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: 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: JCI Insight

Article Title: CDK12 regulates cellular metabolism to promote glioblastoma growth

doi: 10.1172/jci.insight.190780

Figure Lengend Snippet: ( A ) Schematic experimental design and RNA-seq sample information. ( B ) This plot shows the normalized enrichment score (NES) of Reactome_Citric acid cycle (TCA cycle) and Respiratory Electron_Transport gene sets derived from GSEA. ( C and D ) GSEA and heatmap of Oxidative Phosphorylation genes in RNA-seq data for DMSO and SR treatment. ( E and F ) The qPCR and Western blot results of oxidative phosphorylation– and fatty acid oxidation–related genes after SR treatment in GBM12, GBM22, and U251 cells. The qPCR data are presented as mean ± SD. ( G ) Western blot results of oxidative phosphorylation– and fatty acid oxidation–related genes after loss of CDK12 in GBM12, GBM22, and U251 cells. ( H ) PPARD mRNA expression (log 2 ) in non-tumor and GBM tissues. Data are presented as box-and-whisker plots; the horizontal line within each box represents the median value, and the whiskers denote the minimum and maximum values. Statistical significance was determined using a Student’s t test (* P < 0.05). ( I ) Viability of GBM12 cells expressing shPPARD ( – ) compared to shNTS was assessed for 0, 2, 4, or 6 days. Data are presented as mean ± SD. ( J ) GBM12 cells were transfected with scrambled siRNA (scRNA) or siRNAs against PPARD and PGC1A. Thereafter, cells were exposed to increasing concentrations of SR-4835 and analyzed for cellular viability. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 by unpaired, 2-tailed t test ( E and H ), 1-way ANOVA with Dunnett’s multiple-comparison test ( I ), or 2-way ANOVA with Tukey’s multiple-comparison test ( J ).

Article Snippet: The scramble shRNA (shNTS, non-target sequence) plasmid was purchased from Addgene (catalog 1864).

Techniques: RNA Sequencing, Derivative Assay, Phospho-proteomics, Western Blot, Expressing, Whisker Assay, Transfection, Comparison

Journal: Cell Research

Article Title: Pericytes augment glioblastoma cell resistance to temozolomide through CCL5-CCR5 paracrine signaling

doi: 10.1038/s41422-021-00528-3

Figure Lengend Snippet: a Apoptosis analysis of GBM-1 cells with indicated treatments. GBM cells were stimulated with CCL5 (10 ng/mL) or vehicle (PBS) followed by the administration of TMZ (500 μmol/L) or DMSO. ns, not significant. ** P < 0.01. b Cell survival analysis of GBM-1 cells with indicated treatments. CM from pericytes expressing shNT or shCCL5 (sh-1 or sh-2) was collected and added to GBM cells. GBM cells were treated with TMZ (500 μmol/L) after CM addition. ** P < 0.01. c Cell survival analysis of GBM-1 cells with indicated treatments. Pericyte CM was pretreated with neutralizing anti-CCL5 antibody or IgG before being added to GBM cells. GBM cells were treated with TMZ (500 μmol/L) after pericyte CM addition. ** P < 0.01. d Apoptosis analysis of GBM-1 cells with indicated treatments. GBM cells expressing shNT or shCCR5 (sh-1 or sh-2) were pretreated with pericyte CM or control GBM cell CM followed by TMZ (500 μmol/L) treatment. * P < 0.05; ** P < 0.01. e Schematic diagram of MVC-mediated inhibition of CCL5-CCR5 signaling. The binding of MVC to CCR5 leads to a conformational change of its extracellular domain to prevent ligand-stimulated CCR5 activation. f Apoptosis analysis of GBM-1 cells with indicated treatments. GBM-1 cells were pretreated with MVC (500 nmol/L) or DMSO for 1 h, followed by pericyte CM stimulation. Apoptosis and cell survival analyses were performed 48 h after TMZ treatment. ns, not significant. ** P < 0.01. g DNA damage after the indicated treatments was assessed by comet assay. Scale bars, 50 μm. h Quantification of percentage of cells with comet tails with indicated treatments. ns, not significant. * P < 0.05; ** P < 0.01. i , j Immunoblot analysis of phosphorylated AKT (Ser473) and AKT ( i ) or phosphorylated DNA-PKcs (Ser2056) and DNA-PKcs ( j ) in GBM-1 cells with indicated treatments. k Immunoblot analysis of phosphorylated AKT (Ser473), AKT, phosphorylated DNA-PKcs (Ser2056), DNA-PKcs, γ-H2AX in GBM-1 cells with indicated treatments. DNA-PKi (DNA-PKcs inhibitor) represents KU-57788; AKTi (AKT inhibitor) represents MK-2206. Experiments in a – k were independently performed three times.

Article Snippet: Human CCR5-specific shRNA vectors, CCL5-specific shRNA vectors and shNT vectors were purchased from Ribobio (China) with shRNA sequences listed in Supplementary information, Table .

Techniques: Expressing, Inhibition, Binding Assay, Activation Assay, Single Cell Gel Electrophoresis, Western Blot