non targeting control shntc  (Addgene inc)

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: Oncotarget

Article Title: KIF7 attenuates prostate tumor growth through LKB1-mediated AKT inhibition

doi: 10.18632/oncotarget.17421

Figure Lengend Snippet: (A) KIF7-CC inhibited AKT phosphorylation at Ser 473 , increased LKB1, LKB1 phosphorylation at Ser 428 and PTEN phosphorylation at Ser 380 /Thr 382/383 by western blot. KIF7-CC over-expression increased both nuclear and cytoplasmic LKB1 expression in PC3, C4-2B and 22Rv1 cells by western blot. As PTEN was slightly expressed in LNCaP-derived C4-2B cells, increasing protein amount and extending exposure are needed for PTEN detection in C4-2B cells. (B) correlation of KIF7 with LKB1 from cBioPortal. Downregulation of LKB1 by shRNAs restored the inhibitory effect of KIF7-CC on AKT phosphorylation at Ser 473 (C) cell proliferation (D) and colony formation (E) in PC3, C4-2B and 22Rv1 cells by western blot, cell number counting as well as colony formation assays, respectively. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with KIF7-CC shNTC, mean ± SD was presented.

Article Snippet: LKB1 knockdown in the KIF7-CC over-expression clones from PC3, C4-2B and 22Rv1 cells was performed by infection with lentivirus that expressed human LKB1-specific short hairpin RNAs (shH and shM) (61231 & 61242) or non-target control shRNA (shNTC) (1864), which were packaged with psPAX2 (12260) and pMD2.G (12259) from Addgene (Cambrige, MA; http://www.addgene.org ) using lipofectamine 2000.

Techniques: Phospho-proteomics, Western Blot, Over Expression, Expressing, Derivative Assay

Journal: Oncotarget

Article Title: KIF7 attenuates prostate tumor growth through LKB1-mediated AKT inhibition

doi: 10.18632/oncotarget.17421

Figure Lengend Snippet: Stable knockdown of LKB1 by shRNAs and the non-target control (shNTC) in PC3 and 22Rv1 KIF7-CC over-expression cells together with their parental controls were subcutaneously implanted into the nude mice. (A&D) tumor growth was monitored at various time points. (B&E) representative tumor images from PC3 and 22Rv1 cell-derived xenografts were shown. (C&F) bar-char summarizing the tumor weights from different groups; *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with the KIF7-CC shNTC group. Data represent the mean ± SD of different group, there are 6 mice in each group. (G) schematic representation depicting inhibitory effect of KIF7-CC on AKT through LKB1/PTEN signaling.

Article Snippet: LKB1 knockdown in the KIF7-CC over-expression clones from PC3, C4-2B and 22Rv1 cells was performed by infection with lentivirus that expressed human LKB1-specific short hairpin RNAs (shH and shM) (61231 & 61242) or non-target control shRNA (shNTC) (1864), which were packaged with psPAX2 (12260) and pMD2.G (12259) from Addgene (Cambrige, MA; http://www.addgene.org ) using lipofectamine 2000.

Techniques: Knockdown, Control, Over Expression, Derivative Assay