Fig 1: Depletion of ADRM1 impairs mitochondrial functions and leads to mitochondrial fragmentation. STHdhQ7/Q7 striatal cells were transfected with pLKO.ADRM1 #1, pLKO.ADRM1 #2, or pLKO.Luc shRNA construct. pLKO.Luc that targeted luciferase gene was used as a negative control. (A) Flow cytometric analysis of mitochondrial membrane potential by JC-1 staining in STHdhQ7/Q7 striatal cells depleted for ADRM1. Quantification of the JC-1 associated red/green fluorescent intensity ratio in STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. The ratio of red to green JC-1 signals of cells expressing control pLKO.Luc. was arbitrarily set to be 1. (B) The ATP content of the STHdhQ7/Q7 striatal cells transfected the indicated plasmids. The ATP content of cells expressing control pLKO.Luc. was arbitrarily set to be 1. (C) The total cell number of the STHdhQ7/Q7 striatal cells transfected with the indicated plasmids were measured by the MTT assay. The percentage number of cells expressing pLKO.Luc only was arbitrarily set to be 100%. (D) The normalized ATP content of STHdhQ7/Q7 striatal cells transfected with the pLKO.ADRM1 #1, pLKO.ADRM1 #2, or pLKO.Luc plasmid. (E) Flow cytometric analysis of ROS accumulation in STHdhQ7/Q7 striatal cells depleted for ADRM1. Quantification of percentage of the cells with positive ROS in STHdhQ7/Q7 striatal cells transfected with indicated plasmids. (F) Quantification of percentage of the cells with different mitochondrial morphology in STHdhQ7/Q7 striatal cells transfected with indicated plasmids. Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences with P < 0.05 are labeled with one asterisk (*); with P < 0.01 are labeled with two asterisks (**); N.S., no significance.
Fig 2: Overexpression of ADRM1 restores mitochondrial functions and reduces HAP40-induced mitochondrial fragmentation. (A) Immunoblot detection for HAP40 and ADRM1 in STHdhQ7/Q7 striatal cells transfected with the FLAG or FLAG-HAP40 plasmid with or without the FLAG-ADRM1 plasmid. Tubulin was used as an internal control for protein loading. (B) Flow cytometric analysis of mitochondrial membrane potential by JC-1 staining in STHdhQ7/Q7 striatal cells in the presence of FLAG or FLAG-ADRM1 plasmids. Quantification of the JC-1 associated red/green fluorescent intensity ratio in STHdhQ7/Q7 striatal cells transfected with indicated plasmids. The ratio of red to green JC-1 signals of cells expressing FLAG only was arbitrarily set to be 1. (C) Flow cytometric analysis of mitochondrial membrane potential by JC-1 in STHdhQ7/Q7 striatal cells in the presence of FLAG-HAP40 and FLAG or FLAG-ADRM1 plasmids. Quantification of the JC-1 associated red/green fluorescent intensity ratio in STHdhQ7/Q7 striatal cells transfected with the FLAG-HAP40 and FLAG or FLAG-ADRM1 plasmids. The ratio of red to green JC-1 signals of cells co-expressing FLAG and FLAG-HAP40 was arbitrarily set to be 1. (D) The cellular ATP content of STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. The ATP content was measured 48 hrs after transfection. The ATP content of cells co-expressing FLAG and HAP40-mCherry was arbitrarily set to be 1. (E) The total cell number of the STHdhQ7/Q7 striatal cells transfected with the indicated plasmids was measured by the MTT assay. Cell number was measured 48 hours after transfection. The percentage number of cells co-expressing FLAG and HAP40-mCherry was arbitrarily set to be 100%. (F) The normalized ATP content of STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. (G) Flow cytometric analysis of ROS accumulation in STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. Quantification of percentage of the cells with positive ROS in STHdhQ7/Q7 striatal cells transfected with indicated plasmids. (H) Quantification of percentage of the cells with different mitochondrial morphology in STHdhQ7/Q7 striatal cells co-transfected with the mito-eGFP and FLAG or FLAG-ADRM1 plasmids. (I) Quantification of percentage of the cells with different mitochondrial morphology in STHdhQ7/Q7 striatal cells co-transfected with the mito-eGFP and FLAG-HAP40 plasmids in the presence or absence of the FLAG or FLAG-ADRM1 plasmid. Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences with P < 0.05 are labeled with one asterisk (*); N.S., no significance.
Fig 3: Mdivi-1 treatment relieves mitochondrial dysfunction caused by down-regulation of ADRM1. Quantification of percentage of the cells with different mitochondrial morphology in STHdhQ7/Q7 striatal cells co-transfected with the mito-eGFP and pLKO.ADRM1 #1 (A) or pLKO.ADRM1 #2 (B) shRNA construct in the presence or absence of the Mdivi-1 treatment. (C) Quantification of JC-1 associated red/green fluorescent intensity ratio in STHdhQ7/Q7striatal cells transfected with pLKO.ADRM1 #1, pLKO.ADRM1 #2, or pLKO.Luc shRNA construct with or without Mdivi-1. (D) Flow cytometric analysis of ROS accumulation in STHdhQ7/Q7 striatal cells depleted for ADRM1 with or without Mdivi-1. Quantification of percentage of the cells with positive ROS in STHdhQ7/Q7 striatal cells transfected with indicated plasmids. Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences with P < 0.05 are labeled with one asterisk (*); N.S., no significance.
Fig 4: Reducing ADRM1 increases the phosphorylation state of Drp1. (A) Immunoblot detection for ADRM1, Parkin, p-Drp1Ser616, Drp1, Mfn1, and tubulin in STHdhQ7/Q7 striatal cells transfected with the pLKO.ADRM1 #1, pLKO.ADRM1 #2, or pLKO.Luc. (B-E and G) Quantification analyses on protein levels with the indicated protein normalized to the tubulin. (F) Quantification analyses on phosphorylated Drp1Ser616 normalized to total Drp1 protein in STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences with P < 0.05 are labeled with one asterisk (*); with P < 0.01 are labeled with two asterisks (**); N.S., no significance.
Fig 5: Overexpression of HAP40 increases the level and phosphorylation state of Drp1. (A) Immunoblot detection for Mfn1, Mfn2, OPA1, Drp1, p-Drp1Ser616, Fis1, PHB1, Parkin, PINK1, ADRM1, GAPDH, and tubulin in STHdhQ7/Q7 striatal cells transfected with the FLAG or FLAG-HAP40 plasmid. (B-K) Quantification analyses on protein levels with the indicated protein normalized to the tubulin (B-H) or GAPDH (I-K). Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences compared with the control (P < 0.05) are labeled with asterisk (*); N.S., no significance.
Supplier Page from Abcam for Anti-ADRM1/ARM-1 antibody [EPR11450(B)]