Journal: Biochemical and biophysical research communications

Article Title: Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells

doi: 10.1016/j.bbrc.2016.07.096

Figure Lengend Snippet: (A) VLDL-apo B and VLDL-B100 secretion were measured following 18 h incubation of McA cells in cDMEM (black bars) and in 1% BSA/DMEM (gray bars) by immuno slot blotting. (B) Representative standard curve generated from an immuno slot blot (duplicate standards) where VLDL protein is plotted against average chemiluminescence signal in arbitrary units (AU) generated using anti-rat B100 monoclonal antibody and HRP-anti-mouse IgG. (C) Immunoblots of McA cell lysates from four independent experiments comparing McA cells incubated in cDMEM with 1% BSA/DMEM. Anti-GAPDH blotting display equal protein loading. (D) Relative expression of Apob, Sort1 and Atf3 mRNA in McA cells incubated in 1% BSA/DMEM as a percentage of mRNA present in McA cells incubated in cDMEM. Results are averages from 4 independent experiments.* Indicates means are significantly different.

Article Snippet: McA cells were transfected using Fugene6 according to manufacturer’s protocol (Promega Corp., Madison WI) using three different pGIPZ based vectors expressing shRNAi targeting rat Sort1 mRNA (V2LMM_58553, V3LMM_450660, V3LMM_450662), and one scrambled, non-silencing control (GE Healthcare Dharmacon, Lafayette, CO).

Techniques: Incubation, Generated, Dot Blot, Western Blot, Expressing

Journal: Biochemical and biophysical research communications

Article Title: Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells

doi: 10.1016/j.bbrc.2016.07.096

Figure Lengend Snippet: (A) Following siRNA-mediated KD of sortilin, three clones of McA cells were selected representing high (H, McA60), medium (M, McA62) and low (L, McA53) sortilin expression evaluated by immunoblotting compared with wild type (WT) McA cells. The effect of increasing sortilin KD on VLDL-B100 secretion by McA cells incubated in cDMEM (B) or incubated in 1% BSA/DMEM (C). Results in (B) and (C) are averages of triplicate plates for each condition (n = 2 studies). * Indicates means are significantly different from the SCR McA cell line.

Article Snippet: McA cells were transfected using Fugene6 according to manufacturer’s protocol (Promega Corp., Madison WI) using three different pGIPZ based vectors expressing shRNAi targeting rat Sort1 mRNA (V2LMM_58553, V3LMM_450660, V3LMM_450662), and one scrambled, non-silencing control (GE Healthcare Dharmacon, Lafayette, CO).

Techniques: Clone Assay, Expressing, Western Blot, Incubation

Journal: Biochemical and biophysical research communications

Article Title: Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells

doi: 10.1016/j.bbrc.2016.07.096

Figure Lengend Snippet: (A) McA cell lines with variable sortilin KD were incubated with vehicle (black bars) or with 10 μM cpd984 (gray bars) for 18 h (3-100 mm plates per condition). Viability of McA cells was not compromised by incubations with cpd984 as there was no significant release of LDH into the medium compared with control incubations. VLDL was isolated from media of each plate and VLDL-B100 was quantified by immuno slot blotting. Results presented are averages of the 3 plates ± S.D. (B) McA cells were incubated for 18 h in 1% BSA/DMEM ± 10 μM cpd984. McA cells were then stimulated with a time course of 250 nM insulin (0, 5, 10 and 15 min). IRβ and AKT, pY-IRβ, and p-AKT (S473) were evaluated by immunoblotting using protein and phosphospecific antibodies.

Article Snippet: McA cells were transfected using Fugene6 according to manufacturer’s protocol (Promega Corp., Madison WI) using three different pGIPZ based vectors expressing shRNAi targeting rat Sort1 mRNA (V2LMM_58553, V3LMM_450660, V3LMM_450662), and one scrambled, non-silencing control (GE Healthcare Dharmacon, Lafayette, CO).

Techniques: Incubation, Control, Isolation, Western Blot

Journal: Molecular cancer therapeutics

Article Title: Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer

doi: 10.1158/1535-7163.MCT-16-0017

Figure Lengend Snippet: A, ERK knockdown by siRNA was performed in VACO432 (BRAFV600E/WT), as well as isogenic VACO432 VT1 (BRAFWT/−) cells with ectopic BRAFV600E vs empty vector (EV). Protein expression was determined by immunoblotting. B, Ectopic expression of HA-tagged wild type (WT) MCL-1, and phosphorylation-mimicking [T92D/T163D (DD)] or unphosphorylated [T92A/T163A (AA)] MCL-1 mutants was performed in HT29 cells. C, Cell lines were treated with cycloheximide (5 mmol/L) for the indicated times and protein expression against HA-tagged MCL-1 was analyzed by immunoblotting. The level of MCL-1 expression was then quantified by densitometry and normalized using tubulin expression.

Article Snippet: The non-targeting shRNA expression vector was obtained from Addgene (Cat #1864).

Techniques: Knockdown, Plasmid Preparation, Expressing, Western Blot, Phospho-proteomics

Journal: Molecular cancer therapeutics

Article Title: Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer

doi: 10.1158/1535-7163.MCT-16-0017

Figure Lengend Snippet: A, RKO and HT29 cells were transduced with lentiviral MCL-1 (#50 or #443) vs control shRNA (#293). Cells with stable expression were then incubated with cobimetinib for 48h at the indicated doses. Apoptosis was analyzed by annexin V+ staining that was quantified using flow cytometry. Mean values were derived from triplicate experiments and bars represent S.D. *p<0.05. B, Apoptosis was also analyzed by expression of cleavage (CL) of PARP and CASPASE-3 by immunoblotting in both cell lines. C, HT29 cells containing stable expression of control (#293) or MCL-1 (#50) shRNA were grown as tumor xenografts in SCID mice. Xenograft-bearing mice with dosed with either vehicle or cobimetinib (15 mg/kg every 3 days by oral gavage) for 14 consecutive days. Xenograft mean tumor volumes are plotted against days of treatment for vehicle- and cobimetinib-treated mice. Error bars represent SEM. Statistical significance(*, P < 0.05) is shown for comparison of MCL-1 (#50) shRNA + cobimetinib vs control (#293) shRNA + cobimetinib. D, Pre-treatment expression of MCL-1 in tumors from xenograft-bearing mice was evaluated by immunoblotting (upper panel). Post-treatment expression of pERK/ERK, MCL-1, cleaved PARP and cleaved caspase-3 in tumor xenografts from the 4 groups of tumor-bearing mice were evaluated by immunoblotting (lower panel).

Article Snippet: The non-targeting shRNA expression vector was obtained from Addgene (Cat #1864).

Techniques: Transduction, Control, shRNA, Expressing, Incubation, Staining, Flow Cytometry, Derivative Assay, Western Blot, Comparison

Journal: Molecular cancer therapeutics

Article Title: Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer

doi: 10.1158/1535-7163.MCT-16-0017

Figure Lengend Snippet: A, RKO and HT29 cell lines were treated with A-1210477, cobimetinib, or in combination for 16 h. Immunoprecipitation (IP) was performed in whole cell lysates (WCL) using conformation-specific antibodies against BAK or BAX (6A7). Normal rabbit (for BAK) and mouse (for BAX) IgG served as antibody controls. B, Cells were treated with A-1210477, cobimetinib, or their combination for 3 h and IP was then performed in WCL using an anti-MCL-1 antibody. Co-precipitated protein complexes were probed for BIM, BAK or BAX by immunoblotting. Normal rabbit IgG served as an antibody control. C, RKO and HT29 cell lines with stable expression of control (#293) or BIM (#49) shRNA were incubated with A-1210477, cobimetinib. or their combination for 16 h. IP was then performed in WCLs using conformation-specific antibodies against BAK or BAX in these cell lines. The effect of BIM shRNA on BIM protein expression was confirmed by immunoblotting.

Article Snippet: The non-targeting shRNA expression vector was obtained from Addgene (Cat #1864).

Techniques: Immunoprecipitation, Western Blot, Control, Expressing, shRNA, Incubation

Journal: bioRxiv

Article Title: The carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

doi: 10.1101/2025.03.18.644053

Figure Lengend Snippet: (A) qRT-PCR quantification of CNDP1 mRNA, relative to GAPDH and normalized to shNTC, in 10-230 BM (top) and 12-273 (bottom) cells expressing indicated shRNA. (B) Top. Proliferation curve performed by analyzing % confluency extracted from Incucyte image analysis normalized to day 0 of 10-230 BM cells expressing indicated tet-On shRNA (n=3, 96 h). Bottom. Proliferation curve performed by serial fixing and crystal violet staining of 12-273BM cells expressing indicated tet-On shRNA. Representative experiment shown of n=3 biological replicates. Statistics derived from one-way ANOVA testing between groups on the final time point. (C) Bar plots representing % cells distributed along cell cycle phases assessed by Edu differential staining (n=2) in 10-230 and 12-273 BM cells expressing indicated tet-On shRNA. Statistical analysis by one-way ANOVA with Dunnett multiple hypothesis testing correction. (D) Left. qRT-PCR quantification of CNDP1 mRNA, relative to hPPIA and then normalized to siNTC, in 10-230 BM. Right. Proliferation ratio of 10-230 BM cells transfected with indicated siRNAs after 72 h of culture normalized to 0h (n=3), indicated as % confluency extracted from Incucyte image analysis. Multiple biological replicates represented. Statistical analysis by one-way ANOVA. (E). Left. qRT-PCR quantification of CNDP1 mRNA, relative to hPPIA and then normalized to sINTC, in 12-273 BM. Right. Proliferation ratio of 12-273 BM cells transfected with indicated siRNAs after 96 h culture normalized to 0h (n=4), indicated as % confluency extracted from Incucyte image analysis technology. Multiple biological replicates represented. Statistical analysis by one-way ANOVA. See

Article Snippet: Tet-pLKO-puro was purchased from Addgene (RRID:Addgene_98398). shRNAs were cloned as previously described 115) into Tet-pLKO-puro using AgeI (NEB, Cat#R3552S) and EcoRI (Thermo, Cat#FD0275) restriction sites. pLKO tet-on scrambled non-targeting control (shNTC) was purchased from Addgene (RRID:Addgene_47541).

Techniques: Quantitative RT-PCR, Expressing, shRNA, Staining, Derivative Assay, Transfection

Journal: bioRxiv

Article Title: The carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

doi: 10.1101/2025.03.18.644053

Figure Lengend Snippet: (A) Carnosine accumulation measured by ELISA represented as normalized values to Carnosine concentration in shNTC conditions (30 mins under culture) in 12-273 transduced with indicated tetON-shRNAs treated with doxycycline for 24h. Mean of two technical replicates shown. (B) Proliferation ratio of 10-230 BM cells cultured with 10 μm, 100 μm and 50 mM Carnosine concentrations during 0.5, 2, 6 and 24 h of culture normalized to not treated (NT) of their respective time points (n=3, technical replicates). Confluency obtained by treating the cells with Resazurin and measuring absorbance after 3 hours of treatment at 570 nm. Statistical analysis by one-way ANOVA. (C) Carnosine accumulation shown as normalized units to STC media in conditioned media and pellet after 30 minutes of treatment. (D) Proliferation ratio of SKMEL-239 cells transfected with indicated shRNAs after 96 h of culture normalized to 0h, indicated as % confluency extracted from Incucyte image analysis. (E) Proliferation ratio performed by serial fixing and crystal violet staining of Yumm3.2 BrafV600E/wt Cdkn2a-/- Pten-/- control or ectopically expressing Cndp1-HA after 96h as indicated. (F) Immunoblot showing Cndp1-HA expression in conditioned media and pellets of Yumm3.2 BrafV600E/wt Cdkn2a-/- Pten-/- control or ectopically expressing Cndp1-HA. See also Document S1 for details. (G) L-Histidine liberation assay as carnosinase activity read-out in pellets of Yumm3.2 BrafV600E/wt Cdkn2a-/- Pten-/- control or ectopically expressing Cndp1-HA. Three technical replicates shown. One representative technical replicate shown for all the above assays.

Article Snippet: Tet-pLKO-puro was purchased from Addgene (RRID:Addgene_98398). shRNAs were cloned as previously described 115) into Tet-pLKO-puro using AgeI (NEB, Cat#R3552S) and EcoRI (Thermo, Cat#FD0275) restriction sites. pLKO tet-on scrambled non-targeting control (shNTC) was purchased from Addgene (RRID:Addgene_47541).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Transduction, Cell Culture, Transfection, Staining, Control, Expressing, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: The carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

doi: 10.1101/2025.03.18.644053

Figure Lengend Snippet: (A) Workflow of RNA-sequencing and LC/MS-based nonpolar metabolomics integrative studio in 10-230BM cells transduced with indicated tetON-shRNA. (B) Differential gene expression/metabolite abundance between shNTC vs shCNDP1-1 and shCNDP1-2 by indicated adjusted p values. MetaboAnalyst executed hypergeometric testing by combining p-values for metabolites and transcripts according to their relative proportion in each pathway. (RNA-seq: DESeq2, Metabolomics: T-test, pathways enrichment FDR < 0.05). See Tables S5-7 (C) Heatmap of t-Aminoacyl synthetases expression across different conditions paired with a heatmap of expression of their corresponding amino acids. MS/MS-based proteomics and LC/MS-based metabolomics were conducted on lysates of 10-230 BM cells expressing the indicated shRNA treated with doxycycline for 72 hours in 10% dialyzed serum DMEM. Differentially expressed proteins were identified by unpaired, two-tailed t-test comparing 2 groups normalized abundance: shCNDP1-1 and shCNDP1-2 vs shNTC (Data represented by z-score as indicated). (D) Integrated Gene Set Enrichment analysis (GSEA) of 12-273 and 10-230 BM shNTC vs both shCNDP1 tetON-shRNA RNA sequencing represented by Normalized Enrichment Score using Reactome Gene Ontology Analysis (RNA-seq: DESeq2, adjust p value < 0.001 and FC > 1.5). See Table S6. (E) Differential expression testing by DESeq2 nominated ATF4 targets in both shCNDP1 vs shNTC conditions in 10-230 BM RNA sequencing. (Data represented by z-score as indicated). (F) Pathway analysis via the Seq-n-Slide pipeline tested for pathway enrichment in differentially expressed genes between shCNDP1 and shNTC 10-230 BM RNA sequencing (Adjust p value indicated). (G) Representative immunoblots of phosphoSerine51 eIF2a, total-eIF2a, ATF4 and housekeeping (HK) protein Vinculin in lysates of 10-230 BM transduced with indicated tetON-shRNA. See Document S1 for details. (H) Proliferation ratio of 12-273 BM cells transfected with indicated siRNAs for CNDP1 silencing after 96 h of culture normalized to 0h (n=4), either treated with vehicle (DMSO) or ISRIB (phospho-eIF2a inhibitor, 1 uM) performed by analyzing % confluency extracted from Incucyte image analysis. Statistical analysis by one-way ANOVA. Proliferation ratio for 12-230 BM siNTC/siCNDP1 also shown in E. See also

Article Snippet: Tet-pLKO-puro was purchased from Addgene (RRID:Addgene_98398). shRNAs were cloned as previously described 115) into Tet-pLKO-puro using AgeI (NEB, Cat#R3552S) and EcoRI (Thermo, Cat#FD0275) restriction sites. pLKO tet-on scrambled non-targeting control (shNTC) was purchased from Addgene (RRID:Addgene_47541).

Techniques: RNA Sequencing, Liquid Chromatography with Mass Spectroscopy, Transduction, shRNA, Gene Expression, Expressing, Tandem Mass Spectroscopy, Two Tailed Test, Quantitative Proteomics, Western Blot, Transfection

Journal: bioRxiv

Article Title: The carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

doi: 10.1101/2025.03.18.644053

Figure Lengend Snippet: (A) 10-230 and 12-273 BM transduced with indicated tetON-shRNAs and treated with doxycycline during 72h, labeled with AHA (APC) for 4 hours and analyzed by flow cytometry. Cycloheximide (CHX, 50 ug/ml) was used as a positive control of translation shutdown. Statistics rendered by one-way ANOVA with Dunnet’s multiple hypothesis testing correction. (B) Left. Representative immunoblots of phospho-S6K1 p70 (Threonine 389), S6K1 p70, phosphoSer65 4E-BP, total 4EBP and housekeeping (HK) protein Hsp90 in lysates of 10-230 BM transduced with indicated tetON-shRNA. Right. Representative immunoblots of phospho-S6K1 p70 (Threonine 389), S6K1 p70, phosphoSer65 4E-BP, 4EBP and housekeeping (HK) protein Hsp90 in lysates of 10-230 BM cells transfected with siRNA NTC or CNDP1 as indicated. Band correspondent to p70 S6K1 is indicated. Results for HRI, phosphoSer51 and total eiF2α expression from same samples shown in and , respectively. Ratio of protein phosphorylation shown normalized to total protein and HK protein levels. See Document S1 for details. (C) GENI (gene set enrichment identifier124) was applied to TCGA melanoma samples (n = 472) using the Reactome 2022 pathway database. Five anticorrelating gene sets with FDR < 0.05 were represented. (D) Genes differentially expressed in shCNDP1 (combined) vs shNTC polysome heavy chain (log fc Translation vs log fc Transcription; p<0.05, lfc > 1) at translation, transcription and both transcription and translation levels in 10-230 BM cells. See Table 11. (E) Integrated Gene Enrichment analysis of Heavy Chain RNA sequencing of 10-230 BM shCNDP1 (combined) vs shNTC represented by Normalized Enrichment Score using Reactome Gene Ontology Analysis (RNA-seq: adjust p value < 0.05). See also

Article Snippet: Tet-pLKO-puro was purchased from Addgene (RRID:Addgene_98398). shRNAs were cloned as previously described 115) into Tet-pLKO-puro using AgeI (NEB, Cat#R3552S) and EcoRI (Thermo, Cat#FD0275) restriction sites. pLKO tet-on scrambled non-targeting control (shNTC) was purchased from Addgene (RRID:Addgene_47541).

Techniques: Transduction, Labeling, Flow Cytometry, Positive Control, Western Blot, shRNA, Transfection, Expressing, Phospho-proteomics, RNA Sequencing

Journal: bioRxiv

Article Title: The carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

doi: 10.1101/2025.03.18.644053

Figure Lengend Snippet: (A) Integrated Gene Enrichment analysis of 12-273 BM heavy chain both shCNDP1 vs shNTC RNA sequencing represented by Normalized Enrichment Score using Reactome Gene Ontology Analysis (RNA-seq: adjust p value < 0.05). (B) Venn diagram including common transcripts changing as indicated at translation level (top) and transcription and translation level (bottom) in both 10-230 and 12-273 BM shCNDP1 (combined) vs shNTC polysome heavy chain (adjusted p value <0.05, n.i - non-identified). See Table S11. (C) Representative immunoblot of eIFs proteins and phophoSerine51 4E-BP1, 4E-BP1 and housekeeping (HK) protein Vinculin in lysates of 12-273 BM cells transfected with siRNA NTC or CNDP1 as indicated. See Document S1 for details.

Article Snippet: Tet-pLKO-puro was purchased from Addgene (RRID:Addgene_98398). shRNAs were cloned as previously described 115) into Tet-pLKO-puro using AgeI (NEB, Cat#R3552S) and EcoRI (Thermo, Cat#FD0275) restriction sites. pLKO tet-on scrambled non-targeting control (shNTC) was purchased from Addgene (RRID:Addgene_47541).

Techniques: RNA Sequencing, Western Blot, Transfection

Journal: bioRxiv

Article Title: The carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

doi: 10.1101/2025.03.18.644053

Figure Lengend Snippet: (A-E) Quantification of total mitochondria per cell (A) , mitochondria area (B) , circularity (C) , cristae number per mitochondria (D) and cristae area per mitochondria area (E) in 12-273 BM. Statistical analysis by one-way ANOVA. (F) Seahorse MitoStress analysis of OCR in 10-230 BM transduced with indicated tetON-shRNA and MDA-231 Brm2 treated as indicated. Statistical analysis by one-way ANOVA. Representative replicate shown (n=3, p<0.05 or indicated). (G) Normalized distributions of glutamine, glutamate, and TCA cycle metabolites, including α-ketoglutarate (αKG), succinate, fumarate, malate and citrate, in 10-230 BM cells treated with 10 10 μm, 100 μm and 50 mM Carnosine for 30 minutes are shown. (n = 3); data are shown as the mean ± SD. Statistical analysis by one-way ANOVA. P value indicated. Cells were cultured with [U- 13 C5] glutamine for 6 h before metabolite extraction and gas chromatography-mass spectrometry (GC-MS) analyses. (H) Carnosine accumulation measured by ELISA represented as normalized values to carnosine concentration in dialyzed media supplemented with indicated concentrations in 10-230 BM cells cultured with 10 μm, 100 μm and 50 mM Carnosine concentrations during 0.5, 2 h. Mean of two technical replicates shown. Results for 0.5 h also shown in Figure S2C. (I) 2logFold Change of HMOX protein abundance measured by proteomics (MS/MS) in 10-230 BM cells transduced with indicated tetON-shRNAs. Differentially expressed proteins identified by unpaired, two-tailed t-test comparing 2 groups: shCNDP1-1 and shCNDP1-2 versus shNTC. (J) 2logFoldChange of LCN2 transcript expression of both shCNDP1 tetON-shRNA vs shNTC in 10-230 BM and 12-230 BM cells. RNA sequencing data. p value < 0.05. (K) 2logFold Change of ATP7B and SLC46A3 transcript of Heavy Chain RNA sequencing of 10-230 BM shCNDP1 vs shNTC. p value < 0.05. (L) AUCell (Area Under the Curve) measurement of copper homeostasis gene signature (WikiPaths, WP3286) in CNDP1 high expression versus CNDP1 low expression in MBM cells from Biermann et al., 2022 data mining. Statistical analysis by one-way ANOVA, p<0.001. (M) Representative immunoblots of OGDH, PDH and housekeeping (HK) protein β-actin in lysates of 10-230 BM cells transfected with indicated sh-RNAs. See document S1 for details. (N) R Pearson correlation between melanoma cell lines classified by their MITF expression and resistance to Elesclomol treatment expressed as AUC of cell viability after treatment. Data mining from Depmap.

Article Snippet: Tet-pLKO-puro was purchased from Addgene (RRID:Addgene_98398). shRNAs were cloned as previously described 115) into Tet-pLKO-puro using AgeI (NEB, Cat#R3552S) and EcoRI (Thermo, Cat#FD0275) restriction sites. pLKO tet-on scrambled non-targeting control (shNTC) was purchased from Addgene (RRID:Addgene_47541).

Techniques: Transduction, shRNA, Cell Culture, Extraction, Gas Chromatography, Mass Spectrometry, Gas Chromatography-Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Concentration Assay, Quantitative Proteomics, Tandem Mass Spectroscopy, Two Tailed Test, Expressing, RNA Sequencing, Western Blot, Transfection

Journal: bioRxiv

Article Title: Dysregulation of Acid Ceramidase-mediated Sphingolipid Metabolism Contributes to Tumor Progression in Tuberous Sclerosis Complex

doi: 10.1101/2022.09.25.509382

Figure Lengend Snippet: 621-101 (TSC2-) and 621-103 (TSC2+) cells were treated with an ASAH1 inhibitor 17a (a) or carmofur (b) with indicated concentrations for 72 hr. Cell viability was measured using MTT assay (n=6/treatment group). (c) TSC2-null 621-101 cells were transfected with three independent ASAH1-siRNAs or control-siRNA for 48 hr. siRNA knockdown efficiency was determined by RT-PCR (n=3/group). (d) Cells were treated with ceramide, and then stained with Annexin V: FITC Apoptosis Detection Kit (BD#556547). Cell death was analyzed by flow cytometry (n=3). (e) The percentage of apoptotic (Annexin V+) cells was determined (percentage of Annexin V: FITC-positive cells in total cell number). (f) Cell viability was assessed 48 hr post siASAH1 RNA transfection in 621-101 cells using MTT assay. (g) siRNA knockdown efficiency was determined by immunoblotting. β-actin was used as a loading control. (h) 621-101 cells were infected with lentiviruses containing shRNA for vector pLKO, or ASAH1 , and then selected with puromycin for two weeks. Stable cells were harvested, and then stained with Annexin V: FITC Apoptosis Detection Kit. Cell death was analyzed by flow cytometry (n=3). (i) The percentage of apoptotic (Annexin V+) cells was determined (percentage of Annexin V: FITC-positive cells in total cell number). (j) Cell death was measured using PI exclusion assay. Relative cell death was compared between pLOK.1 and shRNA- ASAH1 cells. (k) Cell viability was measured using MTT assay (n=8-16/treatment group). *p<0.05; **p<0.01, Student t-test.

Article Snippet: 293T packaging cells were transfected with ASAH1 shRNA, or non-Targeting shRNA vectors using Mirus Trans-IT TKO Transfection reagent (Mirus).

Techniques: MTT Assay, Transfection, Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Staining, Flow Cytometry, Western Blot, Infection, shRNA, Plasmid Preparation, Exclusion Assay

Journal: Journal of cellular and molecular medicine

Article Title: Placental growth factor silencing ameliorates liver fibrosis and angiogenesis and inhibits activation of hepatic stellate cells in a murine model of chronic liver disease.

doi: 10.1111/jcmm.13158

Figure Lengend Snippet: Fig. 1 Experimental study design. Fibrosis was induced in mice by carbon tetrachlo- ride (CCl4) for 8 weeks, and mice were treated with PlGF siRNA or non-targeting control (NTC) siRNA four cycles via tail vein injection starting 2 weeks after initiat- ing CCl4 injections.

Article Snippet: Retroviral HIF-1a shRNA (shHIF-1a) and non-targeting control shRNA (shNTC) were purchased from OriGene (Rockville, MD, USA).

Techniques: Control, Injection

Journal: Journal of cellular and molecular medicine

Article Title: Placental growth factor silencing ameliorates liver fibrosis and angiogenesis and inhibits activation of hepatic stellate cells in a murine model of chronic liver disease.

doi: 10.1111/jcmm.13158

Figure Lengend Snippet: Fig. 8 PlGF knockdown by siRNA inhibits the proliferation and activation of hepatic stellate cells (HSCs) via the PI3K/Akt signalling pathway. (A) Measurement of cell proliferation of rat HSCs using CCK-8 assay. Cells were transfected with PlGF siRNA or NTC siRNA; cells were treated with rPlGF administration (50 ng/ml) or co-incubation with PI3K inhibitor LY294002 for 5 days. aP < 0.001 compared with mimics control (PBS), bP < 0.001 compared with PlGF+LY294002. (B) The mRNA levels of a-SMA in rat HSCs with stimulation PlGF (50 ng/ml) for 6 or 24 hrs. (C) Rep- resentative Western blot of a-SMA expression in rat HSC treated with rPlGF (50 ng/ml) or PBS for 6–24 hrs and quantification compared to b-actin content. (D) Western blot for PI3K, phospho-Akt (p-Akt), Akt and a-SMA in rat HSCs, and b-tubulin or b-actin as loading controls. Cells were trea- ted with rPlGF administration (50 ng/mL) or co-incubation with PI3K inhibitor LY294002 for 8 hrs. (E) The Western blot results of part (D) were quantified by densitometry.

Article Snippet: Retroviral HIF-1a shRNA (shHIF-1a) and non-targeting control shRNA (shNTC) were purchased from OriGene (Rockville, MD, USA).

Techniques: Knockdown, Activation Assay, CCK-8 Assay, Transfection, Incubation, Control, Western Blot, Expressing

Journal: Nature cell biology

Article Title: Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity

doi: 10.1038/ncb2332

Figure Lengend Snippet: MBd enrichment increases reprogramming efficiency and enhances in vitro tumorigenicity. (a-c) Reprogramming is more efficient after MBd enrichment. Differentiated cells (dH1f) and embryonic fibroblasts (IMR90) are reprogrammed after stable expression of either NBR1-specific shRNA (shNBR1) or non-targeting shRNA (shNT). Emerging iPSC colonies are scored based on Tra-1-60 expression37. (a, b) Cells depleted of NBR1 to increase MBd levels show an increase in iPSC colony formation (a, dH1f: 3.1±0.5-fold, n=15, p=0.00035; IMR90: 3.4±0.8-fold, n=3, p=0.02; data are mean ± s.e.m.) but insignificant changes in autophagic activity (c) over shNT control. (b) Representative plates with Tra-1-60-immunostained iPSC colonies. Immunoblot (c, top) and densitometry (c, bottom; percent of autophagic flux) show representative result (n=3); α-tubulin, loading control. (d) MCF-7 side-population (SP) cells have a significantly higher percentage of MBd+ cells over the non-SP population (MP; p=0.0015, n=3; data are mean ± s.d.). (e, f) MBd enrichment in cancer cells leads to increased anchorage-independent growth. MKLP1-GFP-expressing HeLa cells are separated into “MBd high” and “MBd low” subpopulations. An increase in the “MBd high” over “MBd low” ratio is associated with an increase in soft-agar colony formation (e). No significant difference was observed when the enrichment of MBd high subpopulation was less than 3-fold. More soft-agar colonies are formed when MBds are enriched by NBR1-depletion (shNBR1) in HeLa (f, left; p=0.0012, n=3) and mouse 134-4 cells (f, right; p=0.0086, n=3); control, shNT. Data are mean ± s.d., and the colony number (e, f) is the sum of INT-violet-stained colonies from 10 random fields. (g) Model for MBd fate in cells. The newly-formed MBd is preferentially inherited by the daughter cell with the older centrosome (top panel). The inherited MBd (black ring) is recognized by binding of the NBR1 autophagic receptor (grey circle) with the MB protein Cep55 (magenta). The MBd is then encapsulated by the autophagosome (yellow circle), and degraded after fusion of autophagosome and lysosome (red circle) in differentiated cells. This pathway prevents MBd-accumulation. In contrast, stem cells efficiently accumulate MBds through successive divisions and evasion of NBR1-mediated autophagy. Additionally, differentiated and stem cells possess overall high and low autophagic activity, respectively.

Article Snippet: Embryonic fibroblasts (IMR90), adult fibroblasts (hFib2) and dH1f cells were transduced with either NBR1-specfic or non-targeting shRNA vector, and puromycin-selected to establish NBR1-depleted (shNBR1) and control (shNT) lines. dH1f (2.5×10 4 /assay) were reprogrammed with lentiviral vectors 69 (Addgene #21162 and 21164) expressing OCT4, SOX2, KLF4 and c-MYC 34 , 35 , 68 whereas the reprogramming of IMR90 and hFib2 cells (5×10 4 /assay) also included lentiviral vectors expressing Nanog and Lin28 55 , 69 (Addgene #21163). iPSC colonies were quantified on day 21 based on Tra-1-60 expression using ImageJ, as reported 35 , 68 , and with parameters: ≥148 (threshold), 0.5-1 (circularity), and either 10-infinity or 30-infinity (size).

Techniques: In Vitro, Expressing, shRNA, Activity Assay, Western Blot, Staining, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Regulation of LPCAT3 mRNA expression by PPARδ activation. A, HepG2 cells were seeded in a 12-well cell culture plate. After 24 h, cells were transduced with Ad-shLacZ (50 MOI) or Ad-shPPARδ (50 MOI) adenovirus in duplicate wells per transduction. Fresh cell culture medium was replaced 5 h later, and cells were incubated for a further 48 h. Total RNA was collected from transduced cells for quantitative PCR analysis. Bars, mean ± S.E. (error bars) of two RNA samples with triplicate measurement per RNA sample. **, p < 0.01; ***, p < 0.001. The data shown are representative of two independent assays. B, HepG2 cells were transduced with Ad-GFP (50 MOI) or Ad-PPARδ (50 MOI) adenoviruses in duplicate wells per transduction. Fresh cell culture medium was replaced 5 h later, and cells were incubated for a further 24 h and then subsequently treated with 1 μm GW0742 or DMSO. Total RNA was collected from transduced cells for quantitative PCR analysis. Bars, mean ± S.E. of two RNA samples with triplicate measurement per RNA sample. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The data shown are representative of two independent assays. C, real-time PCR quantification of LPCAT3 mRNA expression from HepG2, Huh7, and Hepa 1-6 cells treated with GW0742 (1 μm), L165041 (20 μm), or DMSO for 24 h. LPCAT3 expression levels were normalized to GAPDH mRNA levels, where the relative expression of LPCAT3 mRNA in DMSO-treated cells was set at 1. D and E, qRT-PCR quantification of LPCAT3 or ACSL4 mRNA expression from HepG2 cells treated with L165041 at the indicated concentrations. Bars, mean ± S.E. of triplicate measurements of each RNA sample. The data shown are representative of two independent assays. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with DMSO-treated samples. F, C57BL/6J mice fed a normal chow diet were injected with Ad-sh-mPPARδ (n = 4) or Ad-shLacZ (n = 4). Ten days after injection, mice were sacrificed for liver tissue collection. qRT-PCR was used to determine the relative expression levels (mean ± S.E., n = 4/group) of individual mRNAs after normalization with GAPDH mRNA levels. *, p < 0.05; **, p < 0.001, compared with the vehicle group, which was set at 1.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Expressing, Activation Assay, Cell Culture, Transduction, Incubation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Injection

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Up-regulation of human LPCAT3 promoter activity in HepG2 cells by PPARδ agonists and LXR agonist. A, diagrammatic representation of human LPCAT3 promoter luciferase reporter construct. B, relative luciferase activities from HepG2 cells transfected with pLPCAT3-Luc promoter luciferase plasmid or pGL3-basic vector. Data represent summarized results (mean ± S.E. (error bars)) of 4–6 replicates/treatment and are expressed as ratio of luciferase/β-gal activity from each sample, where the relative luminescence from cells transfected with promoterless pGL3-basic vector and treated with DMSO is set to 1. *, p < 0.05 compared with DMSO-treated samples. The data shown are representative of three separate transfection experiments.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Activity Assay, Luciferase, Construct, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Mapping the functional PPRE site in human LPCAT3 promoter. A, diagrammatic representation of human LPCAT3 promoter luciferase reporter constructs of wild type and PPRE mutants. B, relative luciferase activities from control and L165041-treated Huh7 cells transfected with LPCAT3 promoter wild type and PPRE site-mutated reporter constructs or pGL3-basic vector. Data represent summarized results (mean ± S.E.) of 4 replicates/treatment and are expressed as the ratio of luciferase/β-gal activity from each sample. ***, p < 0.001 compared with DMSO-treated samples. C, after transfection, cells were treated with DMSO as control, 1 μm GW3965, 10 μm L165041, or GW3965 + L165041 for 24 h before cell lysis for the luciferase and β-gal activity assay. The graph represents relative luciferase activities from control and treated HepG2 cells transfected with LPCAT3 promoter wild type and PPRE site-mutated reporter constructs or pGL3-basic vector. Data represent summarized results (mean ± S.E. (error bars)) of 4 replicates/treatment and are expressed as the ratio of luciferase/β-gal activity from each sample. *, p < 0.05; ***, p < 0.001 compared with DMSO-treated samples. The data shown are representative of two separate transfection experiments.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Functional Assay, Luciferase, Construct, Control, Transfection, Plasmid Preparation, Activity Assay, Lysis

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: EMSA and ChIP analyses of PPARδ association with PPRE1 site of LPCAT3 promoter in vitro and in vivo. A, chemiluminescent signal from cross-linked nylon membrane, where biotin-5′-end-labeled LPCAT3-PPRE1 (lanes 1–4) was incubated with 200 ng of PPARδ and 100 ng of RXRα recombinant proteins in the absence (lane 2) or presence of a 100-fold molar excess of unlabeled wild type probe (lane 3) or mutated probe (lane 4). B, ChIP analysis on HepG2 cells treated with DMSO or L165041 (10 and 20 μm), where rabbit anti-PPARδ or isotype IgG antibody-immunoprecipitated DNA samples and input DNA samples were PCR-amplified with primers specific for the LPCAT3-PPRE1 and LPCAT3-PPRE2 promoter region. The PCR products were separated on a 2% agarose gel and stained with ethidium bromide. C, real-time quantitative PCR was performed using the two primer sets and chromatin DNA contained in the immunoprecipitates. Values from IgG-immunoprecipitated samples were arbitrarily set to 1.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: In Vitro, In Vivo, Membrane, Labeling, Incubation, Recombinant, Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Administering L165041 to mice increases hepatic Lpcat3 mRNA and LPCAT enzyme activity along with elevations of Acsl4 and other PPARδ-target genes. Male C57BL/6 mice were gavaged daily with 40 mg/kg L165041 (n = 5) or vehicle (n = 5). After 7 days of treatment, the liver was excised, and the following were measured. A, all isoforms of Lpcat mRNAs. B, D, and F, qRT-PCR was conducted to determine the relative expression levels (mean ± S.E. (error bars), n = 5/group) of individual mRNAs after normalization with GAPDH mRNA levels. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with the vehicle group, which was set at 1. C, total LPCAT activity (mean ± S.E., n = 5/group). ***, p < 0.001, compared with the control group. E, Western blotting analysis of hepatic ACSL4 protein levels.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Depletion of hepatic LPCAT3 expression in mice fed a chow diet attenuated PPARδ-mediated activation of hepatic genes in the FA metabolic pathway. Male C57BL/6J mice (n = 5 animals/treatment) were retro-orbitally injected with 3 × 109 IFU/mouse of Ad-shLacZ or Ad-shLPCAT3 adenovirus particles. Three days after injection, mice were orally treated with L165041 (40 mg/kg) or vehicle for 7 days. Four-h-fasted serum samples were collected at the experimental termination, and the liver was excised for the total LPCAT activity assay (A and B) and gene expression analysis (C). Statistical analysis was performed using Student's t test for the enzyme assay (A and B), and one-way ANOVA with Dunnett post hoc test was performed in gene expression analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with the vehicle group injected with Ad-shLacZ. V, vehicle; L, L165041. Error bars, S.E.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Expressing, Activation Assay, Injection, Activity Assay, Gene Expression, Enzymatic Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Effects of hepatic LPCAT3 knockdown on serum and hepatic lipid levels of mice with or without L165041 treatment. Male C57BL/6J mice (5 mice/group) were injected with 3 × 109 IFU/mouse of Ad-shLacZ or Ad-shLPCAT3 adenovirus particles. Three days after injection, mice were orally treated with L165041 (40 mg/kg) or vehicle for 7 days. Four-h-fasted serum samples were collected at the experimental termination, and the liver was excised. Serum TG (A), TC (B), PL (C), and NEFA (D) were measured. Liver lipids were extracted and used to measure hepatic TG (E), TC (F), PL (G), and NEFA (H). Statistical analysis was performed using one-way ANOVA with Dunnett's post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with the vehicle group injected with Ad-shLacZ. V, vehicle; L, L165041. Error bars, S.E.

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Knockdown, Injection

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: qRT-PCR primer, EMSA probes, promoter cloning primers, and ChIP primer sequences

Article Snippet: An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Cloning

Journal: Frontiers in Immunology

Article Title: Prognostic stratification of sepsis through DNA damage response based RiskScore system: insights from single-cell RNA-sequencing and transcriptomic profiling

doi: 10.3389/fimmu.2024.1345321

Figure Lengend Snippet: Validation of characteristic genes in vivo (A) Relative expression levels of DDR-related genes in control and sepsis groups (n=4 in each group). (B) Relative expression levels of ARL4C in control, sepsis, sepsis+Ad-shNC, and sepsis+Ad-shARL4C group (n=8 in each group). (C) Expression levels of inflammatory factors (IL-1β, TNF-α, IL-10, and IL-18) in the peripheral blood of rat with sepsis+Ad-shNC and sepsis+Ad-shARL4C (n=8 in each group). (D) Survival status of rats in each group (n=10 in each group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. NS, no significant difference.

Article Snippet: In summary, adenoviral vectors containing shRNA sequences specifically targeting the ARL4C gene in rats (Ad-shARL4C) alongside a non-targeting control shRNA sequence (Ad-shNC), both sourced from RiboBio in Guangzhou, China, were utilized.

Techniques: Biomarker Discovery, In Vivo, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Prognostic stratification of sepsis through DNA damage response based RiskScore system: insights from single-cell RNA-sequencing and transcriptomic profiling

doi: 10.3389/fimmu.2024.1345321

Figure Lengend Snippet: Validation of ARL4C in vitro . (A) Flow cytometry detected the apoptosis rate in the Control, Sepsis, Sepsis+Lv-shNC, and Sepsis+Lv-shARL4C groups. (n=4 in each group) (B) Flow cytometry detected the ROS production in the Control, Sepsis, Sepsis+Lv-shNC, and Sepsis+Lv-shARL4C groups. (n=4 in each group), *** p < 0.001, **** p < 0.001. NS, no significant difference.

Article Snippet: In summary, adenoviral vectors containing shRNA sequences specifically targeting the ARL4C gene in rats (Ad-shARL4C) alongside a non-targeting control shRNA sequence (Ad-shNC), both sourced from RiboBio in Guangzhou, China, were utilized.

Techniques: Biomarker Discovery, In Vitro, Flow Cytometry, Control