Phosphorylation of p38 MAPK (P-p38) and ERK1/2 induced by dbcAMP (100 µM) and hCG (10 IU/ml) in human placental syncytiotrophoblasts.

<p>Upper panels of each bar graph are the representative blots. The bar graphs are the average data of three experiments. *P<0.05, **P<0.01,***P<0.001 versus 0 min.</p

Primer sequences used for PCR.

<p>Primer sequences used for PCR.</p

(A) Effect of SB203580 (p38 MAPK inhibitor, 10 µM) on the basal and dbcAMP (100 µM) and hCG (10 IU/ml)-induced HSD11B2 protein level in human placental syncytiotrophoblasts.

<p>*p<0.05, **p<0.01, ***p<0.001 versus control; ## p<0.01 ### p<0.001 versus treatment with dbcAMP and hCG, n = 4. (B) Effect of SB203580 (p38 MAPK inhibitor, 10 µM) on the basal and dbcAMP, 100 µM) and hCG (10 IU/ml)-induced SP1 protein level in human placental syncytiotrophoblasts. ***p<0.001 vs control; ### p<0.001 versus treatment with dbcAMP and hCG (n = 4).</p

(A) Effect of PD98059(ERK1/2 MAPK inhibitor, 50 µM) on the basal and dbcAMP (100 µM) and hCG (10 IU/ml)-induced HSD11B2 mRNA expression in human placental syncytiotrophoblasts.

<p>*p<0.05, **p<0.01, ***p<0.001 versus control (n = 4) (B) Effect of PD98059 (ERK1/2 MAPK inhibitor, 50 µM) on the basal and dbcAMP(100 µM) and hCG (10 IU/ml)-induced SP1 mRNA expression in human placental syncytiotrophoblasts. *p<0.05, **p<0.01, ***p<0.001 versus control (n = 4).</p

(A) Effect of SB203580 (p38 MAPK inhibitor, 10 µM) on the basal and dbcAMP (100 µM) and hCG (10 IU/ml)-induced HSD11B2 mRNA expression in human placental syncytiotrophoblasts.

<p>*p<0.05, **p<0.01, ***p<0.001 versus control; ## p<0.01 ### p<0.001 versus treatment with db-cAMP and hCG n = 4; (B) Effect of SB203580 (p38 MAPK inhibitor, 10 µM) on the basal and dbcAMP (db cAMP, 100 µM) and hCG (10 IU/ml)-induced SP1 mRNA expression in human placental syncytiotrophoblasts. *p<0.05, **p<0.01, ***p<0.001 versus control;# p<0.05, ## p<0.01 versus treatment with dbcAMP and hCG (n = 4).</p

LRPPRC maintains levels of Bcl-2 and suppresses basal levels of autophagy through Parkin.

<p>(<b>A</b>) Immunoblot analyses of Bcl-2 levels in 293T cells overexpressing Parkin in the absence or presence of LRPPRC siRNA. (<b>B</b>) Plots of relative intensities of Bcl-2 as shown in (<b>A</b>). The intensities in samples overexpressing GFP were set to 1. Data were the average and standard deviation of at least three repeats and the differences were compared based on paired T-test. *, p value ≤0.05. (<b>C</b>) Immunoblot analyses of LC3-II levels in 293T cells treated with either random or LRPPRC-specific siRNAs and/or overexpressing GFP or GFP-Parkin in the presence of Bafilomycin A1. (<b>D</b>) Plots of relative intensities of LC3-II as shown in (<b>C</b>). The intensities in samples treated with random siRNA and overexpressing GFP were set to 1. Data were the average and standard deviation of at least three repeats and the differences were compared based on paired T-test. *, p value ≤0.05.</p

LRPPRC controls the stability of Parkin.

<p>(<b>A</b>–<b>D</b>) Immunoblot analyses showing the impact of different levels of Parkin on the levels of LRPPRC. Equal amount of cell lysates from 293T cells treated with random (Mock) or Parkin-specific siRNA (<b>A</b>) or HeLa cells overexpressing GFP or GFP-Parkin (<b>C</b>) were analyzed by immunoblot and LRPPRC levels under different conditions were quantified (B,D). n.s., not significant. (<b>E</b>) Immunoblot analyses showing the impact of different levels of Parkin on the levels of LRPPRC under mitophagy stress. Equal amount of cell lysates from HeLa cells overexpressing GFP or GFP-Parkin treated with CCCP for different times were analyzed by immunoblot. (<b>F,G</b>) Plots of relative intensities of LRPPRC (<b>E</b>) or Tom20 (<b>F</b>) in HeLa cells treated as in (<b>E</b>). The intensities in samples at time zero were set to 1. (<b>H</b>) An immunoblot analysis showing that suppression of LRPPRC resulted in degradation of Parkin. 293T cells were treated with Mock or LRPPRC siRNA for 72 hrs. Equal amount of lysates were analyzed. (<b>I,J</b>) Plots of relative intensities of LRPPRC (<b>I</b>) or Parkin (<b>J</b>) in 293T cells treated with Mock or LRPPRC siRNA. The intensities in samples treated with MOCK siRNA were set to 1. Data were the average and standard deviation of at least three repeats and the differences were compared based on paired T-test. *, p value ≤0.05; ***, p value ≤0.0001. (<b>K</b>) Immunoblot analyses showing that overexpression of LRPPRC enhanced the stability of Parkin. COS7 cells were transiently transfected with plasmids carry only GFP or GFP-LRPPRC for 24 hrs, detached and distributed equally to 8 wells. Each well was treated with cycloheximide (CHX) for different times (hrs). Equal amount of lysates as indicated by total protein concentration and β-actin control were analyzed. (<b>L</b>) Plots of relative intensities of Parkin in COS7 cells treated as in (<b>K</b>). The intensities in samples at time zero were set to 1.</p

Colocalization among Parkin, Tom20-indicated mitochondria and RFP-LC3-labelled autophagosomes at different times under mitophagy stress.

<p>HeLa cells stably expressing RFP-LC3 transiently expressed GFP-Parkin treated with 10 µM CCCP for different times were fixed and stained with antibodies against Tom20 (blue). Mitochondrial aggregates associating with Parkin signals were shown as cyan in the Merge panels. Bar = 5 µm.</p

LRPPRC interacts with mitophagy stress-induced mitochondrion-translocated mitophagy initiator Parkin.

<p>(<b>A</b>) Immunoblot analyses of interaction between LRPPRC and Parkin or Pink 1. Same amount of 293T cell lysates were used to perform immunoprecipitation with same amount of anti-LRPPRC antibody or mouse IgG control under identical procedure. (<b>B</b>–<b>D</b>) Coimmunoprecipitation analyses of LRPPRC-Parkin interaction under mitophagy stress. Lysates with equal amount of total proteins prepared from 293T cells untreated or similarly treated with 10 µM CCCP for 2.5 hrs were immunoprecipitated with anti-LRPPRC or Parkin antibody (<b>B</b>). IgG served as control antibody. The relative amounts of Parkin bound on LRPPRC (<b>C</b>) or LRPPRC bound on Parkin (<b>D</b>) were quantified against the precipitated amounts of LRPPRC (<b>C</b>) or Parkin (<b>D</b>). The level in the absence of CCCP is set to 1. **, p value ≤0.01. (<b>E</b>) A fluorescent imaging analysis showing the colocalization of LRPPRC with GFP-Parkin. CCCP, HeLa cells transiently expressing GFP-Parkin for 48 hrs were treated with 10 µM CCCP for 2.5 hrs before fixation. Bottom panel is the amplification of the square in the middle panel. Bar = 10 µm. (<b>F,G</b>) Coimmunoprecipitation analyses of LRPPRC-Parkin interaction in HeLa cells overexpressing GFP-Parkin untreated or similarly treated with CCCP as shown in (<b>E</b>). GFP-Parkin is coimmunoprecipitated with anti-LRPPRC antibody (<b>F</b>), and the relative amounts of GFP-Parkin bound on LRPPRC are quantified against the precipitated amounts of LRPPRC (<b>G</b>). The level in the absence of CCCP is set to 1. **, p value ≤0.01. (<b>H,I</b>) Fluorescent imaging analyses showing the colocalization of GFP-Parkin and LRPPRC with RFP-LC3 punctate foci (<b>H</b>) or LAMP2-labelled lysosomes (<b>I</b>) 3 or 12 hrs after exposure to CCCP in the absence (Ctrl) or presence of Bafilomycin A1 (BAF). Bar = 5 µm in (<b>H</b>) and 2 µm in (<b>I</b>).</p

Colocalization among Parkin, Tom20-indicated mitochondria and LAMP2-labelled lysosomes at different times under mitophagy stress.

<p>HeLa cells transiently expressing GFP-Parkin treated with 10 µM CCCP for different times were fixed and stained with antibodies against LAMP2 (red) and Tom20 (blue). Lysosome-contained Parkin-associated mitochondrial aggregates were shown as white in the Merge panels. All experiments were carried out in the absence of lysosomal inhibitor. Bar = 2 µm.</p