Journal: Frontiers in Pharmacology

Article Title: A peptide derived from TID1S rescues frataxin deficiency and mitochondrial defects in FRDA cellular models

doi: 10.3389/fphar.2024.1352311

Figure Lengend Snippet: TID1 physically interacts with frataxin both in vivo in mouse cortex and in vitro in cortical neurons. In both cortical homogenates (A) and neuronal lysates (B) , frataxin was immunoprecipitated by a TID1L/S antibody but not control IgG. Similarly, a specific TID1L antibody but not control IgG immunoprecipitated frataxin from cortical homogenates (C) and HEK293 cells (D) but to a much lesser extent. In an in vitro binding assay, purified glutathione S- transferase (GST)-frataxin but not purified GST pulled down TID1L (E) . In HEK293 cells transfected with frataxin-HA and TID1S-Flag, immunofluorescence was performed using anti-HA and anti-Flag antibodies. Frataxin colocalized with TID1S (F) . Frataxin also colocalized with endogenously expressed TID1L stained with an anti-TID1L antibody (F) , further supported the interaction between TID1 and frataxin.

Article Snippet: After 4 washes, 2 μg human TID1L fused to a C-terminal Myc/DDK tag (Origene, Rockville, MD, Catalog #TP315039) was added and incubated overnight at 4°C.

Techniques: In Vivo, In Vitro, Immunoprecipitation, Control, Binding Assay, Purification, Transfection, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: A peptide derived from TID1S rescues frataxin deficiency and mitochondrial defects in FRDA cellular models

doi: 10.3389/fphar.2024.1352311

Figure Lengend Snippet: TID1L protein levels are reduced in FRDA patient-derived cells. FRDA patient buccal cells, skin fibroblasts, platelets or PBMCs were lysed and subjected to Western blotting with the indicated antibodies. The amount of immunoreactivity in the lysates was quantified as a percentage of the controls. Representative blots and bar graphs demonstrate reduced frataxin and TID1L in buccal cells (A,B) ( n = 14 for controls and n = 16 for patients), platelets (C,D) ( n = 16 for controls and n = 17 for patients) and fibroblasts (G,H) ( n = 15 for controls and n = 20 for patients). While frataxin was significantly reduced in PBMCs, no change in TID1L was detected in PBMCs (E,F) ( n = 18 for both controls and patients). ∗ p < 0.05; ∗∗ p < 0.01. Data were shown as mean ± SE.

Article Snippet: After 4 washes, 2 μg human TID1L fused to a C-terminal Myc/DDK tag (Origene, Rockville, MD, Catalog #TP315039) was added and incubated overnight at 4°C.

Techniques: Derivative Assay, Western Blot

Journal: Frontiers in Pharmacology

Article Title: A peptide derived from TID1S rescues frataxin deficiency and mitochondrial defects in FRDA cellular models

doi: 10.3389/fphar.2024.1352311

Figure Lengend Snippet: Effect of TID1 overexpression on frataxin protein levels. HEK293 cells transfected with frataxin and TID1 plasmid DNAs were lysed and subjected to Western blotting with the indicated antibodies. The amount of immunoreactivity in the lysates was quantified as a percentage of vector control. Representative blots and bar graphs demonstrate increased frataxin precursor and decreased intermediate and mature frataxin following TID1L or TID1S overexpression (A,B) ( n = 8). TID1L H121Q and TID1S H121Q had similar effects as wildtype TID1L and TID1S (A,B) ( n = 6), respectively. No change was found in GRP75 levels upon TID1L or TID1S overexpression (C) . ∗ p < 0.05, ∗∗ p < 0.01. Data were shown as mean ± SE.

Article Snippet: After 4 washes, 2 μg human TID1L fused to a C-terminal Myc/DDK tag (Origene, Rockville, MD, Catalog #TP315039) was added and incubated overnight at 4°C.

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: A peptide derived from TID1S rescues frataxin deficiency and mitochondrial defects in FRDA cellular models

doi: 10.3389/fphar.2024.1352311

Figure Lengend Snippet: Mechanistic study of TID1 overexpression-caused changes in frataxin levels. HEK293 cells transfected with frataxin and TID1 plasmid DNAs were subject to mitochondria fractionation followed by Western blotting analysis. Frataxin precursor is predominantly localized in the mitochondrial fraction upon TID1L or TID1S overexpression (A) . Tom20 was used as a mitochondrial marker (A) . Treatment with MG132 (10 μM) also had no effect on TID1L or TID1S overexpression-caused decrease in intermediate and mature frataxin (B) . Frataxin G130V mutant transfected HEK293 cells were used as a positive control (B) . Neither TID1L nor TID1S interacted with MPP, in contrast to GRP75 (C) .

Article Snippet: After 4 washes, 2 μg human TID1L fused to a C-terminal Myc/DDK tag (Origene, Rockville, MD, Catalog #TP315039) was added and incubated overnight at 4°C.

Techniques: Over Expression, Transfection, Plasmid Preparation, Fractionation, Western Blot, Marker, Mutagenesis, Positive Control

Journal: Frontiers in Pharmacology

Article Title: A peptide derived from TID1S rescues frataxin deficiency and mitochondrial defects in FRDA cellular models

doi: 10.3389/fphar.2024.1352311

Figure Lengend Snippet: TID1S overexpression decreases mature frataxin in human skin fibroblasts. Human skin fibroblasts from healthy individuals were transduced with lentivirus carrying pHAGE-TID1S gene or vector control for 5 days before Western blotting or immunofluorescence. TID1S transduction decreased mature frataxin (A, B) and ATP levels (C) ( n = 5). TID1S transduction also caused mitochondria fractionation in fibroblasts (D) . Vector control was stained with an anti-GFP antibody and TID1S was stained with an anti-TID1L/S antibody. ∗ p < 0.05, ∗∗ p < 0.01. Data were shown as mean ± SE.

Article Snippet: After 4 washes, 2 μg human TID1L fused to a C-terminal Myc/DDK tag (Origene, Rockville, MD, Catalog #TP315039) was added and incubated overnight at 4°C.

Techniques: Over Expression, Transduction, Plasmid Preparation, Control, Western Blot, Immunofluorescence, Fractionation, Staining

Journal: Cancer research

Article Title: Genome-wide RNAi Screen identifies PMPCB as a therapeutic vulnerability in EpCAM+ hepatocellular carcinoma

doi: 10.1158/0008-5472.CAN-18-3015

Figure Lengend Snippet: The functional effect of PMPCB knockdown in HUH7 cells. (A) The relative expression level of PMPCB and EpCAM genes with two independent PMPCB shRNAs. eGFP shRNA was used as a control. (B) Flow cytometer analysis of HUH7 cells stained with anti-EpCAM antibody 5 days after the expression of each shRNA. (C) The number of colonies cultured for 10 days after two independent PMPCB shRNAs were induced. (D) The number of spheroids cultured for 14 days after two independent PMPCB shRNAs were induced. (E) The proliferation curves of cells with eGFP shRNA and PMPCB shRNA were plotted. (F) Flow cytometry analyses of CD133 and CD90 in HUH7 cells after 72 hr of PMPCB shRNA. The proportion of each marker is shown. (G) Oxygen consumption in the cells with eGFP shRNA and PMPCB shRNA under basal conditions, following the addition of oligomycin (2.5μM), FCCP or antimycin A and Rotenone. (H) Relative ATP level of the cells with PMPCB shRNA. (I) DCFDA level was measured to see the ROS activity in the cells with PMPCB shRNA compared with the control cells. All graphs are represented as mean±SD (triplicates). *p<0.05, student’s t-test between sheGFP vs. shPMPCB-1 or shPMPCB-2.

Article Snippet: Taqman Gene Expression Assays (Life Technologies) including primers and a probe specific for each gene used for the analyses were as follows: PMPCB, Hs00188704_m1; EpCAM, Hs00901885_m1; MYC, Hs00153408_m1; Cyclin D1, Hs00765553_m1.

Techniques: Functional Assay, Knockdown, Expressing, shRNA, Control, Flow Cytometry, Staining, Cell Culture, Marker, Activity Assay

Journal: Cancer research

Article Title: Genome-wide RNAi Screen identifies PMPCB as a therapeutic vulnerability in EpCAM+ hepatocellular carcinoma

doi: 10.1158/0008-5472.CAN-18-3015

Figure Lengend Snippet: Apoptosis induced by PMPCB shRNA was dependent on EpCAM expression. (A) Flow cytometer analysis of EpCAM positive cells (HUH7 and HUH1) and negative cells (MHCC97) stained with anti-EpCAM antibody and 7-AAD 5 days after PMPCB shRNA transduction. (B) The proportion of 7-AAD positive cells in each cell line was shown. (C) The proportion of 7-AAD positive cells with eGFP shRNA or PMPCB shRNA treated with or without Z-VAD, caspase inhibitor in HUH7 cell line. All graphs are represented as mean±SD (triplicates). *p<0.05, student’s t-test.

Article Snippet: Taqman Gene Expression Assays (Life Technologies) including primers and a probe specific for each gene used for the analyses were as follows: PMPCB, Hs00188704_m1; EpCAM, Hs00901885_m1; MYC, Hs00153408_m1; Cyclin D1, Hs00765553_m1.

Techniques: shRNA, Expressing, Flow Cytometry, Staining, Transduction

Journal: Cancer research

Article Title: Genome-wide RNAi Screen identifies PMPCB as a therapeutic vulnerability in EpCAM+ hepatocellular carcinoma

doi: 10.1158/0008-5472.CAN-18-3015

Figure Lengend Snippet: In vivo tumor suppressive effect of PMPCB shRNA. (A) Live luminescence imaging of nude mice 4 weeks after the subcutaneous inoculation of HUH7-Luc cells with eGFP shRNA or shPMPCB (upper panels: n=5, cells were injected at both side of each mouse). Macroscopic images of tumors resected immediately after the live imaging (lower panels). (B) The average volume of the tumors resected. (C) Representative microscopic (hematoxylin and eosin stain) images of tumors with eGFP shRNA and shPMPCB (a,c:40×(scale bars: 200uM) and b,d:200× (scale bars are 20μM)). (D) TUNEL staining of tumors with eGFP shRNA (a,c) and shPMPCB (b,d). (a,b:100× (scale bars are 50uM); c,d:400× (scale bars are 20μM)). (E) Apoptotic index of tumors with eGFP shRNA or PMPCB shRNA. All graphs are represented as mean±SD (triplicates). *p<0.05, student’s t-test.

Article Snippet: Taqman Gene Expression Assays (Life Technologies) including primers and a probe specific for each gene used for the analyses were as follows: PMPCB, Hs00188704_m1; EpCAM, Hs00901885_m1; MYC, Hs00153408_m1; Cyclin D1, Hs00765553_m1.

Techniques: In Vivo, shRNA, Imaging, Injection, H&E Stain, TUNEL Assay, Staining

Journal: Cancer research

Article Title: Genome-wide RNAi Screen identifies PMPCB as a therapeutic vulnerability in EpCAM+ hepatocellular carcinoma

doi: 10.1158/0008-5472.CAN-18-3015

Figure Lengend Snippet: Role of PMPCB in Wnt/β-catenin pathway. (A) The relative TCF4 reporter activity on HUH7 cells (upper left). The relative mRNA expression of EpCAM (upper right), MYC (lower left) and CCDN1 (lower right) in HUH7 cells. All graphs are represented as mean±SD (triplicates). *p<0.05, student’s t-test. (B) Representative western blot analysis for the whole lysates of HUH7 and HUH1 cells. CTR: cells with eGFP shRNA, KD: cells with PMPCB shRNA. (C) Flow cytometer analysis of HUH7 cells with eGFP shRNA (upper left), PMPCB shRNA (lower left) or PMPCB shRNA with mutant CTNNB1 expression (upper right) stained with anti-EpCAM antibody and 7-AAD. The proportion of 7-AAD positive cells are shown as a bar graph (lower right). (D) Representative flow cytometer analysis of HUH7 cells with eGFP shRNA and MeBIO (upper left), eGFP shRNA and BIO (upper right), PMPCB shRNA and MeBIO (lower left) or PMPCB shRNA and BIO (lower right) stained with anti-EpCAM antibody and eFlour 520.

Article Snippet: Taqman Gene Expression Assays (Life Technologies) including primers and a probe specific for each gene used for the analyses were as follows: PMPCB, Hs00188704_m1; EpCAM, Hs00901885_m1; MYC, Hs00153408_m1; Cyclin D1, Hs00765553_m1.

Techniques: Activity Assay, Expressing, Western Blot, shRNA, Flow Cytometry, Mutagenesis, Staining

Journal: Cancer research

Article Title: Genome-wide RNAi Screen identifies PMPCB as a therapeutic vulnerability in EpCAM+ hepatocellular carcinoma

doi: 10.1158/0008-5472.CAN-18-3015

Figure Lengend Snippet: Wnt/β-catenin/FOXO pathway contributes to EpCAM+ dependency on PMPCB. (A-B) ProteinSimple analysis of FOXO1 and FOXO3A and (C) phospho_FOXO for the whole lysates, cytosol lysates and nuclear lysates of HUH7 and HepB3 cells. CTR: cells with eGFP shRNA, shPMPCB: cells with PMPCB shRNA. Represented as mean±SD of two independent Simple Western experiments. Chemiluminescence is of each protein is normalized to total protein.

Article Snippet: Taqman Gene Expression Assays (Life Technologies) including primers and a probe specific for each gene used for the analyses were as follows: PMPCB, Hs00188704_m1; EpCAM, Hs00901885_m1; MYC, Hs00153408_m1; Cyclin D1, Hs00765553_m1.

Techniques: shRNA, Simple Western