Journal:

Article Title: Sirt3 promotes the urea cycle and fatty acid oxidation during dietary restriction

doi: 10.1016/j.molcel.2011.01.002

Figure Lengend Snippet: A.) Steady-state kinetic analysis of Sirt3-dependent deacetylation of acetylated OTC-K88 peptide. Sirt3 (filled circles) demonstrates substrate saturation kinetics. No significant activity was observed with Sirt5 (filled boxes) (n=3). B.) Sirt3, but not Sirt5, deacetylates OTC in vitro. Acetylated OTC was prepared as outlined in Experimental Procedure, and was incubated with purified recombinant Sirt3 37 °C or Sirt5 with or without NAD+ at 37 °C for 1 hour. Acetylation status was assessed by western blotting with anti-acetyl-lysine antibody. An anti-MYC western shows equivalent OTC protein levels were used and anti-HIS shows purified Sirt3 and Sirt5. C.) shRNA knockdown of Sirt3 and increase in OTC acetylation levels. HEK293 cells were transiently transfected with control or shRNA 1# or 2# for knockdown Sirt3, cell lysates were analyzed by western blotting with anti-actin or anti-Sirt3 antibodies. D.) OTC was Overexpressed in HEK293 cells of Sirt3 shRNA 2# and cell lysates were analyzed by western blotting with FLAG antibody or anti-acetyl-lysine antibody.

Article Snippet: Mitochondrial lysates from wild type and sirt3 −/− mice were performed with anti-Sirt3 antibody (gift of Dr. Eric Verdin, UCSF) or anti-OTC antibody (Aviva Systems Biology; 1:500) overnight at 4°C, then added with Protein A/G beads for 4h followed by western blotting using anti-acetyl-lysine antibody (generated following the procedure of Zhao et al., 2010 , GeneTel Laboratories LLC, Madison, WI).

Techniques: Activity Assay, In Vitro, Incubation, Purification, Recombinant, Western Blot, shRNA, Knockdown, Transfection, Control

Journal:

Article Title: Sirt3 promotes the urea cycle and fatty acid oxidation during dietary restriction

doi: 10.1016/j.molcel.2011.01.002

Figure Lengend Snippet: A.) Immunoprecipitation of OTC detects Sirt3 when both OTC and Sirt3 are coexpressed in HEK293 cells. B) Immunoprecipitation of Sirt3 detects OTC when both OTC and Sirt3 are coexpressed in HEK293 cells. C.) Sirt3 deacetylates K88 of OTC when coexpressed in HEK293 cells. D.) Sirt3 catalytic activity is necessary for deacetylation of OTC. OTC was cotransfected with Sirt3 or H248Y mutant of Sirt3 in HEK293, isolated by immunoprecipitation with anti-FLAG antibody followed by western blotting with anti-acetyl-lysine antibody. E-F.) OTC activity is stimulated by Sirt3-dependent deacetylation. Proteins from lysates were immunoprecipitated with anti-FLAG and anti-MYC antibodies and were analyzed by western blotting with anti-FLAG, anti-MYC or anti-acetyl-lysine antibodies (E). A significant stimulation of OTC activity was observed with Sirt3, but not with Sirt4 and Sirt5 (F). Error bars represent standard error measurement (SEM) (n = 3), * signifies a p < 0.05.

Article Snippet: Mitochondrial lysates from wild type and sirt3 −/− mice were performed with anti-Sirt3 antibody (gift of Dr. Eric Verdin, UCSF) or anti-OTC antibody (Aviva Systems Biology; 1:500) overnight at 4°C, then added with Protein A/G beads for 4h followed by western blotting using anti-acetyl-lysine antibody (generated following the procedure of Zhao et al., 2010 , GeneTel Laboratories LLC, Madison, WI).

Techniques: Immunoprecipitation, Activity Assay, Mutagenesis, Isolation, Western Blot

Journal:

Article Title: Sirt3 promotes the urea cycle and fatty acid oxidation during dietary restriction

doi: 10.1016/j.molcel.2011.01.002

Figure Lengend Snippet: (A) Top panels: Western blot analysis of Sirt3 and OTC levels in livers from 5-month old wild type or sirt3−/− mice fed either control (CD) or calorie restricted (CR) diet. Each lane presents an individual animal. Lower panels: Endogenous acetylated OTC was isolated by immunoprecipitation with anti-OTC antibody followed by western blotting with anti-acetyl-lysine antibody (n = 3). (B-C) Quantification of total Sirt3 protein (B) and OTC acetylation (C) from (A). Error bars represent standard error measurement (SEM) (n=3) Western blot was normalized with Sirt3 levels or OTC levels quantified and analyzed by Image software (n = 3). D.) OTC activity was measured in wild type and sirt3−/− mice fed control or CR diet. Error bars represent standard error measurement (SEM) (n = 5). E.) Orotic acid was measured in wild type and sirt3−/− mice fed control or CR diet. Error bars represent standard error measurement (SEM) (n = 5). * signifies a p < 0.05.

Article Snippet: Mitochondrial lysates from wild type and sirt3 −/− mice were performed with anti-Sirt3 antibody (gift of Dr. Eric Verdin, UCSF) or anti-OTC antibody (Aviva Systems Biology; 1:500) overnight at 4°C, then added with Protein A/G beads for 4h followed by western blotting using anti-acetyl-lysine antibody (generated following the procedure of Zhao et al., 2010 , GeneTel Laboratories LLC, Madison, WI).

Techniques: Western Blot, Control, Isolation, Immunoprecipitation, Software, Activity Assay

Journal:

Article Title: Sirt3 promotes the urea cycle and fatty acid oxidation during dietary restriction

doi: 10.1016/j.molcel.2011.01.002

Figure Lengend Snippet: Fasting and CR results in the up-regulation of Sirt3 levels. Sirt3 functions include the stimulation of enzymatic activities of AceCS1, β-oxidation enzymes, OTC, and IDH2. The coordinated response facilitates mitochondrial adaptation to dietary challenge.

Article Snippet: Mitochondrial lysates from wild type and sirt3 −/− mice were performed with anti-Sirt3 antibody (gift of Dr. Eric Verdin, UCSF) or anti-OTC antibody (Aviva Systems Biology; 1:500) overnight at 4°C, then added with Protein A/G beads for 4h followed by western blotting using anti-acetyl-lysine antibody (generated following the procedure of Zhao et al., 2010 , GeneTel Laboratories LLC, Madison, WI).

Techniques:

Journal: bioRxiv

Article Title: ATAD1 and the integrated stress response prevent clogging of TOM and damage caused by un-imported mitochondrial proteins

doi: 10.1101/2023.09.06.556408

Figure Lengend Snippet: ( A ) Western blot analysis of OTC-V5 stably expressing HEK293T cells untreated or treated with CCCP (10 µM) and valinomycin (Val) (1 µM) for either 6 or 24 hours. ( B ) Western blot analysis of ATP5G1-HA transiently expressing HEK293T cells untreated or treated with CCCP (10 µM) and valinomycin (1 µM) for either 6 or 24 hours. ( C ) Same as A. Bafilomycin A1 (BafA1) (0.1 µM) and MG132 (20 µM) were added to the cells 6 hours prior to cell harvesting (CCCP/valinomycin treatment was for 24 hours). ( D ) Same as B. BafA1 (0.1 µM) and MG132 (20 µM) were added to the cells 6 hours prior to cell harvesting (CCCP/valinomycin treatment was for 24 hours). ( E ) OTC-V5 stably expressing HEK293T cells were either untreated or treated with CCCP and valinomycin in the absence or presence of ISRIB (0.5 µM) for 6 or 24 hours. Quantifications of OTC-V5 precursor levels normalized to total OTC-V5 and β-tubulin are shown ( n =4). Data are mean ± SD. Paired t-test was used (* = p < 0.05). ( F ) ATP5G1-HA transiently expressing HEK293T cells were either untreated or treated with CCCP and valinomycin in the absence or presence of ISRIB (0.5 µM) for 6 or 24 hours. Quantifications of ATP5G1-HA precursor levels normalized to total ATP5G1-HA and β-tubulin are shown ( n =3). Data are mean ± SD. Paired t-test was used (* = p < 0.05). ( G ) HEK293T cells were transiently transfected with OTC-V5 and either empty vector, COQ7-HA (control), or PISD-FLAG. p , precursor; m , mature protein.

Article Snippet: To construct the OTC-V5 expression plasmid, rat OTC (from Addgene plasmid #71877) was cloned into the lentiviral backbone pLV-EF1a-IRES-Hygro (Addgene plasmid #85134).

Techniques: Western Blot, Stable Transfection, Expressing, Cell Harvesting, Transfection, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: ATAD1 and the integrated stress response prevent clogging of TOM and damage caused by un-imported mitochondrial proteins

doi: 10.1101/2023.09.06.556408

Figure Lengend Snippet: ( A ) Mitochondrial and cytosolic fractions were enriched by differential centrifugation from OTC-V5 stably expressing HEK293T cells untreated or treated with CCCP (10 µM), valinomycin (Val) (1 µM), and ISRIB (0.5 µM) for 24 hours to induce protein import stress. TOMM40 was used as a mitochondrial marker. ( B ) Mitochondrial fractions from A were treated with 0.25 mg/mL or 0.5 mg/mL of proteinase K. TOMM40 was used as a mitochondrial outer membrane marker. COXIV was used as a mitochondrial matrix marker. ( C ) OTC-V5 was immunoprecipitated (IP) from HEK293T cells stably expressing OTC-V5, treated as in A, using antibodies to V5. Quantifications of co-immunoprecipitated TOMM20 and TOMM40 normalized to their respective inputs are presented as fold change, for which the control condition was set to 1 ( n =3). Data are mean ± SD. Paired t-test was used (** = p < 0.01). p , precursor; m , mature protein.

Article Snippet: To construct the OTC-V5 expression plasmid, rat OTC (from Addgene plasmid #71877) was cloned into the lentiviral backbone pLV-EF1a-IRES-Hygro (Addgene plasmid #85134).

Techniques: Centrifugation, Stable Transfection, Expressing, Marker, Membrane, Immunoprecipitation, Control

Journal: bioRxiv

Article Title: ATAD1 and the integrated stress response prevent clogging of TOM and damage caused by un-imported mitochondrial proteins

doi: 10.1101/2023.09.06.556408

Figure Lengend Snippet: ( A-B ) Western blot analysis of wild type and ATAD1 KO HEK293T cells transiently expressing OTC-V5. Mitochondrial protein import stress was induced by CCCP (10 µM), valinomycin (Val) (1 µM), and ISRIB (0.5 µM) for 24 hours (A) or for the indicated time periods (B). Quantifications of OTC-V5 precursor levels normalized to total OTC-V5, as well as mature OTC-V5, to β-tubulin are shown ( n =3). Data are mean ± SD. Paired t-test was used ( ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001). ( C ) ATAD1 KO HEK293T cells were co-transfected with OTC-V5 and either empty vector (EV), ATAD1-HA (WT), or ATAD1 E193Q -HA (EQ). Mitochondrial protein import stress was induced as in A. Bar graph shows densitometry of OTC-V5 precursor levels normalized to total OTC-V5 and vinculin ( n =4). Data are mean ± SD. Paired t-test was used (*** = p < 0.001). ( D-E ) HEK293T cells were untreated or treated for 24 hours with CCCP/valinomycin, ISRIB, or both CCCP/valinomycin and ISRIB (same concentrations as in A). ATAD1 mRNA level was analyzed by means of quantitative reverse transcription polymerase chain reaction (RT-PCR) and quantified by normalization to GAPDH (D). ATAD1 protein level was analyzed by Western blot and quantified by normalization to β-tubulin (E). Quantifications are presented as fold change, for which the untreated condition was set to 1 ( n =3). Data are mean ± SD. Paired t-test was used ( ns = not significant). p , precursor; m , mature protein.

Article Snippet: To construct the OTC-V5 expression plasmid, rat OTC (from Addgene plasmid #71877) was cloned into the lentiviral backbone pLV-EF1a-IRES-Hygro (Addgene plasmid #85134).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: ATAD1 and the integrated stress response prevent clogging of TOM and damage caused by un-imported mitochondrial proteins

doi: 10.1101/2023.09.06.556408

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with either empty vector or ATAD1-HA. Mitochondrial protein import stress was induced by CCCP (10 µM), valinomycin (Val) (1 µM), and ISRIB (0.5 µM) for 24 hours. ATAD1-HA was immunoprecipitated using HA antibodies. The pound symbol (#) indicates an unspecific band. Quantification of co-immunoprecipitated TOMM20 and TOMM40 normalized to their respective inputs are presented as fold change, for which the control condition was set to 1 ( n ≥4). Control conditions were normalized to 1. Data are mean ± SD. Paired t-test was used (** = p < 0.01, **** = p < 0.0001). ( B ) Same as in A. HEK293T cells stably expressing OTC-V5 were used. ( C ) OTC-V5 was immunoprecipitated from ATAD1 KO HEK293T cells stably expressing OTC-V5, transfected with empty vector, ATAD1-HA, or ATAD1 E193Q -HA. Mitochondrial protein import stress was induced as in A. Quantification of co-immunoprecipitated ATAD1 normalized to their respective input are presented as fold change, for which the ATAD1-HA expressing cells was set to 1 ( n =4). Data are mean ± SD. Paired t-test was used (** = p < 0.01). ( D ) ATAD1-HA was immunoprecipitated from ATAD1 KO HEK293T cells transfected with empty vector, ATAD1-HA, or ATAD1 E193Q -HA. Mitochondrial protein import stress was induced as in A. Quantification of the IP is presented on the right. Co-immunoprecipitated TOMM20 was normalized to co-immunoprecipitated ATAD1-HA and TOMM20 input. Levels are presented as fold change, for which the ATAD1-HA expressing cells was set to 1 ( n =4). Data are mean ± SD. Paired t-test was used (** = p < 0.01).

Article Snippet: To construct the OTC-V5 expression plasmid, rat OTC (from Addgene plasmid #71877) was cloned into the lentiviral backbone pLV-EF1a-IRES-Hygro (Addgene plasmid #85134).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Control, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: ATAD1 and the integrated stress response prevent clogging of TOM and damage caused by un-imported mitochondrial proteins

doi: 10.1101/2023.09.06.556408

Figure Lengend Snippet: ( A , C ) Western blot analysis of OTC-V5 stably expressing HEK293T, HeLa, RPE-1, and HT1080 cells untreated (A) or treated with ISRIB (0.5 µM) for 24 hours (C). ( B ) Western blot analysis of p-eIF2α level in HEK293T, HeLa, RPE-1, and HT1080 cell lines. Bar graph shows fold change in p-eIF2α level normalized to total eIF2α level, for which the HEK293T cells was set to 1 ( n =4). Data are mean ± SD. Paired t-test was used (* = p < 0.05, ** = p < 0.01). ( D ) Growth curves of HEK293T and HT1080 cell lines untreated or treated with ISRIB (0.5 µM) for the indicated time periods. Data are mean ± SD of 3 biological replicates, consisting of 3 technical replicates each ( ns = not significant, **** = p < 0.001). ( E ) OTC-V5 stably expressing HT1080 cells were treated with ISRIB (0.5 µM) for 24 hours or transfected with non-targeting control siRNA (NTC) or a pool of HRI siRNAs (si HRI ) for 48 hours. Quantifications of OTC-V5 precursor levels normalized to total OTC-V5 and β-tubulin are presented as fold change, for which the NTC condition was set to 1 ( n =3). Data are mean ± SD. Paired t-test was used (* = p < 0.05, ** = p < 0.01). p , precursor; m , mature protein.

Article Snippet: To construct the OTC-V5 expression plasmid, rat OTC (from Addgene plasmid #71877) was cloned into the lentiviral backbone pLV-EF1a-IRES-Hygro (Addgene plasmid #85134).

Techniques: Western Blot, Stable Transfection, Expressing, Transfection, Control

Journal: bioRxiv

Article Title: ATAD1 and the integrated stress response prevent clogging of TOM and damage caused by un-imported mitochondrial proteins

doi: 10.1101/2023.09.06.556408

Figure Lengend Snippet: ( A ) Western blot analysis of endogenous ATAD1 protein from HEK293T, HeLa, RPE-1, and HT1080 cells. Bar graph shows fold change in ATAD1 level normalized to β-tubulin, for which the HEK293T cells was set to 1 ( n =3). Data are mean ± SD. Paired t-test was used (* = p < 0.05, ** = p < 0.01). ( B ) Western blot analysis of empty vector or ATAD1-HA (ATAD1 ↑ ) stably expressing HT1080 cells transiently transfected with OTC-V5 and treated with ISRIB for 24 hours. p , precursor; m , mature protein. Quantifications of OTC-V5 precursor levels normalized to total OTC-V5 and β-tubulin are presented as fold change, for which the empty vector condition was set to 1 ( n =6). Data are mean ± SD. Paired t-test was used (*** = p < 0.001). ( C ) Growth curves of HEK293T and HT1080 cell lines stably expressing empty vector, ATAD1 ↑ , or ATAD1 E193Q↑ . Data are mean ± SD of 3 biological replicates, consisting of 2 technical replicates each ( ns = not significant, ** = p < 0.01). ( D ) Overall schematic of proposed model. The stalling of un-imported proteins inside TOM can be alleviated by attenuating global protein translation via ISR, as well as removal of un-imported proteins from TOM by ATAD1.

Article Snippet: To construct the OTC-V5 expression plasmid, rat OTC (from Addgene plasmid #71877) was cloned into the lentiviral backbone pLV-EF1a-IRES-Hygro (Addgene plasmid #85134).

Techniques: Western Blot, Plasmid Preparation, Stable Transfection, Expressing, Transfection

Journal: medRxiv

Article Title: Evaluation of Physico-chemical parameters of selected Clotrimazole pessaries from pharmaceutical importers and Accredited Drug Dispensing Outlets in Dar es Salaam, Tanzania

doi: 10.1101/2025.01.21.25320887

Figure Lengend Snippet: Chemical Formulae of Clotrimazole (C 22 H 17 ClN 2 )

Article Snippet: Clotrimazole CRS was procured from the European Directorate for the Quality of Medicines (EDQM) and HealthCare (Strasbourg, France).

Techniques:

Journal: medRxiv

Article Title: Evaluation of Physico-chemical parameters of selected Clotrimazole pessaries from pharmaceutical importers and Accredited Drug Dispensing Outlets in Dar es Salaam, Tanzania

doi: 10.1101/2025.01.21.25320887

Figure Lengend Snippet: Calibration curve indicating a linear relationship between concentration and peak area response for Clotrimazole

Article Snippet: Clotrimazole CRS was procured from the European Directorate for the Quality of Medicines (EDQM) and HealthCare (Strasbourg, France).

Techniques: Concentration Assay