 μg/ml CSA for 24 and 48 hours to harvest protein for western blotting. (A). Quantification of western blots (caspase 7, caspase 9, C-caspase 7, C-caspase 9) was performed using Image J at 24 hours (B), and 48 hours of incubation with CsA (C). C-caspases represent cleaved (active)-caspases; shRNA GADD, shRNA constructs to knockdown GADD45G; shRNA Con, control shRNA constructs targeting no known genes.
Fig 12.
Western blot analysis of caspase activation in HK-2 cells: Effect of silencing of MAGEH1.
HK-2 cells were transfected with siRNAs for 24 hours, and then treated with 12.5 μg/ml and 25 μg/ml CSA for 24 hours to harvest protein for western blotting (A). Quantification of western blots was performed using Image J for caspase 7 and caspase 9 (B), and C-caspase 7 and C-caspase 9 (C). C-caspases represent cleaved (active)-caspases; siRNA MAGEH, siRNA targeting MAGEH1; siRNA Con, control siRNA targeting no known genes.
Fig 13.
Western blot analysis of caspase activation in HK-2 cells: Effect of silencing of GADD45G.
HK-2 cells were transfected with siRNAs for 24 hours, and then treated with 12.5 μg/ml and 25 μg/ml CSA for 24 hours to harvest protein for western blotting (A). Quantification of western blots was performed using Image J for caspase 7 and caspase 9 (B), and C-caspase 7 and C-caspase 9 (C). C-caspases represent cleaved (active)-caspases; siRNA GADD, siRNA targeting GADD45G; siRNA Con, control siRNA targeting no known genes.