human colon cancer cell lines hct116 (ATCC)

ATCC human colon cancer cell lines hct116

Natural compounds inhibit the growth of human colon cancer cells. The chemical structure of aMan ( A ), Pae ( B ), aMan1 ( C ), and Pae1 ( D ). aMan (5, 10, 25, and 40 μmol/L; E ), aMan1 (5, 10, 25, and 40 μmol/L; F ), Pae (100, 200, 300, and 400 μmol/L; G ), or Pae1 (3, 10, 25, and 40 μmol/L; H ) were added to the BjhTERT, <t>HCT116,</t> <t>HT29,</t> and SW48 cell lines at the indicated concentrations for 72 hours. The viability of the cells was quantified by crystal violet assay. I, The IC 50 values of aMan, aMan1, Pae, and Pae1 for the indicated cell lines were calculated at 72 hours. E, F, G, H , and I indicate mean ± SD of three independent experiments. The statistical significance between two groups were analyzed by a two-tailed unpaired t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Human Colon Cancer Cell Lines Hct116, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fxcycle+violet+dna+stain/pmc09398122-39-1-7?v=ATCC
Average 99 stars, based on 1 article reviews

human colon cancer cell lines hct116 - by Bioz Stars, 2026-07

99/100 stars

Buy from Supplier

mouse rnf183 (Integrated DNA Technologies)

Integrated DNA Technologies mouse rnf183

<t>RNF183</t> is predominantly expressed in the renal medulla. A, schematic representation of the predicted domain organization of mouse RNF183 (middle), deletion mutant RNF183 (amino acids 61–158) (top), and GFP-RNF183 (bottom) used in this study. Anti-RNF183 antibody was generated using deletion mutant RNF183. The number on the right indicates the peptide length. B, Western blotting of HEK293 cells transfected with GFP-RNF183. The anti-RNF183 and GFP antibody recognized a GFP-RNF183 at ∼50 kDa. C, confocal images of immunofluorescence with anti-RNF183 antibody (top, red) and GFP signals (middle, green) in HeLa cells transfected with GFP-RNF183. Bar, 10 μm. D, confocal images of immunofluorescence with GFP signals (middle panels, green) and various antibodies for organelle markers (EEA1 and Lamp1; top panels, red) in HeLa cells transfected with GFP-RNF183. Bars, 10 μm. E, RT-PCR analysis of Rnf183 mRNA in 10 tissues from mice. F, tissue immunoblot analysis of RNF183. Tissue lysates containing equal amounts of total protein were analyzed by Western blotting. G and H, expression patterns of RNF183 mRNA and protein in the renal cortex and medulla. Mouse tissue lysates were analyzed by RT-PCR (G) and Western blotting (H). Liver was used as a negative control. Megalin and AKR1B1 were used as positive controls for the renal cortex and medulla, respectively. Arrowhead, full-length megalin (H, middle). The results shown are representative of at least three replicates in independent observations.

Mouse Rnf183, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fxcycle+violet+dna+stain/pmc06322883-551-16-9?v=Integrated+DNA+Technologies
Average 95 stars, based on 1 article reviews

mouse rnf183 - by Bioz Stars, 2026-07

95/100 stars

Buy from Supplier

endothelial growth factors (egm-2 (Lonza)

Lonza endothelial growth factors (egm-2

A, HUVECs were treated with or without DHC (0.3–10 µM) in <t>EGM-2</t> medium. After 72 h of incubation, cells were stained with crystal violet and determined the inhibition of cell proliferation by the absorbance at 550 nm. B, DNA synthesis was assessed by BrdU incorporation assay. C, representative photographs of capillary-like structures formation of CTL and DHC-treated HUVECs on matrigel under microscope (magnification is X100). D, Quantification of the total tube length of capillary-like structures by image analysis software. Data represent the mean ± SEM from three independent experiments. ** P <0.01 and *** P <0.001 versus control. E, effect of DHC on cell migration using a transwell assay. F, the graph shows quantitative analysis of the migrated cell numbers in tranwell assay. Data represent the mean ± SEM from three independent experiments. ## P <0.01 versus basal.

Endothelial Growth Factors (Egm 2, supplied by Lonza, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fxcycle+violet+dna+stain/pmc03281050-140-6-13?v=Lonza
Average 90 stars, based on 1 article reviews

endothelial growth factors (egm-2 - by Bioz Stars, 2026-07

90/100 stars

Buy from Supplier

dna dye fxcycle violet (Thermo Fisher)

Thermo Fisher dna dye fxcycle violet

Dna Dye Fxcycle Violet, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fxcycle+violet+dna+stain/pm38943343-167-4-8?v=Thermo+Fisher
Average 99 stars, based on 1 article reviews

dna dye fxcycle violet - by Bioz Stars, 2026-07

99/100 stars

Buy from Supplier

bovine serum albumin bsa (Jackson Immuno)

Jackson Immuno bovine serum albumin bsa

Bovine Serum Albumin Bsa, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fxcycle+violet+dna+stain/pmc11216748-102-4-9?v=Jackson+Immuno
Average 96 stars, based on 1 article reviews

bovine serum albumin bsa - by Bioz Stars, 2026-07

96/100 stars

Buy from Supplier

crystal violet dna staining (BMG Labtech)

BMG Labtech crystal violet dna staining

Crystal Violet Dna Staining, supplied by BMG Labtech, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fxcycle+violet+dna+stain/pm39707547-115-18-29?v=BMG+Labtech
Average 99 stars, based on 1 article reviews

crystal violet dna staining - by Bioz Stars, 2026-07

99/100 stars

Buy from Supplier

fxcycle violet dna stain (Thermo Fisher)

Thermo Fisher fxcycle violet dna stain

Representative xCELLigence RTCA impendence measurements of ( a ) A549 and ( b ) MCF10A cells treated with 10 μM GC7 (indicated by the dotted line) and continuously monitored in technical duplicate. Cell index is an arbitrary value of impedance generated from proliferating cells. c) Cell index at 48 h 10 μM GC7 treatment in A549 and MCF10A cells. Error bars represent means ± SD ( n = 3 independent experiments). Statistical analysis was carried out using two-tailed unpaired student’s t test (ns = not significant and **** = p < 0.0001). d Basal, maximum and reserve capacity of oxygen consumption rates (OCR) were measured in A549 ( n = 4 technical replicates) and MCF10A ( n = 5 technical replicates) cells. OCR was normalized to total protein and error bars represent means ± SD. e Basal OCR of A549 and MCF10A cells treated with 10 μM GC7 for 6 and 24 h. OCR was normalized to total protein. Data shown represent mean ± SD from technical replicates for untreated ( n = 4), 6 h ( n = 6) and 24 h ( n = 5). f Left, representative xCELLigence RTCA impendence measurements of A549 cells treated with 10 μM GC7 (red), 200 mM ISRIB (grey), GC7 plus ISRIB (blue), or untreated (black). Error bars represent mean ± SD ( n = 3 technical replicates) and treatment point is indicated by red line. Right, cell index at 96 h. Error bars represent means ± SD ( n = 3 independent experiments). g Cell cycle distribution of A549 cells treated with 10 μM GC7 and 200 mM ISRIB for 72 h. <t>DNA</t> was stained using <t>FxCycle</t> violet dye and quantified using the Dean-Jett-Fox model. Error bars represent means ± SD ( n = 3 independent experiments). Cell index from xCELLigence RTCA instrument of ( h ) MEF WT and MEF S51A cells, or A549 following transfection with siRNAs specific to ( i ) HRI, ( j ) PERK, ( k ) OMA1 and ( l ) DELE-1, treated with GC7 for 72 h. Error bars represent means ± SD ( n = 3 independent experiments). Statistical analysis (in f – l ) was carried out using two-way ANOVA with Tukey’s multiple comparisons test (ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). Source data are provided within the Source Data file.

Fxcycle Violet Dna Stain, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fxcycle+violet+dna+stain/pmc12749925-284-6-13?v=Thermo+Fisher
Average 96 stars, based on 1 article reviews

fxcycle violet dna stain - by Bioz Stars, 2026-07

96/100 stars

Buy from Supplier

human pea 15 cdna (OriGene)

OriGene human pea 15 cdna
$ERK translocation is regulated by <t>PEA-15</t> in phosphorylation-dependent manner in LNCaP cells. (a) PEA-15 regulates ERK translocation. LNCaP cells transfected with a non-phosphorylatable mutant (S116G) or a phosphomimetic mutant of PEA-15 (S116D). The nucleocytoplasmic distribution of ERK was assessed in LNCaPmock and LNCaPHsp27 cells by immunofluorescence microscopy. Cells were immunostained with anti-ERK (red) antibodies. DAPI (blue) nuclear counterstaining was used to define the cell nuclei. (b) Levels of exogenous and endogenous PEA-15 proteins were analyzed by immunoblotting with anti-Flag and anti-PEA-15 antibodies. (c) Growth effect of LNCaP cells expressing non-phosphorylatable (S116G) and phosphomimetic (S116D) PEA-15 mutants. Cell growth rates of LNCaP cells were compared by crystal violet assay. (d) Protein levels of ERK, Hsp27, Oct-4 and vinculin were determined in nuclear and cytoplasmic fractions of cell lysates derived from LN-221 versus LNCaPmock cells treated with Scr or siHsp27 by immunoblotting. shRNA-mediated silencing of PEA-15 was examined by immunoblotting. (e) ERK nuclear translocation in LNCaP cells is regulated by PEA-15. The nucleocytoplasmic localization of ERK was analyzed in LN-221 versus LNCaPmock cells treated with Scr or siHsp27 by immunofluorescence microscopy as described above. Cells were immunostained with anti-Hsp27 (green) and anti-ERK (red) antibodies. DAPI (blue) nuclear counterstaining was used to define the cell nuclei. The mean fluorescence ratios FN/C are shown for ERK. Values shown represent mean±S.D. The symbol ‘*' denotes statistical significance (P<0.05)$
Human Pea 15 Cdna, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fxcycle+violet+dna+stain/pmc03354053-482-13-18?v=OriGene
Average 90 stars, based on 1 article reviews

human pea 15 cdna - by Bioz Stars, 2026-07

90/100 stars

Buy from Supplier

c2c12 myoblast cells (ATCC)

ATCC c2c12 myoblast cells
$( A ) Experimental schema for bulk and scRNA-seq from the Pax7 CreERT2 :R26R tdT :Flk1 GF P mice. Bulk RNA-seq performed on MuSCs, ECs and single muscle fibers (SMFs) from uninjured muscle. FACS sorted MuSCs and ECs from uninjured and regenerating TA muscle (3 days following CTX) were run separately on the 10 X single cell platform and aggregated. This panel created with BioRender.com , and published using a BW26O8AXHL license with permission. ( B ) Bulk RNA-seq showing EC signature in MuSCs. Subset dividing genes that are commonly used to delineate cell identity for MuSCs, ECs and SMFs. Last column shows genes that define macrophages (Mφ), which should not be highly expressed in any on our cell types. Red dots indicate MuSCs, green dots indicate ECs and blue dots indicate SMFs. Data show mean ± SD (n=3). ( C ) UMAP from aggregated single cell RNA-seq shows expression of different phases of MuSCs (quiescent MuSCs, activated MuSCs and myoblasts), ECs (tip ECs and ECs) and from likely contaminant cells such as macrophages (Mφ) and smooth muscle cells (SMC). ( D ) UMAP from aggregated data visualized by sample day showing MuSCs segregated by the sample day but overlap in the EC population. Red dots indicate intact (day 0) and blue dots indicate 3 days following CTX. ( E ) Expression of quality control genes such as eGFP , tdTomato , CreERT2, and EC genes such as Cdh5 , Kdr, and Flt1 . ( F ) Genome browser tracks of whole muscle and TU-tagged MuSC nascent RNA (GSE97399, ). Kdr and Pecam1 expression can be found in the MuSC fraction. As control, Myh1 is highly expressed in the whole muscle preparation but largely absent in the MuSC fraction. Sdc4 and Calcr are highly expressed in MuSC and less so in the whole muscle fraction. ( G ) qPCR for Kdr, Flt1, Nrp1 and Nrp2 in EC lines (bEnd.3 and C166), muscle cell line <t>(C2C12)</t> and MuSC-derived myoblasts in growth and differentiation medium (DM) shows low level expression of VEGFRs and VEGF co-receptors. Data show mean ± SD (n=3). ( H ) RNAScope of Flt1 on freshly isolated single muscle fibers from Pax7 tdT mice shows Flt1 expression (green) and tdTomato (red) in MuSCs. Nuclei were counterstained with DAPI (blue). Scale bar indicates 5 µm. ( I ) Immunostaining for PECAM1, VE-cadherin (VE-Cad), VEGFA co-receptors (NRP1 and NRP2) and VEGFA receptors (FLT1 and FLK1) in bEnd.3 EC cell line and MuSC-derived myoblasts (MB). Nuclei were counterstained with DAPI (blue). Scale bar indicates 20 µm. Figure 1—source data 1. Measurement of EC gene signal including VEGF receptor genes in MuSCs.$
C2c12 Myoblast Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fxcycle+violet+dna+stain/pmc11216748-368-10-17?v=ATCC
Average 99 stars, based on 1 article reviews

c2c12 myoblast cells - by Bioz Stars, 2026-07

99/100 stars

Buy from Supplier

full length human sdha cdna (OriGene)

OriGene full length human sdha cdna

(A) a bottom-up coessentiality network analysis through the FIREWORKS portal between SDH subunits <t>(SDHA,</t> SDHB, SDHC, and SDHD) and other critical cellular metabolism genes. A coessentiality network of 20 positively correlated primary nodes with 5 positively correlated secondary nodes was generated. Solid red line represents a primary node that is positively coessential, and a dotted red line represents a secondary node that is positively coessential with a primary node. Note that double looping (two connections between a given gene pair) indicates that the correlation relationship is among the top-ranked for both genes at the specified rank thresholds (here, 20 for primary nodes and 5 for secondary nodes). The green module highlights genes involved in purine metabolism and the blue module highlights genes involved in pyrimidine metabolism. (B) CERES scores from DepMap for SDH subunits, GART (purine metabolism), UMPS (pyrimidine metabolism), and NDUFS1 (Complex I). (C) Immunoblot and relative viability of wildtype, SDHA knockout (SDHA KO), or SDHA KO HeLa cells reconstituted with a SDHA <t>cDNA</t> construct. (D) Immunoblot and relative cell viability of renal cancer cells wildtype or SDHA KO 786-O cells. (E) Time course treatment with 3-NPA (SDHi) in CT-26 (colorectal cancer (3NPA, 500 µM)), CAL-51 (breast cancer (3NPA, 200 µM)), H460 (lung cancer (3NPA, 200 µM)), and PC-3M (prostate cancer (3NPA, 500 µM)) cells. For all plots, data are shown as mean ± s.d. of n = 3 independent experiments. (F) Schematic of the CRISPR-based screen to identify metabolic genes required for growth under the LD 20 concentration of 3-NPA. (G) Rank-order plot highlighting sgRNAs for purine genes in 3- NPA versus DMSO from the CRISPR–Cas9 screen. Data are representative of n = 3 independent biological replicates (C, D). * P < 0.05, by one-way ANOVA with Turkey’s post-hoc test for multiple comparisons (C) or unpaired t-test for pairwise comparisons (E, D).

Full Length Human Sdha Cdna, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fxcycle+violet+dna+stain/bio_rxiv__2025__02__26__640389-153-0-13?v=OriGene
Average 93 stars, based on 1 article reviews

full length human sdha cdna - by Bioz Stars, 2026-07

93/100 stars

Buy from Supplier

a549 human lung adenocarcinoma cells (ATCC)

ATCC a549 human lung adenocarcinoma cells

<t>A549</t> (A) and H1299 (B) cells were incubated with with different concentrations of CVT-11127 (CVT) or DMSO vehicle for 24 h, as described, and cell proliferation was determined by Crystal violet assay. For a similar analysis, H460 cells (C) were treated with 1 µm CVT-11127 for 24 h. For the determination of DNA synthesis, A549 (D) and H460 (E) cells were incubated with 10 µM and 5 µM CVT-11127 or vehicle for 24 h and pulsed with [ 3 H]thymidine (1 µCi/dish) for 2 h at 37°C. Total [ 3 H]-labeled DNA was precipitated, radioactivity was quantified in a scintillation counter and normalized to protein concentration. F, MCF-7 and MDA-MB-231 breast cancer cells, and WS-1 human skin fibroblasts were incubated with 10 µM CVT-11127 (CVT) or DMSO vehicle for 24 h and cell proliferation was assessed by crystal violet staining method. E, H460 cells were incubated for 48 h with 1 µM CVT in presence of 1, 10, 50 and 100 µM sodium oleate complexed with BSA (1∶2 BSA:fatty acid ratio). Cell incubated with DMSO vehicle were considered the control group. Cell growth was estimated by crystal violet staining method. Values represent the mean±S.D. of triplicate determinations. *, p<0.05 or less vs control, by Student's t test.

A549 Human Lung Adenocarcinoma Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fxcycle+violet+dna+stain/pmc02728543-38-0-13?v=ATCC
Average 99 stars, based on 1 article reviews

a549 human lung adenocarcinoma cells - by Bioz Stars, 2026-07

99/100 stars

Buy from Supplier

c166 (ATCC)

ATCC c166
$( A ) Experimental schema for bulk and scRNA-seq from the Pax7 CreERT2 :R26R tdT :Flk1 GF P mice. Bulk RNA-seq performed on MuSCs, ECs and single muscle fibers (SMFs) from uninjured muscle. FACS sorted MuSCs and ECs from uninjured and regenerating TA muscle (3 days following CTX) were run separately on the 10 X single cell platform and aggregated. This panel created with BioRender.com , and published using a BW26O8AXHL license with permission. ( B ) Bulk RNA-seq showing EC signature in MuSCs. Subset dividing genes that are commonly used to delineate cell identity for MuSCs, ECs and SMFs. Last column shows genes that define macrophages (Mφ), which should not be highly expressed in any on our cell types. Red dots indicate MuSCs, green dots indicate ECs and blue dots indicate SMFs. Data show mean ± SD (n=3). ( C ) UMAP from aggregated single cell RNA-seq shows expression of different phases of MuSCs (quiescent MuSCs, activated MuSCs and myoblasts), ECs (tip ECs and ECs) and from likely contaminant cells such as macrophages (Mφ) and smooth muscle cells (SMC). ( D ) UMAP from aggregated data visualized by sample day showing MuSCs segregated by the sample day but overlap in the EC population. Red dots indicate intact (day 0) and blue dots indicate 3 days following CTX. ( E ) Expression of quality control genes such as eGFP , tdTomato , CreERT2, and EC genes such as Cdh5 , Kdr, and Flt1 . ( F ) Genome browser tracks of whole muscle and TU-tagged MuSC nascent RNA (GSE97399, ). Kdr and Pecam1 expression can be found in the MuSC fraction. As control, Myh1 is highly expressed in the whole muscle preparation but largely absent in the MuSC fraction. Sdc4 and Calcr are highly expressed in MuSC and less so in the whole muscle fraction. ( G ) qPCR for Kdr, Flt1, Nrp1 and Nrp2 in EC lines (bEnd.3 and <t>C166),</t> muscle cell line (C2C12) and MuSC-derived myoblasts in growth and differentiation medium (DM) shows low level expression of VEGFRs and VEGF co-receptors. Data show mean ± SD (n=3). ( H ) RNAScope of Flt1 on freshly isolated single muscle fibers from Pax7 tdT mice shows Flt1 expression (green) and tdTomato (red) in MuSCs. Nuclei were counterstained with DAPI (blue). Scale bar indicates 5 µm. ( I ) Immunostaining for PECAM1, VE-cadherin (VE-Cad), VEGFA co-receptors (NRP1 and NRP2) and VEGFA receptors (FLT1 and FLK1) in bEnd.3 EC cell line and MuSC-derived myoblasts (MB). Nuclei were counterstained with DAPI (blue). Scale bar indicates 20 µm. Figure 1—source data 1. Measurement of EC gene signal including VEGF receptor genes in MuSCs.$
C166, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/fxcycle+violet+dna+stain/pmc11216748-16-7-9?v=ATCC
Average 96 stars, based on 1 article reviews

c166 - by Bioz Stars, 2026-07

96/100 stars

Buy from Supplier

Image Search Results

Journal: Molecular Cancer Therapeutics

Article Title: Characterization of Novel α-Mangostin and Paeonol Derivatives With Cancer-Selective Cytotoxicity

doi: 10.1158/1535-7163.MCT-20-0787

Figure Lengend Snippet: Natural compounds inhibit the growth of human colon cancer cells. The chemical structure of aMan ( A ), Pae ( B ), aMan1 ( C ), and Pae1 ( D ). aMan (5, 10, 25, and 40 μmol/L; E ), aMan1 (5, 10, 25, and 40 μmol/L; F ), Pae (100, 200, 300, and 400 μmol/L; G ), or Pae1 (3, 10, 25, and 40 μmol/L; H ) were added to the BjhTERT, HCT116, HT29, and SW48 cell lines at the indicated concentrations for 72 hours. The viability of the cells was quantified by crystal violet assay. I, The IC 50 values of aMan, aMan1, Pae, and Pae1 for the indicated cell lines were calculated at 72 hours. E, F, G, H , and I indicate mean ± SD of three independent experiments. The statistical significance between two groups were analyzed by a two-tailed unpaired t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Article Snippet: Mycoplasma-free human colon cancer cell lines HCT116 (ATCC, CCL-247), HT29 (ATCC, HTB-38), or SW48 (ATCC, CCL-231) were purchased from ATCC.

Techniques: Crystal Violet Assay, Two Tailed Test

Natural compounds induce cell death, apoptosis, and DNA fragmentation in colon cancer cell lines. A, aMan (25 μmol/L) or aMan1 (25 μmol/L) was added to BjhTERT, HCT116, HT29, and SW48 cell lines. After 48 hours, the cells were stained with DAPI, and the dead cells were quantified by flow cytometry. B, Quantification of the dead cells (in percentage) in the indicated different cell lines with the indicated treatments ( n = 3). Pae and Pae1 were used at 300 and 10 μmol/L, respectively. C, Cells treated as in A were stained with Annexin V to quantify cell apoptosis by flow cytometry. D, Quantification of the apoptotic cells (in percentage) in the indicated different cell lines with the indicated treatments ( n = 3). Pae was used at 300 μmol/L, and Pae1 was used at 10 μmol/L. E, F, aMan1 (25 μmol/L) and Pae1 (10 μmol/L) was incubated with SW48 cells. After 72 hours, chromatin degradation was visualized by agarose gel electrophoresis ( E ) and confocal microscopy ( F ; white arrows). E and F show representative view of a single experiment; however, the same result was observed in at least three independent replicates. B and E indicate mean ± SD of three independent experiments. The significance between the two groups was analyzed by a two-tailed unpaired t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Journal: Molecular Cancer Therapeutics

Article Title: Characterization of Novel α-Mangostin and Paeonol Derivatives With Cancer-Selective Cytotoxicity

doi: 10.1158/1535-7163.MCT-20-0787

Figure Lengend Snippet: Natural compounds induce cell death, apoptosis, and DNA fragmentation in colon cancer cell lines. A, aMan (25 μmol/L) or aMan1 (25 μmol/L) was added to BjhTERT, HCT116, HT29, and SW48 cell lines. After 48 hours, the cells were stained with DAPI, and the dead cells were quantified by flow cytometry. B, Quantification of the dead cells (in percentage) in the indicated different cell lines with the indicated treatments ( n = 3). Pae and Pae1 were used at 300 and 10 μmol/L, respectively. C, Cells treated as in A were stained with Annexin V to quantify cell apoptosis by flow cytometry. D, Quantification of the apoptotic cells (in percentage) in the indicated different cell lines with the indicated treatments ( n = 3). Pae was used at 300 μmol/L, and Pae1 was used at 10 μmol/L. E, F, aMan1 (25 μmol/L) and Pae1 (10 μmol/L) was incubated with SW48 cells. After 72 hours, chromatin degradation was visualized by agarose gel electrophoresis ( E ) and confocal microscopy ( F ; white arrows). E and F show representative view of a single experiment; however, the same result was observed in at least three independent replicates. B and E indicate mean ± SD of three independent experiments. The significance between the two groups was analyzed by a two-tailed unpaired t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Article Snippet: Mycoplasma-free human colon cancer cell lines HCT116 (ATCC, CCL-247), HT29 (ATCC, HTB-38), or SW48 (ATCC, CCL-231) were purchased from ATCC.

Techniques: Staining, Flow Cytometry, Incubation, Agarose Gel Electrophoresis, Confocal Microscopy, Two Tailed Test

Natural compounds arrest colon cancer cell lines at the G 1 or G 2 –M stage of the cell cycle. A, aMan (25 μmol/L) or aMan1 (25 μmol/L), or ( C ) Pae (300 μmol/L), or Pae1 (10 μmol/L) was added to BjhTERT, HCT116, HT29, and SW48 cell lines for 48 hours. Then the cells were incubated with 5-ethynyl-2′-deoxyuridine (EdU), a nucleoside analogue of thymidine that incorporates into DNA during active DNA synthesis. After incubation, the cells were stained with azide conjugated to Alexa fluor and DAPI and were analyzed by FACS. B–D, Quantification of the distribution in each cell-cycle phase of cells treated with the indicated compounds. DMSO was used as control vehicle. A and C indicate single experiments from two independent replicates. B and D represent mean ± SD of two independent experiments.

Journal: Molecular Cancer Therapeutics

Article Title: Characterization of Novel α-Mangostin and Paeonol Derivatives With Cancer-Selective Cytotoxicity

doi: 10.1158/1535-7163.MCT-20-0787

Figure Lengend Snippet: Natural compounds arrest colon cancer cell lines at the G 1 or G 2 –M stage of the cell cycle. A, aMan (25 μmol/L) or aMan1 (25 μmol/L), or ( C ) Pae (300 μmol/L), or Pae1 (10 μmol/L) was added to BjhTERT, HCT116, HT29, and SW48 cell lines for 48 hours. Then the cells were incubated with 5-ethynyl-2′-deoxyuridine (EdU), a nucleoside analogue of thymidine that incorporates into DNA during active DNA synthesis. After incubation, the cells were stained with azide conjugated to Alexa fluor and DAPI and were analyzed by FACS. B–D, Quantification of the distribution in each cell-cycle phase of cells treated with the indicated compounds. DMSO was used as control vehicle. A and C indicate single experiments from two independent replicates. B and D represent mean ± SD of two independent experiments.

Article Snippet: Mycoplasma-free human colon cancer cell lines HCT116 (ATCC, CCL-247), HT29 (ATCC, HTB-38), or SW48 (ATCC, CCL-231) were purchased from ATCC.

Techniques: Incubation, DNA Synthesis, Staining, Control

aMan1 and Pae1 induce higher percentage of apoptosis in HCT116 wild-type cell than TP53 knockout cells. A, aMan1 (25 μmol/L) or and Pae1 (10 μmol/L) was added to HCT116 wild-type and TP53 knockout cells. After 48 hours, the cells were stained with Annexin V and analyzed by flow cytometry. B, Quantification of apoptosis (in percentage) in WT and KO cell lines with the indicated treatments. C, MTT assay in WT and KO cell lines with the indicated treatments. D, Melting point differences between Pae1 treated versus DMSO control treated in SW48 cells. E and F, Melting curves of NOTCH1 ( D ) and IFRD2 ( E ). G, The FPKM (fragment per kilo base pair transcript per million sequenced reads) of human normal and colorectal cancer tumor samples were downloaded from The Cancer Genome Atlas (TCGA) database. H, MTT assay in SW48 cells with the indicated treatments. The P value is calculated using two-tailed unpaired t test. B, C, and H shows mean ± SD of at least three independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001).

Journal: Molecular Cancer Therapeutics

Article Title: Characterization of Novel α-Mangostin and Paeonol Derivatives With Cancer-Selective Cytotoxicity

doi: 10.1158/1535-7163.MCT-20-0787

Figure Lengend Snippet: aMan1 and Pae1 induce higher percentage of apoptosis in HCT116 wild-type cell than TP53 knockout cells. A, aMan1 (25 μmol/L) or and Pae1 (10 μmol/L) was added to HCT116 wild-type and TP53 knockout cells. After 48 hours, the cells were stained with Annexin V and analyzed by flow cytometry. B, Quantification of apoptosis (in percentage) in WT and KO cell lines with the indicated treatments. C, MTT assay in WT and KO cell lines with the indicated treatments. D, Melting point differences between Pae1 treated versus DMSO control treated in SW48 cells. E and F, Melting curves of NOTCH1 ( D ) and IFRD2 ( E ). G, The FPKM (fragment per kilo base pair transcript per million sequenced reads) of human normal and colorectal cancer tumor samples were downloaded from The Cancer Genome Atlas (TCGA) database. H, MTT assay in SW48 cells with the indicated treatments. The P value is calculated using two-tailed unpaired t test. B, C, and H shows mean ± SD of at least three independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001).

Article Snippet: Mycoplasma-free human colon cancer cell lines HCT116 (ATCC, CCL-247), HT29 (ATCC, HTB-38), or SW48 (ATCC, CCL-231) were purchased from ATCC.

Techniques: Knock-Out, Staining, Flow Cytometry, MTT Assay, Control, Two Tailed Test

RNF183 is predominantly expressed in the renal medulla. A, schematic representation of the predicted domain organization of mouse RNF183 (middle), deletion mutant RNF183 (amino acids 61–158) (top), and GFP-RNF183 (bottom) used in this study. Anti-RNF183 antibody was generated using deletion mutant RNF183. The number on the right indicates the peptide length. B, Western blotting of HEK293 cells transfected with GFP-RNF183. The anti-RNF183 and GFP antibody recognized a GFP-RNF183 at ∼50 kDa. C, confocal images of immunofluorescence with anti-RNF183 antibody (top, red) and GFP signals (middle, green) in HeLa cells transfected with GFP-RNF183. Bar, 10 μm. D, confocal images of immunofluorescence with GFP signals (middle panels, green) and various antibodies for organelle markers (EEA1 and Lamp1; top panels, red) in HeLa cells transfected with GFP-RNF183. Bars, 10 μm. E, RT-PCR analysis of Rnf183 mRNA in 10 tissues from mice. F, tissue immunoblot analysis of RNF183. Tissue lysates containing equal amounts of total protein were analyzed by Western blotting. G and H, expression patterns of RNF183 mRNA and protein in the renal cortex and medulla. Mouse tissue lysates were analyzed by RT-PCR (G) and Western blotting (H). Liver was used as a negative control. Megalin and AKR1B1 were used as positive controls for the renal cortex and medulla, respectively. Arrowhead, full-length megalin (H, middle). The results shown are representative of at least three replicates in independent observations.

Journal: The Journal of Biological Chemistry

Article Title: NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells

doi: 10.1074/jbc.RA118.002896

Figure Lengend Snippet: RNF183 is predominantly expressed in the renal medulla. A, schematic representation of the predicted domain organization of mouse RNF183 (middle), deletion mutant RNF183 (amino acids 61–158) (top), and GFP-RNF183 (bottom) used in this study. Anti-RNF183 antibody was generated using deletion mutant RNF183. The number on the right indicates the peptide length. B, Western blotting of HEK293 cells transfected with GFP-RNF183. The anti-RNF183 and GFP antibody recognized a GFP-RNF183 at ∼50 kDa. C, confocal images of immunofluorescence with anti-RNF183 antibody (top, red) and GFP signals (middle, green) in HeLa cells transfected with GFP-RNF183. Bar, 10 μm. D, confocal images of immunofluorescence with GFP signals (middle panels, green) and various antibodies for organelle markers (EEA1 and Lamp1; top panels, red) in HeLa cells transfected with GFP-RNF183. Bars, 10 μm. E, RT-PCR analysis of Rnf183 mRNA in 10 tissues from mice. F, tissue immunoblot analysis of RNF183. Tissue lysates containing equal amounts of total protein were analyzed by Western blotting. G and H, expression patterns of RNF183 mRNA and protein in the renal cortex and medulla. Mouse tissue lysates were analyzed by RT-PCR (G) and Western blotting (H). Liver was used as a negative control. Megalin and AKR1B1 were used as positive controls for the renal cortex and medulla, respectively. Arrowhead, full-length megalin (H, middle). The results shown are representative of at least three replicates in independent observations.

Article Snippet: The following TaqMan primer and probe sets, purchased from Integrated DNA Technologies (Coralville, IA) were used: mouse Rnf183 (assay ID: Mm.PT.58.42013627), mouse Akr1b1 (assay ID: 214320988), mouse Hspa1b (assay ID: Mm.PT.58.31570020.g), mouse Vegfa (assay ID: Mm.PT.58.31754187), mouse Tnfa (assay ID: Mm.PT.58.29509614), mouse Rnf130 (assay ID: Mm.PT.58.9219868), mouse Rnf150 (assay ID: Mm.PT.58.10273312), mouse Rnf149 (assay ID: Mm.PT.58.11306683), mouse Rnf133 (assay ID: Mm.PT.58.43776563.g), mouse Rnf128 (assay ID: Mm.PT.56a.8462982), mouse Rnf122 (assay ID: Mm.PT.58.8505793), mouse Rnf24 (assay ID: Mm.PT.58.9635653), mouse Rnf13 (assay ID: Mm.PT.58.11306683), mouse Rnf167 (assay ID: Mm.PT.58.32670381), mouse Znf4 (assay ID: Mm.PT.58.32478833.g), mouse Rnf43 (assay ID: Mm.PT.56a.31556976), mouse Znf3 (assay ID: Mm.PT.56a.5645486), mouse Syvn1 (assay ID: Mm.PT.58.14132695), mouse Amfr (assay ID: Mm.PT.58.9856954), mouse Rnf121 (assay ID: Mm.PT.58.6378114), mouse Rnf145 (assay ID: Mm.PT.58.10110397), mouse Rnf139 (assay ID: Mm.PT.58.45812642), mouse Rnf103 (assay ID: Mm.PT.58.9763784), mouse Rnf19A (assay ID: Mm.PT.58.28600163), mouse Rnf19B (assay ID: Mm.PT.58.28736280), mouse Rnf5 (assay ID: Mm.PT.58.14209781), mouse Rnf185 (assay ID: Mm.PT.56a.11642500), mouse Rnf170 (assay ID: Mm.PT.56a.13272002), mouse Rnf186 (assay ID: Mm.PT.56a.29338718.g), mouse Rnf152 (assay ID: Mm.PT.58.29277956.g), mouse Rnf182 (assay ID: Mm.PT.58.30902864.g), mouse Trim59 (assay ID: Mm.PT.58.30417218), mouse Trim13 (assay ID: Mm.PT.56a.9751937), mouse Bfar (assay ID: Mm.PT.56a.28494992), mouse Rnf180 (assay ID: Mm.PT.58.45835696), mouse Rnft1 (assay ID: Mm.PT.58.6836835), mouse Rnf26 (assay ID: Mm.PT.58.5764306.g), mouse Cgrnf1 (assay ID: Mm.PT.58.5664724), mouse Mul1 (assay ID: Mm.PT.58.11670267), and mouse Rnf217 (assay ID: Mm.PT.56a.33323466).

Techniques: Mutagenesis, Generated, Western Blot, Transfection, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

RNF183 is up-regulated in response to hypertonic stress. A, the hypertonic response of Rnf183 (top) and the other transmembrane RNF family member (middle, 18 of the C3H2C3 type; bottom, 17 of the C3HC4 type) mRNA levels in mIMCD-3 cells. Akr1b1 and Hspa1b were used as tonicity-dependent positive controls (top). Cells were treated with isotonic or the indicated NaCl- or sucrose-supplemented medium for 12 h and analyzed by qRT-PCR (n = 5). B and D, the tonicity-dependent induction of RNF183 and AKR1B1 protein in mIMCD-3 cells. Cells were cultured under hypertonic conditions by adding the indicated NaCl (B) or sucrose (D) supplementation for 12 h and analyzed by Western blotting. C and E, quantitative analysis of RNF183 (left) and AKR1B1 (right) protein expression in NaCl (B) and sucrose (D) (n = 5). F, the up-regulation patterns of RNF183 and AKR1B1 proteins in four renal cell lines in response to hypertonic stress. NRK-52E, NRK-49F, mIMCD-3, and HEK293 cells were treated with isotonic or 75 mm NaCl–supplemented medium for 12 h and analyzed by Western blotting. HEK293 cells transfected with mouse RNF183 were used as a positive control. Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus isotonic control).

Journal: The Journal of Biological Chemistry

Article Title: NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells

doi: 10.1074/jbc.RA118.002896

Figure Lengend Snippet: RNF183 is up-regulated in response to hypertonic stress. A, the hypertonic response of Rnf183 (top) and the other transmembrane RNF family member (middle, 18 of the C3H2C3 type; bottom, 17 of the C3HC4 type) mRNA levels in mIMCD-3 cells. Akr1b1 and Hspa1b were used as tonicity-dependent positive controls (top). Cells were treated with isotonic or the indicated NaCl- or sucrose-supplemented medium for 12 h and analyzed by qRT-PCR (n = 5). B and D, the tonicity-dependent induction of RNF183 and AKR1B1 protein in mIMCD-3 cells. Cells were cultured under hypertonic conditions by adding the indicated NaCl (B) or sucrose (D) supplementation for 12 h and analyzed by Western blotting. C and E, quantitative analysis of RNF183 (left) and AKR1B1 (right) protein expression in NaCl (B) and sucrose (D) (n = 5). F, the up-regulation patterns of RNF183 and AKR1B1 proteins in four renal cell lines in response to hypertonic stress. NRK-52E, NRK-49F, mIMCD-3, and HEK293 cells were treated with isotonic or 75 mm NaCl–supplemented medium for 12 h and analyzed by Western blotting. HEK293 cells transfected with mouse RNF183 were used as a positive control. Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus isotonic control).

Techniques: Quantitative RT-PCR, Cell Culture, Western Blot, Expressing, Transfection, Positive Control

RNF183 is up-regulated concurrently with NFAT5 activation. A, nuclear translocation of NFAT5 in response to hypertonic stress. Mouse IMCD-3 cells were treated with hypertonic medium (75 mm NaCl– or 150 mm sucrose–supplemented medium) for the indicated time and subjected to immunofluorescence staining with anti-NFAT5 antibody (top panels, green). Bars, 20 μm. The NFAT5 fluorescence intensities of cells were plotted on line graphs (bottom panels). Gray lines correspond to the relative fluorescence of cells marked with gray arrows. B, increase in total NFAT5 protein abundance in response to hypertonic stress. Mouse IMCD-3 cells were treated with 75 mm NaCl–supplemented medium for the indicated time and analyzed by Western blotting. C, quantification of data from B (n = 4). D and E, time-course analysis of mRNA and protein in response to hypertonic stress. Mouse IMCD-3 cells were treated with NaCl-supplemented medium for the indicated time and analyzed by qRT-PCR (D; n = 6) or by Western blotting (E). F, quantitative analysis of RNF183 (left) and AKR1B1 (right) expression in E (n = 5). Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus isotonic control).

Journal: The Journal of Biological Chemistry

Article Title: NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells

doi: 10.1074/jbc.RA118.002896

Figure Lengend Snippet: RNF183 is up-regulated concurrently with NFAT5 activation. A, nuclear translocation of NFAT5 in response to hypertonic stress. Mouse IMCD-3 cells were treated with hypertonic medium (75 mm NaCl– or 150 mm sucrose–supplemented medium) for the indicated time and subjected to immunofluorescence staining with anti-NFAT5 antibody (top panels, green). Bars, 20 μm. The NFAT5 fluorescence intensities of cells were plotted on line graphs (bottom panels). Gray lines correspond to the relative fluorescence of cells marked with gray arrows. B, increase in total NFAT5 protein abundance in response to hypertonic stress. Mouse IMCD-3 cells were treated with 75 mm NaCl–supplemented medium for the indicated time and analyzed by Western blotting. C, quantification of data from B (n = 4). D and E, time-course analysis of mRNA and protein in response to hypertonic stress. Mouse IMCD-3 cells were treated with NaCl-supplemented medium for the indicated time and analyzed by qRT-PCR (D; n = 6) or by Western blotting (E). F, quantitative analysis of RNF183 (left) and AKR1B1 (right) expression in E (n = 5). Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus isotonic control).

Techniques: Activation Assay, Translocation Assay, Immunofluorescence, Staining, Fluorescence, Western Blot, Quantitative RT-PCR, Expressing

NFAT5 knockdown and p38/MAPK inhibitor SB203580 attenuate hypertonicity-induced RNF183 expression. A, inhibition of NFAT5 expression using siRNA. Mouse IMCD-3 cells transfected with NFAT5 siRNA 1 or NC siRNA were treated with isotonic or 75 mm NaCl– or 150 mm sucrose–supplemented medium for 12 h and analyzed by Western blotting. B, quantification of data from A (n = 4). C, effect of NFAT5 knockdown on RNF183 protein expression induced by hypertonicity. D, quantitative analysis of RNF183 in C (n = 4). E, effect of NFAT5 knockdown on Rnf183 (left) and Akr1b1 (right) mRNA (n = 5). F and G, effect of p38/MAPK inhibitor SB203580 on mRNA expression of Rnf183, Akr1b1, and Sgk1 (F) and protein expression of RNF183 (G). Cells were treated with 75 mm NaCl for 12 h in the presence of the indicated concentrations of SB203580 (1 h of preincubation) and analyzed by qRT-PCR (F) (n = 6) or Western blotting (G). Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus NC).

Journal: The Journal of Biological Chemistry

Article Title: NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells

doi: 10.1074/jbc.RA118.002896

Figure Lengend Snippet: NFAT5 knockdown and p38/MAPK inhibitor SB203580 attenuate hypertonicity-induced RNF183 expression. A, inhibition of NFAT5 expression using siRNA. Mouse IMCD-3 cells transfected with NFAT5 siRNA 1 or NC siRNA were treated with isotonic or 75 mm NaCl– or 150 mm sucrose–supplemented medium for 12 h and analyzed by Western blotting. B, quantification of data from A (n = 4). C, effect of NFAT5 knockdown on RNF183 protein expression induced by hypertonicity. D, quantitative analysis of RNF183 in C (n = 4). E, effect of NFAT5 knockdown on Rnf183 (left) and Akr1b1 (right) mRNA (n = 5). F and G, effect of p38/MAPK inhibitor SB203580 on mRNA expression of Rnf183, Akr1b1, and Sgk1 (F) and protein expression of RNF183 (G). Cells were treated with 75 mm NaCl for 12 h in the presence of the indicated concentrations of SB203580 (1 h of preincubation) and analyzed by qRT-PCR (F) (n = 6) or Western blotting (G). Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus NC).

Techniques: Expressing, Inhibition, Transfection, Western Blot, Quantitative RT-PCR

Identification of the Rnf183 enhancer region that responds to hypertonic stress. A, screen capture from the ECR Browser website (http://ecrbrowser.dcode.org/) (57) (please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site) of the human RNF183 gene with ECR in the genomes of mouse, rat, canis, cattle, rhesus, and chimpanzee. The peaks in red show comparative pairwise ECR between human and the indicated mammals. The 319-bp region (−4,367 to −4,049 region; numbers indicate nucleotide positions relative to the transcription start site) is conserved in mammals (blue). B, location and sequence of the conserved NFAT5 DNA–binding site (yellow background with underline; mouse, −3,342 to −3,332 bp; rat, −1,870 to −1,860 bp; human, −4,256 to −4,246 bp). Conserved bases around the binding site are indicated as blue background. NFAT5 consensus sequence (RNRNNTTTCCA) is indicated below the site. C, scheme of the mouse Rnf183 promoter region and reporter constructs. Two NFAT5 DNA–binding sites are indicated in 3.5 kbp upstream of the mouse Rnf183 start site. 3.5 kbp-Luc and 2.0 kbp-Luc constructs consist of ∼3.5 kbp and 2.0 kbp upstream of the mouse Rnf183 transcription start site, respectively. D, effect of hypertonic stress on luciferase activities. Mouse IMCD-3 cells transfected with 3.5 kbp-Luc or 2.0 kbp-Luc constructs were treated with isotonic or 75 mm NaCl–supplemented medium for 24 h. The pGL4-Luc without the promoter was used as a control. Firefly luciferase activities were normalized to Renilla luciferase signals (n = 3). Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). **, p < 0.01; N.S., p > 0.05.

Journal: The Journal of Biological Chemistry

Article Title: NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells

doi: 10.1074/jbc.RA118.002896

Figure Lengend Snippet: Identification of the Rnf183 enhancer region that responds to hypertonic stress. A, screen capture from the ECR Browser website (http://ecrbrowser.dcode.org/) (57) (please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site) of the human RNF183 gene with ECR in the genomes of mouse, rat, canis, cattle, rhesus, and chimpanzee. The peaks in red show comparative pairwise ECR between human and the indicated mammals. The 319-bp region (−4,367 to −4,049 region; numbers indicate nucleotide positions relative to the transcription start site) is conserved in mammals (blue). B, location and sequence of the conserved NFAT5 DNA–binding site (yellow background with underline; mouse, −3,342 to −3,332 bp; rat, −1,870 to −1,860 bp; human, −4,256 to −4,246 bp). Conserved bases around the binding site are indicated as blue background. NFAT5 consensus sequence (RNRNNTTTCCA) is indicated below the site. C, scheme of the mouse Rnf183 promoter region and reporter constructs. Two NFAT5 DNA–binding sites are indicated in 3.5 kbp upstream of the mouse Rnf183 start site. 3.5 kbp-Luc and 2.0 kbp-Luc constructs consist of ∼3.5 kbp and 2.0 kbp upstream of the mouse Rnf183 transcription start site, respectively. D, effect of hypertonic stress on luciferase activities. Mouse IMCD-3 cells transfected with 3.5 kbp-Luc or 2.0 kbp-Luc constructs were treated with isotonic or 75 mm NaCl–supplemented medium for 24 h. The pGL4-Luc without the promoter was used as a control. Firefly luciferase activities were normalized to Renilla luciferase signals (n = 3). Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). **, p < 0.01; N.S., p > 0.05.

Techniques: Sequencing, Binding Assay, Construct, Luciferase, Transfection

Conserved putative transcription factor binding sites in the RNF183 enhancer region Transcription factor–binding sites predicted using the JASPAR software are shown in the mammalian conserved region of RNF183 genes (mouse, −3,466 to −3,136; rat, −1,978 to −1,664; human, −4,367 to −4,049). Conserved binding sites from mice, rats, and humans were selected from the potential binding sites with a >95% chance (relative score) of binding to any of the listed transcription factors ( Table S1 ). The conserved NFAT5 DNA–binding site is indicated in boldface type. NFIC, nuclear factor 1 C-type; ZNF354C, zinc finger protein 354C; NFE2L1-MafG, nuclear factor erythroid 2–related factor 1 and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G complex; NFAT, nuclear factor of activated T cells; Atoh1, atonal homolog1; Prrx2, paired-related homeobox 2.

Journal: The Journal of Biological Chemistry

Article Title: NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells

doi: 10.1074/jbc.RA118.002896

Figure Lengend Snippet: Conserved putative transcription factor binding sites in the RNF183 enhancer region Transcription factor–binding sites predicted using the JASPAR software are shown in the mammalian conserved region of RNF183 genes (mouse, −3,466 to −3,136; rat, −1,978 to −1,664; human, −4,367 to −4,049). Conserved binding sites from mice, rats, and humans were selected from the potential binding sites with a >95% chance (relative score) of binding to any of the listed transcription factors ( Table S1 ). The conserved NFAT5 DNA–binding site is indicated in boldface type. NFIC, nuclear factor 1 C-type; ZNF354C, zinc finger protein 354C; NFE2L1-MafG, nuclear factor erythroid 2–related factor 1 and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G complex; NFAT, nuclear factor of activated T cells; Atoh1, atonal homolog1; Prrx2, paired-related homeobox 2.

Techniques: Binding Assay, Software, Sequencing

Rnf183 enhancer region depends on NFAT5. A, effect of NFAT5 knockdown on hypertonicity-induced luciferase activities. Cells cotransfected with 3.5 kbp-Luc and siRNA (NFAT5 1 or NC) were treated with isotonic or 75 mm NaCl– or sucrose–supplemented medium for 24 h (n = 3). B, effect of rescue with FLAG-NFAT5 on luciferase activities. Cells pretransfected with siRNA (NFAT5 3 or NC) were cotransfected with 3.5 kbp-Luc and either empty or pFLAG-NFAT5 vectors after 12 h of knockdown. After 24 h of cotransfection, cells were treated with isotonic or 75 mm NaCl–supplemented medium for an additional 24 h (n = 4). C, effect of p38/MAPK inhibitor SB203580 on Rnf183 luciferase activities. Cells transfected with 3.5 kbp-Luc were treated with 75 mm NaCl for 24 h in the presence of the indicated concentrations of SB203580 (1 h of preincubation) (n = 3). D, effect of overexpression with FLAG-DN-NFAT5 and FLAG-NFAT5 on luciferase activities. Cells cotransfected with 3.5 kbp-Luc and either empty, pFLAG-DN-NFAT5, or pFLAG-NFAT5 vectors were analyzed after 48 h of transfection (n = 4). Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Journal: The Journal of Biological Chemistry

Article Title: NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells

doi: 10.1074/jbc.RA118.002896

Figure Lengend Snippet: Rnf183 enhancer region depends on NFAT5. A, effect of NFAT5 knockdown on hypertonicity-induced luciferase activities. Cells cotransfected with 3.5 kbp-Luc and siRNA (NFAT5 1 or NC) were treated with isotonic or 75 mm NaCl– or sucrose–supplemented medium for 24 h (n = 3). B, effect of rescue with FLAG-NFAT5 on luciferase activities. Cells pretransfected with siRNA (NFAT5 3 or NC) were cotransfected with 3.5 kbp-Luc and either empty or pFLAG-NFAT5 vectors after 12 h of knockdown. After 24 h of cotransfection, cells were treated with isotonic or 75 mm NaCl–supplemented medium for an additional 24 h (n = 4). C, effect of p38/MAPK inhibitor SB203580 on Rnf183 luciferase activities. Cells transfected with 3.5 kbp-Luc were treated with 75 mm NaCl for 24 h in the presence of the indicated concentrations of SB203580 (1 h of preincubation) (n = 3). D, effect of overexpression with FLAG-DN-NFAT5 and FLAG-NFAT5 on luciferase activities. Cells cotransfected with 3.5 kbp-Luc and either empty, pFLAG-DN-NFAT5, or pFLAG-NFAT5 vectors were analyzed after 48 h of transfection (n = 4). Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Techniques: Luciferase, Cotransfection, Transfection, Over Expression

Rnf183 is a direct target of NFAT5. A, location and site-directed mutagenesis of the conserved NFAT5 DNA–binding site. The sequences from the pGL4–3.5 kbp-Luc of the WT and mutant are shown. Mutagenized bases are indicated by lowercase letters. B, effect of conserved site mutation on luciferase activities. Cells transfected with WT or mutant were cultured in isotonic or 75 mm NaCl–supplemented medium for 24 h. The pGL4-Luc was used as a control. Firefly luciferase activities were normalized to Renilla luciferase signals (n = 4). C, effect of NFAT5 overexpression on luciferase activities. Cells transfected with WT, mutant, empty, or pFLAG-NFAT5 were cultured in an isotonic medium for 36 h (n = 5). D, schematic representation of the Rnf183 promoter and the annealing sites of the primer set used in the ChIP assays. E, ChIP assay analysis of the NFAT5 binding to the Rnf183 promoter in mIMCD-3 cells. ChIP assays were performed with the indicated antibodies. Immunoprecipitated (IP) DNA and input DNA were subjected to RT-PCR. IgG was used as a control. F, quantitative analysis of RT-PCR in E. Results were normalized to input DNA (n = 3). Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus isotonic control).

Journal: The Journal of Biological Chemistry

Article Title: NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells

doi: 10.1074/jbc.RA118.002896

Figure Lengend Snippet: Rnf183 is a direct target of NFAT5. A, location and site-directed mutagenesis of the conserved NFAT5 DNA–binding site. The sequences from the pGL4–3.5 kbp-Luc of the WT and mutant are shown. Mutagenized bases are indicated by lowercase letters. B, effect of conserved site mutation on luciferase activities. Cells transfected with WT or mutant were cultured in isotonic or 75 mm NaCl–supplemented medium for 24 h. The pGL4-Luc was used as a control. Firefly luciferase activities were normalized to Renilla luciferase signals (n = 4). C, effect of NFAT5 overexpression on luciferase activities. Cells transfected with WT, mutant, empty, or pFLAG-NFAT5 were cultured in an isotonic medium for 36 h (n = 5). D, schematic representation of the Rnf183 promoter and the annealing sites of the primer set used in the ChIP assays. E, ChIP assay analysis of the NFAT5 binding to the Rnf183 promoter in mIMCD-3 cells. ChIP assays were performed with the indicated antibodies. Immunoprecipitated (IP) DNA and input DNA were subjected to RT-PCR. IgG was used as a control. F, quantitative analysis of RT-PCR in E. Results were normalized to input DNA (n = 3). Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus isotonic control).

Techniques: Mutagenesis, Binding Assay, Luciferase, Transfection, Cell Culture, Over Expression, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

RNF183 expression provides protection against hypertonicity-induced apoptosis. A, inhibition of Rnf183 expression using siRNA. Mouse IMCD-3 cells transfected with RNF183 siRNA or NC siRNA were analyzed using qRT-PCR (n = 3). B and C, effect of RNF183 knockdown on hypertonicity-induced apoptosis. Cells transfected with the indicated siRNAs were treated with isotonic or 200 mm NaCl–supplemented medium for 4 h and analyzed using Western blotting (B) or immunofluorescence staining (C) using anti-cleaved caspase-3–specific antibody. B, the accompanying bar graph summarizes the quantification of relative amounts of cleaved caspase-3 (n = 3). C, the cleaved caspase-3 fluorescence–positive cells (red) are marked with white arrows. Bars, 100 μm. The number of cleaved caspase-3–positive cells was counted in five different fields of view and divided by the number of DAPI-stained cells (blue) to yield the ratio of cleaved caspase-3 positive cells; the results are summarized in the accompanying bar graph (n = 3). D, effect of RNF183 knockdown on cell viability under hypertonic conditions. Cells transfected with the indicated siRNAs were treated with isotonic or 200 mm NaCl–supplemented medium for 12 h and analyzed using a crystal violet assay (n = 3). Data were analyzed using a t test (A and D) or one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction (B and C). Values represent mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus NC).

Journal: The Journal of Biological Chemistry

Article Title: NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells

doi: 10.1074/jbc.RA118.002896

Figure Lengend Snippet: RNF183 expression provides protection against hypertonicity-induced apoptosis. A, inhibition of Rnf183 expression using siRNA. Mouse IMCD-3 cells transfected with RNF183 siRNA or NC siRNA were analyzed using qRT-PCR (n = 3). B and C, effect of RNF183 knockdown on hypertonicity-induced apoptosis. Cells transfected with the indicated siRNAs were treated with isotonic or 200 mm NaCl–supplemented medium for 4 h and analyzed using Western blotting (B) or immunofluorescence staining (C) using anti-cleaved caspase-3–specific antibody. B, the accompanying bar graph summarizes the quantification of relative amounts of cleaved caspase-3 (n = 3). C, the cleaved caspase-3 fluorescence–positive cells (red) are marked with white arrows. Bars, 100 μm. The number of cleaved caspase-3–positive cells was counted in five different fields of view and divided by the number of DAPI-stained cells (blue) to yield the ratio of cleaved caspase-3 positive cells; the results are summarized in the accompanying bar graph (n = 3). D, effect of RNF183 knockdown on cell viability under hypertonic conditions. Cells transfected with the indicated siRNAs were treated with isotonic or 200 mm NaCl–supplemented medium for 12 h and analyzed using a crystal violet assay (n = 3). Data were analyzed using a t test (A and D) or one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction (B and C). Values represent mean ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus NC).

Techniques: Expressing, Inhibition, Transfection, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Fluorescence, Crystal Violet Assay

Rescue of RNF183 knockdown attenuates hypertonicity-induced apoptosis. A, resistance of 3×FLAG-RNF183 to RNF183 siRNA. Mouse IMCD-3 cells were transfected with RNF183 siRNA or NC siRNA. After 12 h, the cells were electroporated with empty or 3×FLAG-RNF183-pcDNA vectors and were analyzed by Western blotting 48 h after electroporation. B and C, effect of rescue of RNF183 knockdown on hypertonicity-induced apoptosis (B) and cell viability (C). Cells transfected with RNF183 siRNA or NC siRNA were electroporated with empty or 3×FLAG-RNF183-pcDNA vectors. After 48 h of electroporation, cells were treated with 200 mm NaCl–supplemented medium for 4 (B) and 12 h (C) and then analyzed using Western blotting (B) and a crystal violet assay (C) (n = 3), respectively. The accompanying bar graph summarizes the quantification of the relative amounts of cleaved caspase-3 (n = 3). Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). *, p < 0.05.

Journal: The Journal of Biological Chemistry

Article Title: NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells

doi: 10.1074/jbc.RA118.002896

Figure Lengend Snippet: Rescue of RNF183 knockdown attenuates hypertonicity-induced apoptosis. A, resistance of 3×FLAG-RNF183 to RNF183 siRNA. Mouse IMCD-3 cells were transfected with RNF183 siRNA or NC siRNA. After 12 h, the cells were electroporated with empty or 3×FLAG-RNF183-pcDNA vectors and were analyzed by Western blotting 48 h after electroporation. B and C, effect of rescue of RNF183 knockdown on hypertonicity-induced apoptosis (B) and cell viability (C). Cells transfected with RNF183 siRNA or NC siRNA were electroporated with empty or 3×FLAG-RNF183-pcDNA vectors. After 48 h of electroporation, cells were treated with 200 mm NaCl–supplemented medium for 4 (B) and 12 h (C) and then analyzed using Western blotting (B) and a crystal violet assay (C) (n = 3), respectively. The accompanying bar graph summarizes the quantification of the relative amounts of cleaved caspase-3 (n = 3). Data were analyzed by one-way ANOVA, followed by post hoc tests using t tests with Bonferroni correction. Values represent mean ± S.D. (error bars). *, p < 0.05.

Techniques: Transfection, Western Blot, Electroporation, Crystal Violet Assay

A, HUVECs were treated with or without DHC (0.3–10 µM) in EGM-2 medium. After 72 h of incubation, cells were stained with crystal violet and determined the inhibition of cell proliferation by the absorbance at 550 nm. B, DNA synthesis was assessed by BrdU incorporation assay. C, representative photographs of capillary-like structures formation of CTL and DHC-treated HUVECs on matrigel under microscope (magnification is X100). D, Quantification of the total tube length of capillary-like structures by image analysis software. Data represent the mean ± SEM from three independent experiments. ** P <0.01 and *** P <0.001 versus control. E, effect of DHC on cell migration using a transwell assay. F, the graph shows quantitative analysis of the migrated cell numbers in tranwell assay. Data represent the mean ± SEM from three independent experiments. ## P <0.01 versus basal.

Journal: PLoS ONE

Article Title: Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

doi: 10.1371/journal.pone.0031195

Figure Lengend Snippet: A, HUVECs were treated with or without DHC (0.3–10 µM) in EGM-2 medium. After 72 h of incubation, cells were stained with crystal violet and determined the inhibition of cell proliferation by the absorbance at 550 nm. B, DNA synthesis was assessed by BrdU incorporation assay. C, representative photographs of capillary-like structures formation of CTL and DHC-treated HUVECs on matrigel under microscope (magnification is X100). D, Quantification of the total tube length of capillary-like structures by image analysis software. Data represent the mean ± SEM from three independent experiments. ** P <0.01 and *** P <0.001 versus control. E, effect of DHC on cell migration using a transwell assay. F, the graph shows quantitative analysis of the migrated cell numbers in tranwell assay. Data represent the mean ± SEM from three independent experiments. ## P <0.01 versus basal.

Article Snippet: Endothelial cell basal medium (EBM) and endothelial growth factors (EGM-2) were purchased from Clonetics (BioWhittaker, Walkersville, MD).

Techniques: Incubation, Staining, Inhibition, DNA Synthesis, BrdU Incorporation Assay, Microscopy, Software, Migration, Transwell Assay

A, after starvation for 24 hr, HUVECs were pretreated with or without DHC for 60 min and then incubated with EGM-2 at indicated time. Cells were harvested, analyzed by western blot and probe with antibodies. B, cyclin D1 expression was inhibited by DHC in a concentration dependent manner. Actin expression served as a loading control. Data represent from three independent experiments.

Journal: PLoS ONE

Article Title: Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

doi: 10.1371/journal.pone.0031195

Figure Lengend Snippet: A, after starvation for 24 hr, HUVECs were pretreated with or without DHC for 60 min and then incubated with EGM-2 at indicated time. Cells were harvested, analyzed by western blot and probe with antibodies. B, cyclin D1 expression was inhibited by DHC in a concentration dependent manner. Actin expression served as a loading control. Data represent from three independent experiments.

Article Snippet: Endothelial cell basal medium (EBM) and endothelial growth factors (EGM-2) were purchased from Clonetics (BioWhittaker, Walkersville, MD).

Techniques: Incubation, Western Blot, Expressing, Concentration Assay

A, Western blot analysis of Akt phosphorylation inhibited by DHC with the indicated times and concentrations. B, HUVECs were transfected with either vector or myr-Akt, starved for 24 hr, and then pretreated with DHC for 1 hr followed by incubation in EGM-2 medium for 10 min. Cells were harvested and analyzed protein expression by western blot. C and D, western blot analysis of cyclin D1 expression and GSK-3β phosphorylation in HUVECs transfected with the indicated plasmids and then treated with DHC (3 and 5 µM) and Ly294002 (10 µM) for 6 hr. Data represent from three independent experiments.

Journal: PLoS ONE

Article Title: Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

doi: 10.1371/journal.pone.0031195

Figure Lengend Snippet: A, Western blot analysis of Akt phosphorylation inhibited by DHC with the indicated times and concentrations. B, HUVECs were transfected with either vector or myr-Akt, starved for 24 hr, and then pretreated with DHC for 1 hr followed by incubation in EGM-2 medium for 10 min. Cells were harvested and analyzed protein expression by western blot. C and D, western blot analysis of cyclin D1 expression and GSK-3β phosphorylation in HUVECs transfected with the indicated plasmids and then treated with DHC (3 and 5 µM) and Ly294002 (10 µM) for 6 hr. Data represent from three independent experiments.

Article Snippet: Endothelial cell basal medium (EBM) and endothelial growth factors (EGM-2) were purchased from Clonetics (BioWhittaker, Walkersville, MD).

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

A, crystal violet assay. Treatment of vector group with DHC (3 µM) in EGM-2 medium significantly decreased cell proliferation numbers compared with untreated vector group. Akt overexpression significantly increased cell proliferation in DHC-treated group. B, tube formation assay. Treatment of vector group with DHC (3 µM) abrogated tube formation compared with vector control group. Akt overexpression partial reversed the inhibitory effect. C, crystal violet assay. HUVECs were treated with DHC (3 µM) and/or LiCl (10 mM). NaCl (10 mM) was as an osmolality control. Data represent from three independent experiments.

Journal: PLoS ONE

Article Title: Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

doi: 10.1371/journal.pone.0031195

Figure Lengend Snippet: A, crystal violet assay. Treatment of vector group with DHC (3 µM) in EGM-2 medium significantly decreased cell proliferation numbers compared with untreated vector group. Akt overexpression significantly increased cell proliferation in DHC-treated group. B, tube formation assay. Treatment of vector group with DHC (3 µM) abrogated tube formation compared with vector control group. Akt overexpression partial reversed the inhibitory effect. C, crystal violet assay. HUVECs were treated with DHC (3 µM) and/or LiCl (10 mM). NaCl (10 mM) was as an osmolality control. Data represent from three independent experiments.

Article Snippet: Endothelial cell basal medium (EBM) and endothelial growth factors (EGM-2) were purchased from Clonetics (BioWhittaker, Walkersville, MD).

Techniques: Crystal Violet Assay, Plasmid Preparation, Over Expression, Tube Formation Assay

A, Western blot analysis of phosphorylation of mTOR, p70S6K, eIF4E and 4EBP in HUVECs treated with DHC for the indicated times and concentrations. B, after transfected with the indicated plasmids, HUVECs were starved for 24 hr and then pretreated with DHC followed by 10 min of EGM-2 incubation. Phosphorylation of mTOR and 4EBP were analyzed by western blot. Data represent from three independent experiments.

Journal: PLoS ONE

Article Title: Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

doi: 10.1371/journal.pone.0031195

Figure Lengend Snippet: A, Western blot analysis of phosphorylation of mTOR, p70S6K, eIF4E and 4EBP in HUVECs treated with DHC for the indicated times and concentrations. B, after transfected with the indicated plasmids, HUVECs were starved for 24 hr and then pretreated with DHC followed by 10 min of EGM-2 incubation. Phosphorylation of mTOR and 4EBP were analyzed by western blot. Data represent from three independent experiments.

Article Snippet: Endothelial cell basal medium (EBM) and endothelial growth factors (EGM-2) were purchased from Clonetics (BioWhittaker, Walkersville, MD).

Techniques: Western Blot, Transfection, Incubation

Journal: eLife

Article Title: Endothelial cell signature in muscle stem cells validated by VEGFA-FLT1-AKT1 axis promoting survival of muscle stem cell

doi: 10.7554/eLife.73592

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , Bovine serum albumin (BSA) , Jackson Immuno Research , 10001620 , FACS/Immunostaining (1–2%).

Techniques: Plasmid Preparation, RNAscope, Sequencing, Imaging, Staining, Crystal Violet Assay, Viability Assay, cDNA Synthesis, Isolation, DNA Extraction, Western Blot, Extraction, Protein Extraction, Modification, Recombinant, Transfection, Infection, Immunostaining, Software, Microscopy, Cell Culture

Journal: Nature Communications

Article Title: Inhibiting translation elongation by reducing eIF5A activity induces feedback inhibition of initiation, limiting tumour cell proliferation

doi: 10.1038/s41467-025-66531-z

Figure Lengend Snippet: Representative xCELLigence RTCA impendence measurements of ( a ) A549 and ( b ) MCF10A cells treated with 10 μM GC7 (indicated by the dotted line) and continuously monitored in technical duplicate. Cell index is an arbitrary value of impedance generated from proliferating cells. c) Cell index at 48 h 10 μM GC7 treatment in A549 and MCF10A cells. Error bars represent means ± SD ( n = 3 independent experiments). Statistical analysis was carried out using two-tailed unpaired student’s t test (ns = not significant and **** = p < 0.0001). d Basal, maximum and reserve capacity of oxygen consumption rates (OCR) were measured in A549 ( n = 4 technical replicates) and MCF10A ( n = 5 technical replicates) cells. OCR was normalized to total protein and error bars represent means ± SD. e Basal OCR of A549 and MCF10A cells treated with 10 μM GC7 for 6 and 24 h. OCR was normalized to total protein. Data shown represent mean ± SD from technical replicates for untreated ( n = 4), 6 h ( n = 6) and 24 h ( n = 5). f Left, representative xCELLigence RTCA impendence measurements of A549 cells treated with 10 μM GC7 (red), 200 mM ISRIB (grey), GC7 plus ISRIB (blue), or untreated (black). Error bars represent mean ± SD ( n = 3 technical replicates) and treatment point is indicated by red line. Right, cell index at 96 h. Error bars represent means ± SD ( n = 3 independent experiments). g Cell cycle distribution of A549 cells treated with 10 μM GC7 and 200 mM ISRIB for 72 h. DNA was stained using FxCycle violet dye and quantified using the Dean-Jett-Fox model. Error bars represent means ± SD ( n = 3 independent experiments). Cell index from xCELLigence RTCA instrument of ( h ) MEF WT and MEF S51A cells, or A549 following transfection with siRNAs specific to ( i ) HRI, ( j ) PERK, ( k ) OMA1 and ( l ) DELE-1, treated with GC7 for 72 h. Error bars represent means ± SD ( n = 3 independent experiments). Statistical analysis (in f – l ) was carried out using two-way ANOVA with Tukey’s multiple comparisons test (ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). Source data are provided within the Source Data file.

Article Snippet: Fixed cells were incubated with either FxCycle Violet DNA stain (4,6-diamidino- 2-phenylindole dihydrochloride) (Thermo Fisher Scientific) in 0.1% BSA/PBS overnight at 4 °C, or 20 μg/ml propidium iodide (PI) in 1% Triton X-100/PBS and 0.1 mg/ml RNase A overnight at room temperature.

Techniques: Generated, Two Tailed Test, Staining, Transfection

$ERK translocation is regulated by PEA-15 in phosphorylation-dependent manner in LNCaP cells. (a) PEA-15 regulates ERK translocation. LNCaP cells transfected with a non-phosphorylatable mutant (S116G) or a phosphomimetic mutant of PEA-15 (S116D). The nucleocytoplasmic distribution of ERK was assessed in LNCaPmock and LNCaPHsp27 cells by immunofluorescence microscopy. Cells were immunostained with anti-ERK (red) antibodies. DAPI (blue) nuclear counterstaining was used to define the cell nuclei. (b) Levels of exogenous and endogenous PEA-15 proteins were analyzed by immunoblotting with anti-Flag and anti-PEA-15 antibodies. (c) Growth effect of LNCaP cells expressing non-phosphorylatable (S116G) and phosphomimetic (S116D) PEA-15 mutants. Cell growth rates of LNCaP cells were compared by crystal violet assay. (d) Protein levels of ERK, Hsp27, Oct-4 and vinculin were determined in nuclear and cytoplasmic fractions of cell lysates derived from LN-221 versus LNCaPmock cells treated with Scr or siHsp27 by immunoblotting. shRNA-mediated silencing of PEA-15 was examined by immunoblotting. (e) ERK nuclear translocation in LNCaP cells is regulated by PEA-15. The nucleocytoplasmic localization of ERK was analyzed in LN-221 versus LNCaPmock cells treated with Scr or siHsp27 by immunofluorescence microscopy as described above. Cells were immunostained with anti-Hsp27 (green) and anti-ERK (red) antibodies. DAPI (blue) nuclear counterstaining was used to define the cell nuclei. The mean fluorescence ratios FN/C are shown for ERK. Values shown represent mean±S.D. The symbol ‘*' denotes statistical significance (P<0.05)$

Journal: Cell Death and Differentiation

Article Title: Hsp27 silencing coordinately inhibits proliferation and promotes Fas-induced apoptosis by regulating the PEA-15 molecular switch

doi: 10.1038/cdd.2011.184

Figure Lengend Snippet: ERK translocation is regulated by PEA-15 in phosphorylation-dependent manner in LNCaP cells. (a) PEA-15 regulates ERK translocation. LNCaP cells transfected with a non-phosphorylatable mutant (S116G) or a phosphomimetic mutant of PEA-15 (S116D). The nucleocytoplasmic distribution of ERK was assessed in LNCaPmock and LNCaPHsp27 cells by immunofluorescence microscopy. Cells were immunostained with anti-ERK (red) antibodies. DAPI (blue) nuclear counterstaining was used to define the cell nuclei. (b) Levels of exogenous and endogenous PEA-15 proteins were analyzed by immunoblotting with anti-Flag and anti-PEA-15 antibodies. (c) Growth effect of LNCaP cells expressing non-phosphorylatable (S116G) and phosphomimetic (S116D) PEA-15 mutants. Cell growth rates of LNCaP cells were compared by crystal violet assay. (d) Protein levels of ERK, Hsp27, Oct-4 and vinculin were determined in nuclear and cytoplasmic fractions of cell lysates derived from LN-221 versus LNCaPmock cells treated with Scr or siHsp27 by immunoblotting. shRNA-mediated silencing of PEA-15 was examined by immunoblotting. (e) ERK nuclear translocation in LNCaP cells is regulated by PEA-15. The nucleocytoplasmic localization of ERK was analyzed in LN-221 versus LNCaPmock cells treated with Scr or siHsp27 by immunofluorescence microscopy as described above. Cells were immunostained with anti-Hsp27 (green) and anti-ERK (red) antibodies. DAPI (blue) nuclear counterstaining was used to define the cell nuclei. The mean fluorescence ratios FN/C are shown for ERK. Values shown represent mean±S.D. The symbol ‘*' denotes statistical significance (P<0.05)

Article Snippet: Lentiviral FLAG-tagged PEA-15 (pLB-U-FPEA-15WT) was derived by PCR and Gateway cloning using the Human PEA-15 cDNA clone (TC108132, OriGene Technologies Inc., Rockville, MD, USA) as template and attB1 and attB2 containing forward 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGATTATAAAGATGATGACGATAAAGCTGAGTACGGGACCCTCCTG-3′ and reverse 5′-GGGGACCACTTTGTACAAAGCTGGGTCTCAGGCCTTCTTCGGTGG-3′ primers, respectively.

Techniques: Translocation Assay, Transfection, Mutagenesis, Immunofluorescence, Microscopy, Western Blot, Expressing, Crystal Violet Assay, Derivative Assay, shRNA, Fluorescence

Hsp27 regulates PEA-15 phosphorylation in an Akt-dependent manner in LNCaP cells. (a) Constitutive overexpression of Hsp27 induces increased phosphorylation of PEA-15 at Ser-116. Cell lysates derived from LNCaPmock and LNCaPHsp27 cells were analyzed for total Akt, phospho-Akt (Ser-473), total PEA-15, phospho-PEA-15 (Ser-116) and phospho-Foxo-1 (Ser-256) by immunoblotting. Vinculin immunoblotting was performed as a loading control. (b) siRNA-mediated silencing of Hsp27 leads to decreased levels of phospho-PEA-15 (Ser-116). LNCaP cells were transfected with various doses (5–20 nM) of Scr or siHsp27. Forty-eight hours after transfection, immunoblotting was performed to analyze the total amounts and phosphorylation states of the indicated proteins. (c and d) Hsp27 interacts with Akt and PEA-15. Lysates from LNCaPmock or LNCaPHsp27 cells or from LNCaP cells following transfection with 20 nM Scr or siHsp27 for 48 h were subjected to anti-PEA-15 or anti-Akt immunoprecipitation (IP) and subsequently immunoblotted (IB) with either anti-Hsp27 and anti-PEA-15, or anti-Hsp27 and anti-Akt antibodies. (e) Hsp27 regulates Akt activity. In vitro Akt kinase assays were performed using the Akt kinase assay kit (Cell Signaling Technology). (f) Hsp27 regulates Akt protein stability. Cells were treated with 10 μg/ml CHX for the indicated times. DMSO was used as a control. Akt and Hsp27 protein levels were analyzed by immunoblotting. Graphs represent densitometric analyses of the levels of total Akt protein from cells treated with CHX for varying lengths of time. Data are presented as means±S.D. from three independent experiments (lower panel). The symbol ‘*' denotes statistical significance (P<0.05)

Journal: Cell Death and Differentiation

Article Title: Hsp27 silencing coordinately inhibits proliferation and promotes Fas-induced apoptosis by regulating the PEA-15 molecular switch

doi: 10.1038/cdd.2011.184

Figure Lengend Snippet: Hsp27 regulates PEA-15 phosphorylation in an Akt-dependent manner in LNCaP cells. (a) Constitutive overexpression of Hsp27 induces increased phosphorylation of PEA-15 at Ser-116. Cell lysates derived from LNCaPmock and LNCaPHsp27 cells were analyzed for total Akt, phospho-Akt (Ser-473), total PEA-15, phospho-PEA-15 (Ser-116) and phospho-Foxo-1 (Ser-256) by immunoblotting. Vinculin immunoblotting was performed as a loading control. (b) siRNA-mediated silencing of Hsp27 leads to decreased levels of phospho-PEA-15 (Ser-116). LNCaP cells were transfected with various doses (5–20 nM) of Scr or siHsp27. Forty-eight hours after transfection, immunoblotting was performed to analyze the total amounts and phosphorylation states of the indicated proteins. (c and d) Hsp27 interacts with Akt and PEA-15. Lysates from LNCaPmock or LNCaPHsp27 cells or from LNCaP cells following transfection with 20 nM Scr or siHsp27 for 48 h were subjected to anti-PEA-15 or anti-Akt immunoprecipitation (IP) and subsequently immunoblotted (IB) with either anti-Hsp27 and anti-PEA-15, or anti-Hsp27 and anti-Akt antibodies. (e) Hsp27 regulates Akt activity. In vitro Akt kinase assays were performed using the Akt kinase assay kit (Cell Signaling Technology). (f) Hsp27 regulates Akt protein stability. Cells were treated with 10 μg/ml CHX for the indicated times. DMSO was used as a control. Akt and Hsp27 protein levels were analyzed by immunoblotting. Graphs represent densitometric analyses of the levels of total Akt protein from cells treated with CHX for varying lengths of time. Data are presented as means±S.D. from three independent experiments (lower panel). The symbol ‘*' denotes statistical significance (P<0.05)

Techniques: Over Expression, Derivative Assay, Western Blot, Transfection, Immunoprecipitation, Activity Assay, In Vitro, Kinase Assay

Phosphorylation of PEA-15 at Ser-116 occurs in an Akt-dependent manner in LNCaP cells. (a) Akt silencing downregulates phospho-PEA-15 expression. LNCaP cells were treated with 100 nM Scr or siAkt for 24 h. Forty-eight hours after transfection, protein lysates were analyzed by immunoblotting with the indicated antibodies. (b) LNCaP cells were treated with various concentrations of the potent small-molecule Akt inhibitor, GSK690693C, or control (DMSO). Twenty-four hours after treatment, total amounts and phosphorylation states of the indicated proteins were analyzed by immunoblotting. (c) GSK690693C inhibits the nuclear localization of ERK. Nuclear translocation of ERK was assessed by immunofluorescence microscopy 24 h after treatment with 1 μM GSK690693C or control (DMSO). Cells were immunostained with anti-ERK (red) antibodies. DAPI (blue) nuclear counterstaining was used to define the cell nuclei. The mean fluorescence ratios FN/C are shown for ERK. Values shown represent mean±S.D. The symbol ‘*' denotes statistical significance (P<0.05)

Journal: Cell Death and Differentiation

Article Title: Hsp27 silencing coordinately inhibits proliferation and promotes Fas-induced apoptosis by regulating the PEA-15 molecular switch

doi: 10.1038/cdd.2011.184

Figure Lengend Snippet: Phosphorylation of PEA-15 at Ser-116 occurs in an Akt-dependent manner in LNCaP cells. (a) Akt silencing downregulates phospho-PEA-15 expression. LNCaP cells were treated with 100 nM Scr or siAkt for 24 h. Forty-eight hours after transfection, protein lysates were analyzed by immunoblotting with the indicated antibodies. (b) LNCaP cells were treated with various concentrations of the potent small-molecule Akt inhibitor, GSK690693C, or control (DMSO). Twenty-four hours after treatment, total amounts and phosphorylation states of the indicated proteins were analyzed by immunoblotting. (c) GSK690693C inhibits the nuclear localization of ERK. Nuclear translocation of ERK was assessed by immunofluorescence microscopy 24 h after treatment with 1 μM GSK690693C or control (DMSO). Cells were immunostained with anti-ERK (red) antibodies. DAPI (blue) nuclear counterstaining was used to define the cell nuclei. The mean fluorescence ratios FN/C are shown for ERK. Values shown represent mean±S.D. The symbol ‘*' denotes statistical significance (P<0.05)

Techniques: Expressing, Transfection, Western Blot, Translocation Assay, Immunofluorescence, Microscopy, Fluorescence

Hsp27 regulates Fas-induced apoptosis by modulating FADD binding to PEA-15. (a) Hsp27 suppresses Fas-induced apoptosis. LNCaPmock and LNCaPHsp27 cells were treated with either 2.5 μg/ml CHX alone, 1.0 μg/ml agonistic anti-Fas antibody (CH-11) alone or in combination in 5% charcoal-stripped serum media. Apoptosis was assessed by measuring the percentage cells containing subdiploid DNA (% sub-G1) as determined by propidium iodide staining and flow cytometry 24 h after treatment. (b) Hsp27 silencing promotes Fas-induced apoptosis. At 48 h after transfection of LNCaP cells with 20 nM Scr or 20 nM siHsp27, apoptosis in response to CH-11 was assessed as above. (c) Hsp27 regulates association of PEA-15 with FADD. PEA-15 was immunoprecipitated from cell lysates from LNCaPmock and LNCaPHsp27 cells and from LNCaP cells transfected with 20 nM Scr or 20 nM siHsp27 and cultured for 24 h. Immune complexes were resolved on SDS-PAGE and immunoblotted for FADD and PEA-15. (d) PEA-15 silencing protects from Fas-induced apoptosis. LNCaPmock and LN-221 cells were treated in the presence or absence of 2.5 μg/ml CHX and 1.0 μg/ml CH-11. Apoptosis was assessed 24 h after treatment. (e) Role of PEA-15 in intrinsic apoptosis. LNCaPmock and LN-221 cells were treated with Scr or siHsp27 in the presence or absence of paclitaxel. Percentage of cells undergoing apoptosis was measured as above. The results (a, b, d and e) are the means of three independent experiments. Values shown represent mean±S.D. The symbol ‘*' denotes statistical significance (P<0.05)

Journal: Cell Death and Differentiation

Article Title: Hsp27 silencing coordinately inhibits proliferation and promotes Fas-induced apoptosis by regulating the PEA-15 molecular switch

doi: 10.1038/cdd.2011.184

Figure Lengend Snippet: Hsp27 regulates Fas-induced apoptosis by modulating FADD binding to PEA-15. (a) Hsp27 suppresses Fas-induced apoptosis. LNCaPmock and LNCaPHsp27 cells were treated with either 2.5 μg/ml CHX alone, 1.0 μg/ml agonistic anti-Fas antibody (CH-11) alone or in combination in 5% charcoal-stripped serum media. Apoptosis was assessed by measuring the percentage cells containing subdiploid DNA (% sub-G1) as determined by propidium iodide staining and flow cytometry 24 h after treatment. (b) Hsp27 silencing promotes Fas-induced apoptosis. At 48 h after transfection of LNCaP cells with 20 nM Scr or 20 nM siHsp27, apoptosis in response to CH-11 was assessed as above. (c) Hsp27 regulates association of PEA-15 with FADD. PEA-15 was immunoprecipitated from cell lysates from LNCaPmock and LNCaPHsp27 cells and from LNCaP cells transfected with 20 nM Scr or 20 nM siHsp27 and cultured for 24 h. Immune complexes were resolved on SDS-PAGE and immunoblotted for FADD and PEA-15. (d) PEA-15 silencing protects from Fas-induced apoptosis. LNCaPmock and LN-221 cells were treated in the presence or absence of 2.5 μg/ml CHX and 1.0 μg/ml CH-11. Apoptosis was assessed 24 h after treatment. (e) Role of PEA-15 in intrinsic apoptosis. LNCaPmock and LN-221 cells were treated with Scr or siHsp27 in the presence or absence of paclitaxel. Percentage of cells undergoing apoptosis was measured as above. The results (a, b, d and e) are the means of three independent experiments. Values shown represent mean±S.D. The symbol ‘*' denotes statistical significance (P<0.05)

Techniques: Binding Assay, Staining, Flow Cytometry, Transfection, Immunoprecipitation, Cell Culture, SDS Page

Hsp27 regulates phosphorylation status of Akt and PEA-15, nuclear translocation of ERK and Fas-induced apoptosis in androgen-independent PC-3 cells. (a) PC-3 cells were transfected with various doses (5–20 nM) of Scr or siHsp27. Forty-eight hours after transfection, immunoblotting was performed to analyze the total amounts and phosphorylation states of the indicated proteins. (b) Nuclear translocation of ERK was assessed by immunofluorescence microscopy after transfection of PC-3 cells with 20 nM Scr or 20 nM siHsp27. Cells were fixed 18 h after culturing in low-serum (0.5%) conditions and were assessed by immunofluorescence staining with anti-Hsp27 (green) and anti-ERK (red) antibodies. DAPI (blue) nuclear counterstaining was used for marking cell nuclei. The mean fluorescence ratios FN/C are shown for ERK. Values shown represent mean±S.D. The symbol ‘*' denotes statistical significance (P<0.05). (c) Assessment of the effect of Hsp27 knockdown on Fas-induced apoptosis of PC-3 cells. PC-3 cells were transfected with 20 nM Scr or 20 nM siHsp27. At 48 h after transfection, apoptosis in response to the agonistic anti-Fas antibody, CH-11, was assessed by flow cytometric analyses of the proportion of cells containing subdiploid DNA as described above

Journal: Cell Death and Differentiation

Article Title: Hsp27 silencing coordinately inhibits proliferation and promotes Fas-induced apoptosis by regulating the PEA-15 molecular switch

doi: 10.1038/cdd.2011.184

Figure Lengend Snippet: Hsp27 regulates phosphorylation status of Akt and PEA-15, nuclear translocation of ERK and Fas-induced apoptosis in androgen-independent PC-3 cells. (a) PC-3 cells were transfected with various doses (5–20 nM) of Scr or siHsp27. Forty-eight hours after transfection, immunoblotting was performed to analyze the total amounts and phosphorylation states of the indicated proteins. (b) Nuclear translocation of ERK was assessed by immunofluorescence microscopy after transfection of PC-3 cells with 20 nM Scr or 20 nM siHsp27. Cells were fixed 18 h after culturing in low-serum (0.5%) conditions and were assessed by immunofluorescence staining with anti-Hsp27 (green) and anti-ERK (red) antibodies. DAPI (blue) nuclear counterstaining was used for marking cell nuclei. The mean fluorescence ratios FN/C are shown for ERK. Values shown represent mean±S.D. The symbol ‘*' denotes statistical significance (P<0.05). (c) Assessment of the effect of Hsp27 knockdown on Fas-induced apoptosis of PC-3 cells. PC-3 cells were transfected with 20 nM Scr or 20 nM siHsp27. At 48 h after transfection, apoptosis in response to the agonistic anti-Fas antibody, CH-11, was assessed by flow cytometric analyses of the proportion of cells containing subdiploid DNA as described above

Techniques: Translocation Assay, Transfection, Western Blot, Immunofluorescence, Microscopy, Staining, Fluorescence

Hsp27 knockdown selectively inhibits growth of PTEN-deficient cells. (a) The effects of Hsp27 knockdown on cell growth were compared among 12 cell lines after transfection with 20 nM Scr or 20 nM siHsp27. The determination of cell proliferation was achieved by direct cell count at 2-day intervals up to day 7 after transfection. The date of transfection was considered as day 1. Data are presented as percentage of control. Cell numbers of control cells treated with Scr were considered as 100%. The levels of expression of PTEN and Hsp27 in each of the cell lines were determined by immunoblotting using the indicated antibodies as described above. Vinculin immunoblotting was performed as a protein loading control. (b) The levels of Hsp27, phospho-Akt and phospho-PEA-15 in BPH-1 and MDA468 transfected with 20 nM Scr or siHsp27 were assessed by immunoblotting. (c) Cell lysates from PTEN Tet-on LNCaP cells treated in the presence or absence of 1 μg/ml Dox for 24 h were analyzed for levels of PTEN, phospho-Akt and phospho-PEA-15 by immunoblotting. (d) Twenty-four hours after preculturing with or without of Dox, cells were transfected with 20 nM Scr or siHsp27, and cell growth was determined at the indicated times by direct viable cell counting. (e) Growth inhibitory effect mediated by Hsp27 knockdown in PC-3 cells after PI3K blockade with LY-294002 was determined by staining cell protein with crystal violet and measuring absorbance at 595 nm. After transfection with 20 nM Scr or siHsp27, PC-3 cells were treated with various doses of LY-294002 (2–20 μM) or control (DMSO). Absorbance of control treated with Scr was considered as 100%. The results (a, d and e) are the means of three independent experiments. Values shown represent mean±S.D. The symbol ‘*' denotes statistical significance (P<0.05)

Journal: Cell Death and Differentiation

Article Title: Hsp27 silencing coordinately inhibits proliferation and promotes Fas-induced apoptosis by regulating the PEA-15 molecular switch

doi: 10.1038/cdd.2011.184

Figure Lengend Snippet: Hsp27 knockdown selectively inhibits growth of PTEN-deficient cells. (a) The effects of Hsp27 knockdown on cell growth were compared among 12 cell lines after transfection with 20 nM Scr or 20 nM siHsp27. The determination of cell proliferation was achieved by direct cell count at 2-day intervals up to day 7 after transfection. The date of transfection was considered as day 1. Data are presented as percentage of control. Cell numbers of control cells treated with Scr were considered as 100%. The levels of expression of PTEN and Hsp27 in each of the cell lines were determined by immunoblotting using the indicated antibodies as described above. Vinculin immunoblotting was performed as a protein loading control. (b) The levels of Hsp27, phospho-Akt and phospho-PEA-15 in BPH-1 and MDA468 transfected with 20 nM Scr or siHsp27 were assessed by immunoblotting. (c) Cell lysates from PTEN Tet-on LNCaP cells treated in the presence or absence of 1 μg/ml Dox for 24 h were analyzed for levels of PTEN, phospho-Akt and phospho-PEA-15 by immunoblotting. (d) Twenty-four hours after preculturing with or without of Dox, cells were transfected with 20 nM Scr or siHsp27, and cell growth was determined at the indicated times by direct viable cell counting. (e) Growth inhibitory effect mediated by Hsp27 knockdown in PC-3 cells after PI3K blockade with LY-294002 was determined by staining cell protein with crystal violet and measuring absorbance at 595 nm. After transfection with 20 nM Scr or siHsp27, PC-3 cells were treated with various doses of LY-294002 (2–20 μM) or control (DMSO). Absorbance of control treated with Scr was considered as 100%. The results (a, d and e) are the means of three independent experiments. Values shown represent mean±S.D. The symbol ‘*' denotes statistical significance (P<0.05)

Techniques: Transfection, Cell Counting, Expressing, Western Blot, Staining

A proposed model for Hsp27/Akt/PEA-15 signaling in the regulation of Fas-mediated apoptosis and cell proliferation in PTEN-deficient cells with (right) and without (left) Hsp27 silencing

Journal: Cell Death and Differentiation

Article Title: Hsp27 silencing coordinately inhibits proliferation and promotes Fas-induced apoptosis by regulating the PEA-15 molecular switch

doi: 10.1038/cdd.2011.184

Figure Lengend Snippet: A proposed model for Hsp27/Akt/PEA-15 signaling in the regulation of Fas-mediated apoptosis and cell proliferation in PTEN-deficient cells with (right) and without (left) Hsp27 silencing

Techniques:

$( A ) Experimental schema for bulk and scRNA-seq from the Pax7 CreERT2 :R26R tdT :Flk1 GF P mice. Bulk RNA-seq performed on MuSCs, ECs and single muscle fibers (SMFs) from uninjured muscle. FACS sorted MuSCs and ECs from uninjured and regenerating TA muscle (3 days following CTX) were run separately on the 10 X single cell platform and aggregated. This panel created with BioRender.com , and published using a BW26O8AXHL license with permission. ( B ) Bulk RNA-seq showing EC signature in MuSCs. Subset dividing genes that are commonly used to delineate cell identity for MuSCs, ECs and SMFs. Last column shows genes that define macrophages (Mφ), which should not be highly expressed in any on our cell types. Red dots indicate MuSCs, green dots indicate ECs and blue dots indicate SMFs. Data show mean ± SD (n=3). ( C ) UMAP from aggregated single cell RNA-seq shows expression of different phases of MuSCs (quiescent MuSCs, activated MuSCs and myoblasts), ECs (tip ECs and ECs) and from likely contaminant cells such as macrophages (Mφ) and smooth muscle cells (SMC). ( D ) UMAP from aggregated data visualized by sample day showing MuSCs segregated by the sample day but overlap in the EC population. Red dots indicate intact (day 0) and blue dots indicate 3 days following CTX. ( E ) Expression of quality control genes such as eGFP , tdTomato , CreERT2, and EC genes such as Cdh5 , Kdr, and Flt1 . ( F ) Genome browser tracks of whole muscle and TU-tagged MuSC nascent RNA (GSE97399, ). Kdr and Pecam1 expression can be found in the MuSC fraction. As control, Myh1 is highly expressed in the whole muscle preparation but largely absent in the MuSC fraction. Sdc4 and Calcr are highly expressed in MuSC and less so in the whole muscle fraction. ( G ) qPCR for Kdr, Flt1, Nrp1 and Nrp2 in EC lines (bEnd.3 and C166), muscle cell line (C2C12) and MuSC-derived myoblasts in growth and differentiation medium (DM) shows low level expression of VEGFRs and VEGF co-receptors. Data show mean ± SD (n=3). ( H ) RNAScope of Flt1 on freshly isolated single muscle fibers from Pax7 tdT mice shows Flt1 expression (green) and tdTomato (red) in MuSCs. Nuclei were counterstained with DAPI (blue). Scale bar indicates 5 µm. ( I ) Immunostaining for PECAM1, VE-cadherin (VE-Cad), VEGFA co-receptors (NRP1 and NRP2) and VEGFA receptors (FLT1 and FLK1) in bEnd.3 EC cell line and MuSC-derived myoblasts (MB). Nuclei were counterstained with DAPI (blue). Scale bar indicates 20 µm. Figure 1—source data 1. Measurement of EC gene signal including VEGF receptor genes in MuSCs.$

Journal: eLife

Article Title: Endothelial cell signature in muscle stem cells validated by VEGFA-FLT1-AKT1 axis promoting survival of muscle stem cell

doi: 10.7554/eLife.73592

Figure Lengend Snippet: ( A ) Experimental schema for bulk and scRNA-seq from the Pax7 CreERT2 :R26R tdT :Flk1 GF P mice. Bulk RNA-seq performed on MuSCs, ECs and single muscle fibers (SMFs) from uninjured muscle. FACS sorted MuSCs and ECs from uninjured and regenerating TA muscle (3 days following CTX) were run separately on the 10 X single cell platform and aggregated. This panel created with BioRender.com , and published using a BW26O8AXHL license with permission. ( B ) Bulk RNA-seq showing EC signature in MuSCs. Subset dividing genes that are commonly used to delineate cell identity for MuSCs, ECs and SMFs. Last column shows genes that define macrophages (Mφ), which should not be highly expressed in any on our cell types. Red dots indicate MuSCs, green dots indicate ECs and blue dots indicate SMFs. Data show mean ± SD (n=3). ( C ) UMAP from aggregated single cell RNA-seq shows expression of different phases of MuSCs (quiescent MuSCs, activated MuSCs and myoblasts), ECs (tip ECs and ECs) and from likely contaminant cells such as macrophages (Mφ) and smooth muscle cells (SMC). ( D ) UMAP from aggregated data visualized by sample day showing MuSCs segregated by the sample day but overlap in the EC population. Red dots indicate intact (day 0) and blue dots indicate 3 days following CTX. ( E ) Expression of quality control genes such as eGFP , tdTomato , CreERT2, and EC genes such as Cdh5 , Kdr, and Flt1 . ( F ) Genome browser tracks of whole muscle and TU-tagged MuSC nascent RNA (GSE97399, ). Kdr and Pecam1 expression can be found in the MuSC fraction. As control, Myh1 is highly expressed in the whole muscle preparation but largely absent in the MuSC fraction. Sdc4 and Calcr are highly expressed in MuSC and less so in the whole muscle fraction. ( G ) qPCR for Kdr, Flt1, Nrp1 and Nrp2 in EC lines (bEnd.3 and C166), muscle cell line (C2C12) and MuSC-derived myoblasts in growth and differentiation medium (DM) shows low level expression of VEGFRs and VEGF co-receptors. Data show mean ± SD (n=3). ( H ) RNAScope of Flt1 on freshly isolated single muscle fibers from Pax7 tdT mice shows Flt1 expression (green) and tdTomato (red) in MuSCs. Nuclei were counterstained with DAPI (blue). Scale bar indicates 5 µm. ( I ) Immunostaining for PECAM1, VE-cadherin (VE-Cad), VEGFA co-receptors (NRP1 and NRP2) and VEGFA receptors (FLT1 and FLK1) in bEnd.3 EC cell line and MuSC-derived myoblasts (MB). Nuclei were counterstained with DAPI (blue). Scale bar indicates 20 µm. Figure 1—source data 1. Measurement of EC gene signal including VEGF receptor genes in MuSCs.

Article Snippet: Mouse bEnd.3 EC cells (CRL-229), C166 EC cells (CRL-2581), and C2C12 myoblast cells (CRL-1772) were obtained from American Type Culture Collection (ATCC).

Techniques: RNA Sequencing, Expressing, Control, Derivative Assay, RNAscope, Isolation, Immunostaining

Journal: eLife

Article Title: Endothelial cell signature in muscle stem cells validated by VEGFA-FLT1-AKT1 axis promoting survival of muscle stem cell

doi: 10.7554/eLife.73592

Figure Lengend Snippet:

Article Snippet: Mouse bEnd.3 EC cells (CRL-229), C166 EC cells (CRL-2581), and C2C12 myoblast cells (CRL-1772) were obtained from American Type Culture Collection (ATCC).

(A) a bottom-up coessentiality network analysis through the FIREWORKS portal between SDH subunits (SDHA, SDHB, SDHC, and SDHD) and other critical cellular metabolism genes. A coessentiality network of 20 positively correlated primary nodes with 5 positively correlated secondary nodes was generated. Solid red line represents a primary node that is positively coessential, and a dotted red line represents a secondary node that is positively coessential with a primary node. Note that double looping (two connections between a given gene pair) indicates that the correlation relationship is among the top-ranked for both genes at the specified rank thresholds (here, 20 for primary nodes and 5 for secondary nodes). The green module highlights genes involved in purine metabolism and the blue module highlights genes involved in pyrimidine metabolism. (B) CERES scores from DepMap for SDH subunits, GART (purine metabolism), UMPS (pyrimidine metabolism), and NDUFS1 (Complex I). (C) Immunoblot and relative viability of wildtype, SDHA knockout (SDHA KO), or SDHA KO HeLa cells reconstituted with a SDHA cDNA construct. (D) Immunoblot and relative cell viability of renal cancer cells wildtype or SDHA KO 786-O cells. (E) Time course treatment with 3-NPA (SDHi) in CT-26 (colorectal cancer (3NPA, 500 µM)), CAL-51 (breast cancer (3NPA, 200 µM)), H460 (lung cancer (3NPA, 200 µM)), and PC-3M (prostate cancer (3NPA, 500 µM)) cells. For all plots, data are shown as mean ± s.d. of n = 3 independent experiments. (F) Schematic of the CRISPR-based screen to identify metabolic genes required for growth under the LD 20 concentration of 3-NPA. (G) Rank-order plot highlighting sgRNAs for purine genes in 3- NPA versus DMSO from the CRISPR–Cas9 screen. Data are representative of n = 3 independent biological replicates (C, D). * P < 0.05, by one-way ANOVA with Turkey’s post-hoc test for multiple comparisons (C) or unpaired t-test for pairwise comparisons (E, D).

Journal: bioRxiv

Article Title: Succinate dehydrogenase activity supports de novo purine synthesis

doi: 10.1101/2025.02.26.640389

Figure Lengend Snippet: (A) a bottom-up coessentiality network analysis through the FIREWORKS portal between SDH subunits (SDHA, SDHB, SDHC, and SDHD) and other critical cellular metabolism genes. A coessentiality network of 20 positively correlated primary nodes with 5 positively correlated secondary nodes was generated. Solid red line represents a primary node that is positively coessential, and a dotted red line represents a secondary node that is positively coessential with a primary node. Note that double looping (two connections between a given gene pair) indicates that the correlation relationship is among the top-ranked for both genes at the specified rank thresholds (here, 20 for primary nodes and 5 for secondary nodes). The green module highlights genes involved in purine metabolism and the blue module highlights genes involved in pyrimidine metabolism. (B) CERES scores from DepMap for SDH subunits, GART (purine metabolism), UMPS (pyrimidine metabolism), and NDUFS1 (Complex I). (C) Immunoblot and relative viability of wildtype, SDHA knockout (SDHA KO), or SDHA KO HeLa cells reconstituted with a SDHA cDNA construct. (D) Immunoblot and relative cell viability of renal cancer cells wildtype or SDHA KO 786-O cells. (E) Time course treatment with 3-NPA (SDHi) in CT-26 (colorectal cancer (3NPA, 500 µM)), CAL-51 (breast cancer (3NPA, 200 µM)), H460 (lung cancer (3NPA, 200 µM)), and PC-3M (prostate cancer (3NPA, 500 µM)) cells. For all plots, data are shown as mean ± s.d. of n = 3 independent experiments. (F) Schematic of the CRISPR-based screen to identify metabolic genes required for growth under the LD 20 concentration of 3-NPA. (G) Rank-order plot highlighting sgRNAs for purine genes in 3- NPA versus DMSO from the CRISPR–Cas9 screen. Data are representative of n = 3 independent biological replicates (C, D). * P < 0.05, by one-way ANOVA with Turkey’s post-hoc test for multiple comparisons (C) or unpaired t-test for pairwise comparisons (E, D).

Article Snippet: Full-length human SDHA cDNA in pCMV6-entry vector containing the FLAG-tag was purchased from Origene (RC200349).

Techniques: Generated, Western Blot, Knock-Out, Construct, CRISPR, Concentration Assay

(A) Schematic illustrating the enzymes and the different substrates required for the synthesis of purines and pyrimidines, which are used in the synthesis of nucleic acids. (B) Fractional enrichment (%) of 15 N-labeled purine and pyrimidine intermediates, from wild-type or SDHA KO HeLa cells labeled with [ 15 N-(amide)]-glutamine for 1 h. (C) Fractional enrichment (%) of 15 N- labeled purine and pyrimidine intermediates from HeLa cells treated either with vehicle (DMSO) or 3-NPA (1 mM) and labeled with [ 15 N-(amide)]-glutamine for 1 h. (D) Fractional enrichment (%) of 15 N- 13 C-labeled purine intermediates, from vehicle or 3-NPA (1 mM) treated HeLa cells and labeled with [ 15 N- 13 C 2 ]–glycine for 4 h. (E) [U 14 C]-glycine radiolabeling was performed in wildtype and SDHA KO, and SDHA KO reconstituted with SDHA cDNA HeLa cells for 4 hours. RNA was extracted and radioactivity counts were measured. (F) Fractional enrichment (%) of labeled purine intermediates from vehicle (DMSO) or 3-NPA treated breast (CAL-51) and colorectal (CT-26) cancer cells, labeled with [ 15 N- 13 C 2 ]–glycine for 4 h. (G) Schematic illustrating the generation of CT26 xenograft tumors in the flanks of mice subsequently treated with vehicle or 3-NPA (30 mg/kg) prior to performing [ 15 N- 13 C 2 ]–glycine (0.5 g/kg) tracing in vivo. (H) Fractional enrichment (%) of 15 N- 13 C-labeled glycine and purine intermediates from tumors are shown, n=5. For all plots, data are shown as mean ± s.d. of at least n = 3 independent biological replicates. * P <0.05. by two-tailed Student’s t-test (B-D, F, H) or one-way ANOVA with Tuckey’s post-hoc test for multiple pairwise comparisons (E).

Journal: bioRxiv

Article Title: Succinate dehydrogenase activity supports de novo purine synthesis

doi: 10.1101/2025.02.26.640389

Figure Lengend Snippet: (A) Schematic illustrating the enzymes and the different substrates required for the synthesis of purines and pyrimidines, which are used in the synthesis of nucleic acids. (B) Fractional enrichment (%) of 15 N-labeled purine and pyrimidine intermediates, from wild-type or SDHA KO HeLa cells labeled with [ 15 N-(amide)]-glutamine for 1 h. (C) Fractional enrichment (%) of 15 N- labeled purine and pyrimidine intermediates from HeLa cells treated either with vehicle (DMSO) or 3-NPA (1 mM) and labeled with [ 15 N-(amide)]-glutamine for 1 h. (D) Fractional enrichment (%) of 15 N- 13 C-labeled purine intermediates, from vehicle or 3-NPA (1 mM) treated HeLa cells and labeled with [ 15 N- 13 C 2 ]–glycine for 4 h. (E) [U 14 C]-glycine radiolabeling was performed in wildtype and SDHA KO, and SDHA KO reconstituted with SDHA cDNA HeLa cells for 4 hours. RNA was extracted and radioactivity counts were measured. (F) Fractional enrichment (%) of labeled purine intermediates from vehicle (DMSO) or 3-NPA treated breast (CAL-51) and colorectal (CT-26) cancer cells, labeled with [ 15 N- 13 C 2 ]–glycine for 4 h. (G) Schematic illustrating the generation of CT26 xenograft tumors in the flanks of mice subsequently treated with vehicle or 3-NPA (30 mg/kg) prior to performing [ 15 N- 13 C 2 ]–glycine (0.5 g/kg) tracing in vivo. (H) Fractional enrichment (%) of 15 N- 13 C-labeled glycine and purine intermediates from tumors are shown, n=5. For all plots, data are shown as mean ± s.d. of at least n = 3 independent biological replicates. * P <0.05. by two-tailed Student’s t-test (B-D, F, H) or one-way ANOVA with Tuckey’s post-hoc test for multiple pairwise comparisons (E).

Article Snippet: Full-length human SDHA cDNA in pCMV6-entry vector containing the FLAG-tag was purchased from Origene (RC200349).

Techniques: Labeling, Radioactivity, In Vivo, Two Tailed Test

(A) Immunoblots of HEK 293E WT cells or knockout for the purine enzyme GART ( sgGART ). (B) Fractional enrichment (%) of the indicated metabolites from wildtype or GART KO HEK-293E cells and labeled with [ 15 N- 13 C 2 ]–glycine (400 µM) for 4 h and treated either with vehicle (DMSO) or 3- NPA. (C) Fractional enrichment (%) of the indicated pyrimidine intermediates from wildtype or SDHA KO (sgSDHA) HeLa cells labeled with [ 15 N-(amide)]-glutamine (4 mM) for 2 hours. (D) Fractional enrichment (%) of UMP (M+1) from wildtype or GART KO HEK-293E cells treated with vehicle (DMSO) or 3-NPA (SDHi, 1 mM) and labeled with [ 15 N-(amide)]-glutamine (4 mM) for 2 hours. (E) The relative levels of incorporation of [ 3 H] from [C-2,3- 3 H]-aspartate into RNA are shown. Labeling in HeLa cells was performed for 6 hours treated with either vehicle (DMSO), rotenone (CI inh, 1µM), or 3-NPA (SDHi, 1 mM). Radioactivity measured reflects de novo pyrimidine synthesis activity. (F) Uptake of glycine in wildtype or SDHA KO HeLa cells or wildtype HeLa cells treated with vehicle (DMSO) or 3-NPA for 8 hours and labeled with [U- 14 C]-glycine for 5 min. (G) The relative levels of incorporation of [ 14 C] from [U- 14 C]-glycine into RNA are shown. Labeling was performed in HEK 293E, H460 (lung cancer) and mouse embryonic stem (mES) cells, treated either with DMSO or 3-NPA for 9 hours. Radioactivity measured reflects de novo purine synthesis activity. (H) The relative levels of incorporation of [ 14 C] from [U- 14 C]-glycine into RNA are shown. Labeling in wildtype and SDHA KO HeLa cells was performed for 4 hours. Radioactivity measured reflects de novo purine synthesis activity. (I) Schematic illustrating the generation of CAL-51 breast cancer subcutaneous xenografts in athymic nude mice and the treatment strategy. (J) Normalized peak areas of purine metabolites IMP, AMP, and GMP in vehicle (DMSO) and 3-NPA treated mice (n=5). For all plots, data are shown as mean ± s.d. of n= 3 independent biological replicates. * P < 0.05 were determined by unpaired, two-tailed Student’s t-test for pairwise comparisons (B, G-H, J) or one-way ANOVA with Tuckey post-hoc test for multiple comparisons (D, E).

Journal: bioRxiv

Article Title: Succinate dehydrogenase activity supports de novo purine synthesis

doi: 10.1101/2025.02.26.640389

Figure Lengend Snippet: (A) Immunoblots of HEK 293E WT cells or knockout for the purine enzyme GART ( sgGART ). (B) Fractional enrichment (%) of the indicated metabolites from wildtype or GART KO HEK-293E cells and labeled with [ 15 N- 13 C 2 ]–glycine (400 µM) for 4 h and treated either with vehicle (DMSO) or 3- NPA. (C) Fractional enrichment (%) of the indicated pyrimidine intermediates from wildtype or SDHA KO (sgSDHA) HeLa cells labeled with [ 15 N-(amide)]-glutamine (4 mM) for 2 hours. (D) Fractional enrichment (%) of UMP (M+1) from wildtype or GART KO HEK-293E cells treated with vehicle (DMSO) or 3-NPA (SDHi, 1 mM) and labeled with [ 15 N-(amide)]-glutamine (4 mM) for 2 hours. (E) The relative levels of incorporation of [ 3 H] from [C-2,3- 3 H]-aspartate into RNA are shown. Labeling in HeLa cells was performed for 6 hours treated with either vehicle (DMSO), rotenone (CI inh, 1µM), or 3-NPA (SDHi, 1 mM). Radioactivity measured reflects de novo pyrimidine synthesis activity. (F) Uptake of glycine in wildtype or SDHA KO HeLa cells or wildtype HeLa cells treated with vehicle (DMSO) or 3-NPA for 8 hours and labeled with [U- 14 C]-glycine for 5 min. (G) The relative levels of incorporation of [ 14 C] from [U- 14 C]-glycine into RNA are shown. Labeling was performed in HEK 293E, H460 (lung cancer) and mouse embryonic stem (mES) cells, treated either with DMSO or 3-NPA for 9 hours. Radioactivity measured reflects de novo purine synthesis activity. (H) The relative levels of incorporation of [ 14 C] from [U- 14 C]-glycine into RNA are shown. Labeling in wildtype and SDHA KO HeLa cells was performed for 4 hours. Radioactivity measured reflects de novo purine synthesis activity. (I) Schematic illustrating the generation of CAL-51 breast cancer subcutaneous xenografts in athymic nude mice and the treatment strategy. (J) Normalized peak areas of purine metabolites IMP, AMP, and GMP in vehicle (DMSO) and 3-NPA treated mice (n=5). For all plots, data are shown as mean ± s.d. of n= 3 independent biological replicates. * P < 0.05 were determined by unpaired, two-tailed Student’s t-test for pairwise comparisons (B, G-H, J) or one-way ANOVA with Tuckey post-hoc test for multiple comparisons (D, E).

Article Snippet: Full-length human SDHA cDNA in pCMV6-entry vector containing the FLAG-tag was purchased from Origene (RC200349).

Techniques: Western Blot, Knock-Out, Labeling, Radioactivity, Activity Assay, Two Tailed Test

(A) Schematic illustrating the flow of carbons from [ 13 C 6 ]-glucose into the TCA cycle and aspartate synthesis. (B) Fractional enrichment (%) of the indicated isotopologues from wild-type and SDHA KO HeLa cells labeled with [ 13 C 6 ]-glucose for 2 h. Results show mean ± s.d., n=3 replicates per group. (C) Immunoblot of wildtype or GOT2 knockout ( sgGOT2 ) HeLa cells. GOT2 and β-actin (loading control) are indicated. (D) Fractional enrichment (%) of the indicated isotopologues from wildtype and GOT2 KO HeLa cells labeled with [ 13 C 5 ]-glutamine (4 mM) for 2 h. (E) The relative levels of incorporation of [ 14 C] into RNA are shown. Wild-type and GOT2 KO cells were labeled with [U- 14 C]-glycine for 4 hours, and the radioactivity measured reflects de novo purine synthesis. (F) Fractional enrichment (%) of indicated isotopologues from HeLa cells treated with either vehicle (DMSO) or SDHi (3-NPA) and labeled with [ 15 N- 13 C 2 ]– glycine (400 µM) in the presence or absence of aspartic acid (1 mM). (G) The relative levels of incorporation of [ 14 C] into RNA are shown. Wildtype and SDHA KO cells were labeled for 4 hours in the presence or absence of aspartic acid (1mM). The radioactivity measured reflects de novo purine synthesis activity. (H) The relative levels of incorporation of [ 14 C] into RNA are shown. HeLa cells were labeled for 6 hours and treated with either vehicle (DMSO) or 3-NPA in the presence or absence of aspartic acid (1 mM). (I) Wildtype and SDHA KO cells were cultured in the presence or absence of exogenous aspartate (1 mM) and cell number was measured via crystal violet staining after 96 h. For all plots, data are shown as mean ± s.d. of n = 3 independent biological replicates. * P < 0.05, by unpaired, two-tailed Student’s t-test (B, D) for pairwise comparisons or by one-way ANOVA coupled with Tukey’s post-hoc test for multiple comparison (F-I).

Journal: bioRxiv

Article Title: Succinate dehydrogenase activity supports de novo purine synthesis

doi: 10.1101/2025.02.26.640389

Figure Lengend Snippet: (A) Schematic illustrating the flow of carbons from [ 13 C 6 ]-glucose into the TCA cycle and aspartate synthesis. (B) Fractional enrichment (%) of the indicated isotopologues from wild-type and SDHA KO HeLa cells labeled with [ 13 C 6 ]-glucose for 2 h. Results show mean ± s.d., n=3 replicates per group. (C) Immunoblot of wildtype or GOT2 knockout ( sgGOT2 ) HeLa cells. GOT2 and β-actin (loading control) are indicated. (D) Fractional enrichment (%) of the indicated isotopologues from wildtype and GOT2 KO HeLa cells labeled with [ 13 C 5 ]-glutamine (4 mM) for 2 h. (E) The relative levels of incorporation of [ 14 C] into RNA are shown. Wild-type and GOT2 KO cells were labeled with [U- 14 C]-glycine for 4 hours, and the radioactivity measured reflects de novo purine synthesis. (F) Fractional enrichment (%) of indicated isotopologues from HeLa cells treated with either vehicle (DMSO) or SDHi (3-NPA) and labeled with [ 15 N- 13 C 2 ]– glycine (400 µM) in the presence or absence of aspartic acid (1 mM). (G) The relative levels of incorporation of [ 14 C] into RNA are shown. Wildtype and SDHA KO cells were labeled for 4 hours in the presence or absence of aspartic acid (1mM). The radioactivity measured reflects de novo purine synthesis activity. (H) The relative levels of incorporation of [ 14 C] into RNA are shown. HeLa cells were labeled for 6 hours and treated with either vehicle (DMSO) or 3-NPA in the presence or absence of aspartic acid (1 mM). (I) Wildtype and SDHA KO cells were cultured in the presence or absence of exogenous aspartate (1 mM) and cell number was measured via crystal violet staining after 96 h. For all plots, data are shown as mean ± s.d. of n = 3 independent biological replicates. * P < 0.05, by unpaired, two-tailed Student’s t-test (B, D) for pairwise comparisons or by one-way ANOVA coupled with Tukey’s post-hoc test for multiple comparison (F-I).

Article Snippet: Full-length human SDHA cDNA in pCMV6-entry vector containing the FLAG-tag was purchased from Origene (RC200349).

Techniques: Labeling, Western Blot, Knock-Out, Control, Radioactivity, Activity Assay, Cell Culture, Staining, Two Tailed Test, Comparison

(A) Uptake of glucose measured in wildtype and SDHA KO HeLa cells cultured in 10% dialyzed serum. Cells were labeled with 3 H-2-deoxyglucose over 5 min. The CPM values were normalized to the protein concentration and uptake duration. (B) Schematic illustrating the glutaminolysis pathway and reductive carboxylation. (C) Fractional enrichment (%) of glutamine (M+5), glutamate (M+5), aspartate (M+4), and citrate (M+5) from wildtype and SDHA KO HeLa cells or HeLa treated with either vehicle (DMSO) or 3-NPA (1 mM) cultured in 10% dialyzed serum and labeled with [ 13 C 5 ]–glutamine for the last 2 hours. (D) Uptake of aspartate measured in wildtype and SDHA KO HeLa cells or HeLa cells treated with vehicle or 3-NPA (1 mM) cultured in 10% dialyzed serum. Cells were labeled with 3 H-aspartate over 5 min. The CPM values were normalized to the protein concentration and uptake duration. For all plots, data are shown as mean ± s.d. of n= 3 independent biological replicates. *P < 0.05 were determined by unpaired, two-tailed Student’s t-test (A, C-D).

Journal: bioRxiv

Article Title: Succinate dehydrogenase activity supports de novo purine synthesis

doi: 10.1101/2025.02.26.640389

Figure Lengend Snippet: (A) Uptake of glucose measured in wildtype and SDHA KO HeLa cells cultured in 10% dialyzed serum. Cells were labeled with 3 H-2-deoxyglucose over 5 min. The CPM values were normalized to the protein concentration and uptake duration. (B) Schematic illustrating the glutaminolysis pathway and reductive carboxylation. (C) Fractional enrichment (%) of glutamine (M+5), glutamate (M+5), aspartate (M+4), and citrate (M+5) from wildtype and SDHA KO HeLa cells or HeLa treated with either vehicle (DMSO) or 3-NPA (1 mM) cultured in 10% dialyzed serum and labeled with [ 13 C 5 ]–glutamine for the last 2 hours. (D) Uptake of aspartate measured in wildtype and SDHA KO HeLa cells or HeLa cells treated with vehicle or 3-NPA (1 mM) cultured in 10% dialyzed serum. Cells were labeled with 3 H-aspartate over 5 min. The CPM values were normalized to the protein concentration and uptake duration. For all plots, data are shown as mean ± s.d. of n= 3 independent biological replicates. *P < 0.05 were determined by unpaired, two-tailed Student’s t-test (A, C-D).

Article Snippet: Full-length human SDHA cDNA in pCMV6-entry vector containing the FLAG-tag was purchased from Origene (RC200349).

Techniques: Cell Culture, Labeling, Protein Concentration, Two Tailed Test

(A) Schematic of the TCA cycle and relevant enzymes. (B) Immunoblots of HeLa cells wildtype or knockout for CS ( sgCS ), ACO2 ( sgACO2 ), IDH3α ( sgIDH3α ), OGDH ( sgOGDH ), SUCLG1 ( sgSUCLG1 ), SDHA ( sgSDHA ), FH ( sgFH ), or MDH2 ( sgMDH2 ). (C) The relative levels of incorporation of [ 14 C] into RNA are shown. Wildtype or TCA enzyme KO cells were labeled with [U- 14 C]-glycine for 4 h, and radioactivity counts were measured, indicating de novo purine synthesis activity. (D) Normalized peak areas of succinate and fumarate in the indicated cells cultured in 10% dialyzed serum. (E) Absolute intracellular concentration of succinate after 8 h of 3-NPA (SDHi) treatment or in SDHA KO HeLa cells, compared with vehicle or wildtype conditions. (F) Fractional enrichment (%) of the indicated isotopologues from HeLa cells labeled with [ 15 N- 13 C 2 ]–glycine for 4 h in the presence or absence of exogenous cell permeable succinate (diethyl- succinate (DES)). (G) The relative levels of incorporation of [ 14 C] into RNA are shown. HeLa cells treated with either vehicle or DES (5 mM) for 8 h. For all plots, data are shown as mean ± s.d. of n = 3 independent biological replicates. * P < 0.05, by one-way ANOVA with Tukey’s post-hoc test (C), or unpaired, two-tailed Student’s t-test for pairwise comparisons (E-G).

Journal: bioRxiv

Article Title: Succinate dehydrogenase activity supports de novo purine synthesis

doi: 10.1101/2025.02.26.640389

Figure Lengend Snippet: (A) Schematic of the TCA cycle and relevant enzymes. (B) Immunoblots of HeLa cells wildtype or knockout for CS ( sgCS ), ACO2 ( sgACO2 ), IDH3α ( sgIDH3α ), OGDH ( sgOGDH ), SUCLG1 ( sgSUCLG1 ), SDHA ( sgSDHA ), FH ( sgFH ), or MDH2 ( sgMDH2 ). (C) The relative levels of incorporation of [ 14 C] into RNA are shown. Wildtype or TCA enzyme KO cells were labeled with [U- 14 C]-glycine for 4 h, and radioactivity counts were measured, indicating de novo purine synthesis activity. (D) Normalized peak areas of succinate and fumarate in the indicated cells cultured in 10% dialyzed serum. (E) Absolute intracellular concentration of succinate after 8 h of 3-NPA (SDHi) treatment or in SDHA KO HeLa cells, compared with vehicle or wildtype conditions. (F) Fractional enrichment (%) of the indicated isotopologues from HeLa cells labeled with [ 15 N- 13 C 2 ]–glycine for 4 h in the presence or absence of exogenous cell permeable succinate (diethyl- succinate (DES)). (G) The relative levels of incorporation of [ 14 C] into RNA are shown. HeLa cells treated with either vehicle or DES (5 mM) for 8 h. For all plots, data are shown as mean ± s.d. of n = 3 independent biological replicates. * P < 0.05, by one-way ANOVA with Tukey’s post-hoc test (C), or unpaired, two-tailed Student’s t-test for pairwise comparisons (E-G).

Article Snippet: Full-length human SDHA cDNA in pCMV6-entry vector containing the FLAG-tag was purchased from Origene (RC200349).

Techniques: Western Blot, Knock-Out, Labeling, Radioactivity, Activity Assay, Cell Culture, Concentration Assay, Two Tailed Test

(A) Normalized peak areas of citrate, a-ketoglutarate, glutamate, and aspartate in CS KO, ACO2 KO, IDH3α KO, OGDH KO, SUCLG1 KO, SDHA KO, FH KO and MDH2 KO HeLa cells cultured in 10% dialyzed serum. (B) Schematic demonstrating the entry of monomethylfumarate (MMF) and its conversion to fumarate in the cell. (C) Fractional enrichment (%) of glycine (M+3), AMP (M+3) and GMP (M+3) from HeLa cells treated either with vehicle (DMSO), MMF (5 mM) and labeled with [ 15 N- 13 C 2 ]–glycine for 4 h. (D) Schematic illustrating the entry of cell permeable succinate (diethyl-succinate (DES)) and its cellular conversion into succinate. (E) Normalized peak areas of DES (M+4), and TCA cycle intermediates from cells treated with vehicle (DMSO) or 13 C 4 -diethyl-succinate (1mM) for 4 hours. (f) The relative levels of incorporation of [ 3 H] from [2,3- 3 H]-aspartate into RNA are shown. Labeling was performed in HeLa cells treated with either vehicle (DMSO) or DES (1 mM) for 4 hours. Radioactivity measured reflects de novo pyrimidine synthesis activity. For all plots, data are shown as mean ± s.d. of n = 3 independent biological replicates. *P < 0.05 were determined by one-way ANOVA with Tuckey post-hoc test for multiple comparisons (C), or unpaired, two-tailed Student’s t-test for pairwise comparisons (E, F).

Journal: bioRxiv

Article Title: Succinate dehydrogenase activity supports de novo purine synthesis

doi: 10.1101/2025.02.26.640389

Figure Lengend Snippet: (A) Normalized peak areas of citrate, a-ketoglutarate, glutamate, and aspartate in CS KO, ACO2 KO, IDH3α KO, OGDH KO, SUCLG1 KO, SDHA KO, FH KO and MDH2 KO HeLa cells cultured in 10% dialyzed serum. (B) Schematic demonstrating the entry of monomethylfumarate (MMF) and its conversion to fumarate in the cell. (C) Fractional enrichment (%) of glycine (M+3), AMP (M+3) and GMP (M+3) from HeLa cells treated either with vehicle (DMSO), MMF (5 mM) and labeled with [ 15 N- 13 C 2 ]–glycine for 4 h. (D) Schematic illustrating the entry of cell permeable succinate (diethyl-succinate (DES)) and its cellular conversion into succinate. (E) Normalized peak areas of DES (M+4), and TCA cycle intermediates from cells treated with vehicle (DMSO) or 13 C 4 -diethyl-succinate (1mM) for 4 hours. (f) The relative levels of incorporation of [ 3 H] from [2,3- 3 H]-aspartate into RNA are shown. Labeling was performed in HeLa cells treated with either vehicle (DMSO) or DES (1 mM) for 4 hours. Radioactivity measured reflects de novo pyrimidine synthesis activity. For all plots, data are shown as mean ± s.d. of n = 3 independent biological replicates. *P < 0.05 were determined by one-way ANOVA with Tuckey post-hoc test for multiple comparisons (C), or unpaired, two-tailed Student’s t-test for pairwise comparisons (E, F).

Article Snippet: Full-length human SDHA cDNA in pCMV6-entry vector containing the FLAG-tag was purchased from Origene (RC200349).

Techniques: Cell Culture, Labeling, Radioactivity, Activity Assay, Two Tailed Test

(A) CAL-51 cells treated with 3-NPA (100 µM), with or without exogenous hypoxanthine (100 µM) or inosine (100 µM). Crystal violet assay was performed after 72 h. (B) Schematic of the purine salvage pathway, highlighting 13 C or 3 H incorporation from hypoxanthine into IMP and subsequently into RNA. (C) Normalized peak areas of the indicated metabolites, measured via LC-MS, from HeLa cells labeled with 13 C 5 -hypoxanthine for 3 hours. (D) Incorporation of [ 3 H] from [ 3 H]-hypoxanthine into RNA is shown. Labeling in wildtype, SDHA KO, or SDHA KO reconstituted with SDHA cDNA HeLa cells was performed for 4 h. Radioactivity counts were measured. (E) H460 cells treated with either vehicle, 3-NPA (30 µM), 6-MP (12.5 µM), or the combination, and crystal violet assays were performed every 24 h. (F) CT-26 cells treated with either vehicle, 3- NPA (250 µM), 6-MP (2 µM), or the combination, and crystal violet assays were performed every 24 h. (G) HeLa cells treated with vehicle or diethyl-succinate (DES, 3 mM), 6-MP (3 µM), or the combination, and crystal violet assays were performed every 24 h. (H) Schematic demonstrating the generation of CT-26 subcutaneous allograft tumors in Balb/c mice and the treatment regimen. (l) Mouse body weight at study endpoint, reflecting overall health. (J) Growth curves of CT-26 xenograft tumors treated with vehicle, 3-NPA (30 mg/kg), 6-MP (15 mg/kg), or their combination (n = 6). Data represent mean ± s.d. of at least n = 3 (A, C-F) or n =6 (H, I) biological replicates. * P < 0.05, by unpaired, two-tailed Student’s t-test (C) or one-way ANOVA with Tukey’s post-hoc test (A, D-G, J).

Journal: bioRxiv

Article Title: Succinate dehydrogenase activity supports de novo purine synthesis

doi: 10.1101/2025.02.26.640389

Figure Lengend Snippet: (A) CAL-51 cells treated with 3-NPA (100 µM), with or without exogenous hypoxanthine (100 µM) or inosine (100 µM). Crystal violet assay was performed after 72 h. (B) Schematic of the purine salvage pathway, highlighting 13 C or 3 H incorporation from hypoxanthine into IMP and subsequently into RNA. (C) Normalized peak areas of the indicated metabolites, measured via LC-MS, from HeLa cells labeled with 13 C 5 -hypoxanthine for 3 hours. (D) Incorporation of [ 3 H] from [ 3 H]-hypoxanthine into RNA is shown. Labeling in wildtype, SDHA KO, or SDHA KO reconstituted with SDHA cDNA HeLa cells was performed for 4 h. Radioactivity counts were measured. (E) H460 cells treated with either vehicle, 3-NPA (30 µM), 6-MP (12.5 µM), or the combination, and crystal violet assays were performed every 24 h. (F) CT-26 cells treated with either vehicle, 3- NPA (250 µM), 6-MP (2 µM), or the combination, and crystal violet assays were performed every 24 h. (G) HeLa cells treated with vehicle or diethyl-succinate (DES, 3 mM), 6-MP (3 µM), or the combination, and crystal violet assays were performed every 24 h. (H) Schematic demonstrating the generation of CT-26 subcutaneous allograft tumors in Balb/c mice and the treatment regimen. (l) Mouse body weight at study endpoint, reflecting overall health. (J) Growth curves of CT-26 xenograft tumors treated with vehicle, 3-NPA (30 mg/kg), 6-MP (15 mg/kg), or their combination (n = 6). Data represent mean ± s.d. of at least n = 3 (A, C-F) or n =6 (H, I) biological replicates. * P < 0.05, by unpaired, two-tailed Student’s t-test (C) or one-way ANOVA with Tukey’s post-hoc test (A, D-G, J).

Article Snippet: Full-length human SDHA cDNA in pCMV6-entry vector containing the FLAG-tag was purchased from Origene (RC200349).

Techniques: Crystal Violet Assay, Liquid Chromatography with Mass Spectroscopy, Labeling, Radioactivity, Two Tailed Test

A549 (A) and H1299 (B) cells were incubated with with different concentrations of CVT-11127 (CVT) or DMSO vehicle for 24 h, as described, and cell proliferation was determined by Crystal violet assay. For a similar analysis, H460 cells (C) were treated with 1 µm CVT-11127 for 24 h. For the determination of DNA synthesis, A549 (D) and H460 (E) cells were incubated with 10 µM and 5 µM CVT-11127 or vehicle for 24 h and pulsed with [ 3 H]thymidine (1 µCi/dish) for 2 h at 37°C. Total [ 3 H]-labeled DNA was precipitated, radioactivity was quantified in a scintillation counter and normalized to protein concentration. F, MCF-7 and MDA-MB-231 breast cancer cells, and WS-1 human skin fibroblasts were incubated with 10 µM CVT-11127 (CVT) or DMSO vehicle for 24 h and cell proliferation was assessed by crystal violet staining method. E, H460 cells were incubated for 48 h with 1 µM CVT in presence of 1, 10, 50 and 100 µM sodium oleate complexed with BSA (1∶2 BSA:fatty acid ratio). Cell incubated with DMSO vehicle were considered the control group. Cell growth was estimated by crystal violet staining method. Values represent the mean±S.D. of triplicate determinations. *, p<0.05 or less vs control, by Student's t test.

Journal: PLoS ONE

Article Title: Inhibition of StearoylCoA Desaturase-1 Inactivates Acetyl-CoA Carboxylase and Impairs Proliferation in Cancer Cells: Role of AMPK

doi: 10.1371/journal.pone.0006812

Figure Lengend Snippet: A549 (A) and H1299 (B) cells were incubated with with different concentrations of CVT-11127 (CVT) or DMSO vehicle for 24 h, as described, and cell proliferation was determined by Crystal violet assay. For a similar analysis, H460 cells (C) were treated with 1 µm CVT-11127 for 24 h. For the determination of DNA synthesis, A549 (D) and H460 (E) cells were incubated with 10 µM and 5 µM CVT-11127 or vehicle for 24 h and pulsed with [ 3 H]thymidine (1 µCi/dish) for 2 h at 37°C. Total [ 3 H]-labeled DNA was precipitated, radioactivity was quantified in a scintillation counter and normalized to protein concentration. F, MCF-7 and MDA-MB-231 breast cancer cells, and WS-1 human skin fibroblasts were incubated with 10 µM CVT-11127 (CVT) or DMSO vehicle for 24 h and cell proliferation was assessed by crystal violet staining method. E, H460 cells were incubated for 48 h with 1 µM CVT in presence of 1, 10, 50 and 100 µM sodium oleate complexed with BSA (1∶2 BSA:fatty acid ratio). Cell incubated with DMSO vehicle were considered the control group. Cell growth was estimated by crystal violet staining method. Values represent the mean±S.D. of triplicate determinations. *, p<0.05 or less vs control, by Student's t test.

Article Snippet: A549 human lung adenocarcinoma cells and WS-1 human fibroblasts were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Incubation, Crystal Violet Assay, DNA Synthesis, Labeling, Radioactivity, Protein Concentration, Staining, Control

For the determination of Δ9-desaturating activity in cancer cells, A549 (A, B) and H1299 cells (A, C) were treated for 24 h with 10 µM and 5 µM CVT respectively, or DMSO vehicle. Six hours before harvesting, the cells were pulsed with [ 14 C]18:0 (0.25 µCi/dish). After conversion into methylesters, fatty acids were separated on silver nitrate-impregnated TLC plates. The radioactive spots corresponding to SCD substrate and product ([ 14 C]18∶1), were visualized with a Phosphor Imager (A) and quantified by densitometric analysis (B and C). Values represent the mean±S.D. of triplicate determinations. *, p<0.05, by Student's t test.

Journal: PLoS ONE

Article Title: Inhibition of StearoylCoA Desaturase-1 Inactivates Acetyl-CoA Carboxylase and Impairs Proliferation in Cancer Cells: Role of AMPK

doi: 10.1371/journal.pone.0006812

Figure Lengend Snippet: For the determination of Δ9-desaturating activity in cancer cells, A549 (A, B) and H1299 cells (A, C) were treated for 24 h with 10 µM and 5 µM CVT respectively, or DMSO vehicle. Six hours before harvesting, the cells were pulsed with [ 14 C]18:0 (0.25 µCi/dish). After conversion into methylesters, fatty acids were separated on silver nitrate-impregnated TLC plates. The radioactive spots corresponding to SCD substrate and product ([ 14 C]18∶1), were visualized with a Phosphor Imager (A) and quantified by densitometric analysis (B and C). Values represent the mean±S.D. of triplicate determinations. *, p<0.05, by Student's t test.

Article Snippet: A549 human lung adenocarcinoma cells and WS-1 human fibroblasts were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Activity Assay

A549 cells (A, B) and H460 cells (C, D) were incubated with 10 µM and 1 µM CVT-11127 (CVT), respectively, or DMSO for 24 h. Cellular lipids were extracted and fatty acids were converted to their methylester form by transesterification as described in . Fatty acid methyl ester composition was assessed by gas chromatography and percent distribution of fatty acids was calculated. Values express the ratio 18:1n-9/18:0 (A, C) and 16∶1-n7+18:1n-7/16:0 (B, D), and represent the mean±S.D. of 4–5 samples. *, p<0.01 or less, by Student's t test.

Journal: PLoS ONE

Article Title: Inhibition of StearoylCoA Desaturase-1 Inactivates Acetyl-CoA Carboxylase and Impairs Proliferation in Cancer Cells: Role of AMPK

doi: 10.1371/journal.pone.0006812

Figure Lengend Snippet: A549 cells (A, B) and H460 cells (C, D) were incubated with 10 µM and 1 µM CVT-11127 (CVT), respectively, or DMSO for 24 h. Cellular lipids were extracted and fatty acids were converted to their methylester form by transesterification as described in . Fatty acid methyl ester composition was assessed by gas chromatography and percent distribution of fatty acids was calculated. Values express the ratio 18:1n-9/18:0 (A, C) and 16∶1-n7+18:1n-7/16:0 (B, D), and represent the mean±S.D. of 4–5 samples. *, p<0.01 or less, by Student's t test.

Article Snippet: A549 human lung adenocarcinoma cells and WS-1 human fibroblasts were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Incubation, Gas Chromatography

A549 cells (A) and H1299 cells (B) were incubated with 10 µM and 1 µM CVT-11127 (CVT), respectively, or DMSO for 24 h. Cells were then pulsed with 1 µCi D-[U- 14 C]glucose for up to 24 h. C, A549 cells with a stable knockdown in SCD1 expression (hSCDas) and mock-transfected control cells were subjected to a similar incubation with [ 14 C]glucose. Cellular lipids were extracted and incorporation of [ 14 C]glucose into total lipids was quantified by scintillation counting and normalized to protein concentration. D, basal glucose uptake was assayed in H1299 cells treated with 1 µM CVT or vehicle for 24 h by estimating the uptake of [ 3 H]deoxyglucose. E, H1299 cells were incubated with [ 14 C]glucose in presence or absence of 1 µM CVT for 6 h and levels of total [ 14 C]fatty acids, as well as radiolabeled SFA and MUFA (upper panel), were determined by argentation TLC as described in . F, the rate of lipid synthesis in H1299 cells was assessed by incubation with 1 µM CVT or vehicle and 0.5 µCi/dish of [ 14 C]acetate for 24 h. Lipids were extracted and radioactivity of total lipids was determined by scintillation counting. Values represent the mean±S.D. of triplicate determinations. *, p<0.05 or less, by Student's t test.

Journal: PLoS ONE

Article Title: Inhibition of StearoylCoA Desaturase-1 Inactivates Acetyl-CoA Carboxylase and Impairs Proliferation in Cancer Cells: Role of AMPK

doi: 10.1371/journal.pone.0006812

Figure Lengend Snippet: A549 cells (A) and H1299 cells (B) were incubated with 10 µM and 1 µM CVT-11127 (CVT), respectively, or DMSO for 24 h. Cells were then pulsed with 1 µCi D-[U- 14 C]glucose for up to 24 h. C, A549 cells with a stable knockdown in SCD1 expression (hSCDas) and mock-transfected control cells were subjected to a similar incubation with [ 14 C]glucose. Cellular lipids were extracted and incorporation of [ 14 C]glucose into total lipids was quantified by scintillation counting and normalized to protein concentration. D, basal glucose uptake was assayed in H1299 cells treated with 1 µM CVT or vehicle for 24 h by estimating the uptake of [ 3 H]deoxyglucose. E, H1299 cells were incubated with [ 14 C]glucose in presence or absence of 1 µM CVT for 6 h and levels of total [ 14 C]fatty acids, as well as radiolabeled SFA and MUFA (upper panel), were determined by argentation TLC as described in . F, the rate of lipid synthesis in H1299 cells was assessed by incubation with 1 µM CVT or vehicle and 0.5 µCi/dish of [ 14 C]acetate for 24 h. Lipids were extracted and radioactivity of total lipids was determined by scintillation counting. Values represent the mean±S.D. of triplicate determinations. *, p<0.05 or less, by Student's t test.

Article Snippet: A549 human lung adenocarcinoma cells and WS-1 human fibroblasts were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Incubation, Knockdown, Expressing, Transfection, Control, Protein Concentration, Radioactivity

A, A549 and H1299 cells were incubated with 10 µM and 5 µM CVT-11127 (CVT) respectively, or vehicle (DMSO) in phenol red-free media for 24 h. The lactate concentration in the conditioned media was determined with a colorimetric kit as described under Experimental procedures and normalized to cellular protein. Control and SCD1-deficient (hSCDas) A549 cells were incubated with the indicated concentrations of glucose (GLU) (B) or pyruvate (C) for 24 h and pulsed with [ 3 H]thymidine (1 µCi/dish) for 2 h at 37°C. Total [ 3 H]-labeled DNA was precipitated, radioactivity was quantified in a scintillation counter and normalized to protein concentration. D, growth curve of control and hSCDas A549 cells in media containing either 0.5 or 5.5 mM glucose. Cells were seeded in 12 well plates (14,000 cell/well) and after 24 h regular growing media was switched to glucose-supplemented media. Media were replaced with fresh glucose-supplemented media every 48 h thereafter. At the indicated times, cell population was determined by crystal violet staining as described under Experimental procedures. Values represent the mean±S.D. of triplicate determinations. *, p<0.05, by Student's t test.

Journal: PLoS ONE

Article Title: Inhibition of StearoylCoA Desaturase-1 Inactivates Acetyl-CoA Carboxylase and Impairs Proliferation in Cancer Cells: Role of AMPK

doi: 10.1371/journal.pone.0006812

Figure Lengend Snippet: A, A549 and H1299 cells were incubated with 10 µM and 5 µM CVT-11127 (CVT) respectively, or vehicle (DMSO) in phenol red-free media for 24 h. The lactate concentration in the conditioned media was determined with a colorimetric kit as described under Experimental procedures and normalized to cellular protein. Control and SCD1-deficient (hSCDas) A549 cells were incubated with the indicated concentrations of glucose (GLU) (B) or pyruvate (C) for 24 h and pulsed with [ 3 H]thymidine (1 µCi/dish) for 2 h at 37°C. Total [ 3 H]-labeled DNA was precipitated, radioactivity was quantified in a scintillation counter and normalized to protein concentration. D, growth curve of control and hSCDas A549 cells in media containing either 0.5 or 5.5 mM glucose. Cells were seeded in 12 well plates (14,000 cell/well) and after 24 h regular growing media was switched to glucose-supplemented media. Media were replaced with fresh glucose-supplemented media every 48 h thereafter. At the indicated times, cell population was determined by crystal violet staining as described under Experimental procedures. Values represent the mean±S.D. of triplicate determinations. *, p<0.05, by Student's t test.

Article Snippet: A549 human lung adenocarcinoma cells and WS-1 human fibroblasts were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Incubation, Concentration Assay, Control, Labeling, Radioactivity, Protein Concentration, Staining

A549 and H1299 cells were incubated with 10 µM and 5 µM CVT-11127 (CVT) respectively, or DMSO for 24 h. The levels of phospho-ACC (Ser79) and phospho-AMPKα (Thr172) were determined by Western Blot in CVT-11127-treated cells (A–B) and SCD1-deficient A549 cells (C) and normalized to β-actin content. Cells treated with 20 µM Compound C (CpC) were included as a control. D, regulation of ACC activity by fatty acids. SCD1-deficient (hSCDas) and control A549 cells were serum-starved (0.1% FBS) for 24 h and incubated for 30 min in serum-depleted media with or without 100 µM palmitic (Pal) or oleic (Ole) acid complexed with 0.5% w/v fatty acid-free bovine serum albumin (BSA). Cellular levels of phospho-ACC (Ser79), phospho-AMPK and β-actin were determined by Western blot. Values represent the mean±S.D. of triplicate determinations. *, p<0.05 or less, by Student's t test.

Journal: PLoS ONE

Article Title: Inhibition of StearoylCoA Desaturase-1 Inactivates Acetyl-CoA Carboxylase and Impairs Proliferation in Cancer Cells: Role of AMPK

doi: 10.1371/journal.pone.0006812

Figure Lengend Snippet: A549 and H1299 cells were incubated with 10 µM and 5 µM CVT-11127 (CVT) respectively, or DMSO for 24 h. The levels of phospho-ACC (Ser79) and phospho-AMPKα (Thr172) were determined by Western Blot in CVT-11127-treated cells (A–B) and SCD1-deficient A549 cells (C) and normalized to β-actin content. Cells treated with 20 µM Compound C (CpC) were included as a control. D, regulation of ACC activity by fatty acids. SCD1-deficient (hSCDas) and control A549 cells were serum-starved (0.1% FBS) for 24 h and incubated for 30 min in serum-depleted media with or without 100 µM palmitic (Pal) or oleic (Ole) acid complexed with 0.5% w/v fatty acid-free bovine serum albumin (BSA). Cellular levels of phospho-ACC (Ser79), phospho-AMPK and β-actin were determined by Western blot. Values represent the mean±S.D. of triplicate determinations. *, p<0.05 or less, by Student's t test.

Article Snippet: A549 human lung adenocarcinoma cells and WS-1 human fibroblasts were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Incubation, Western Blot, Control, Activity Assay

A , control and SCD1-deficient A549 cells were incubated with compound C (20 µM), AICAR (0.25 mM) or vehicle for 24 h and the levels of phospho-ACC (Ser79) were estimated by Western Blot. Protein bands were quantified by densitometric analysis and normalized to β-actin content. hSCDas and mock-transfected control cells ( B ), or CVT-11127-treated (CVT) H1299 ( C ) and A549 ( D ) cells were treated with Compound C, AICAR or vehicle and pulsed with [ 3 H]thymidine for 2 h. The radiolabeled DNA was quantified as described in . Results are expressed as fold-change in total [ 3 H]DNA levels over vehicle-treated control. Values represent the mean±S.D. of triplicate determinations. *, p<0.05 vs DMSO under the same conditions; #, p<0.05 vs CVT treated cells incubated with vehicle by Student's t test.

Journal: PLoS ONE

Article Title: Inhibition of StearoylCoA Desaturase-1 Inactivates Acetyl-CoA Carboxylase and Impairs Proliferation in Cancer Cells: Role of AMPK

doi: 10.1371/journal.pone.0006812

Figure Lengend Snippet: A , control and SCD1-deficient A549 cells were incubated with compound C (20 µM), AICAR (0.25 mM) or vehicle for 24 h and the levels of phospho-ACC (Ser79) were estimated by Western Blot. Protein bands were quantified by densitometric analysis and normalized to β-actin content. hSCDas and mock-transfected control cells ( B ), or CVT-11127-treated (CVT) H1299 ( C ) and A549 ( D ) cells were treated with Compound C, AICAR or vehicle and pulsed with [ 3 H]thymidine for 2 h. The radiolabeled DNA was quantified as described in . Results are expressed as fold-change in total [ 3 H]DNA levels over vehicle-treated control. Values represent the mean±S.D. of triplicate determinations. *, p<0.05 vs DMSO under the same conditions; #, p<0.05 vs CVT treated cells incubated with vehicle by Student's t test.

Article Snippet: A549 human lung adenocarcinoma cells and WS-1 human fibroblasts were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Control, Incubation, Western Blot, Transfection

Journal: eLife

Article Title: Endothelial cell signature in muscle stem cells validated by VEGFA-FLT1-AKT1 axis promoting survival of muscle stem cell

doi: 10.7554/eLife.73592

Article Snippet: Cell line ( Mus musculus ) , C166 , American Type Culture Collection (ATCC) , CRL-2581 , EC line.

Techniques: RNA Sequencing, Expressing, Control, Derivative Assay, RNAscope, Isolation, Immunostaining

Journal: eLife

Article Title: Endothelial cell signature in muscle stem cells validated by VEGFA-FLT1-AKT1 axis promoting survival of muscle stem cell

doi: 10.7554/eLife.73592

Figure Lengend Snippet:

Article Snippet: Cell line ( Mus musculus ) , C166 , American Type Culture Collection (ATCC) , CRL-2581 , EC line.

fxcycle violet dna stain Search Results

Image Search Results