Journal: Inhalation toxicology

Article Title: Automated crude oil vapor inhalation exposure system

doi: 10.1080/08958378.2022.2114562

Figure Lengend Snippet: GC-FID temperature ramp.

Article Snippet: During each 6-h exposure run two samples were taken from inside the exposure chamber by a gas chromatograph flame ionizing detector (GC-FID) instrument (Model 8610 C GC-FID; SRI Instruments, Torrance, CA).

Techniques:

Journal: Inhalation toxicology

Article Title: Automated crude oil vapor inhalation exposure system

doi: 10.1080/08958378.2022.2114562

Figure Lengend Snippet: GC-FID spectrum output from a typical crude oil vapor reading at 300 ppm TVOC.

Article Snippet: During each 6-h exposure run two samples were taken from inside the exposure chamber by a gas chromatograph flame ionizing detector (GC-FID) instrument (Model 8610 C GC-FID; SRI Instruments, Torrance, CA).

Techniques:

Journal: Cell Death & Disease

Article Title: p38 α MAPK antagonizing JNK to control the hepatic fat accumulation in pediatric patients onset intestinal failure

doi: 10.1038/cddis.2017.523

Figure Lengend Snippet: The p38 α MAPK activation was decreased in livers of IF patients with steatosis and associated with expression of cholesterol 7- α -hydroxylase (CYP7A1), proliferator-activated receptor α coactivator-1 (PGC-1 α ) and nuclear receptor peroxisome proliferator-activated receptor α (PPAR α ). ( a ) Representative images of p-p38, p-JNK, CYP7A1, PGC-1 α , PPAR α , CPT1A and ACOX1 stainings in the liver tissues of IF patients without steatosis ( n =14) and with steatosis ( n =10). ( b ) Quantification of the results in panel A. ( c ) Western blot analyses of the p-p38, p-JNK, CYP7A1, PGC-1 α , PPAR α , CPT1A and ACOX1 protein levels in the livers of IF patients with or without steatosis. ( d ) Quantification of the protein results in panel C. ( e ) qRT- PCR analyses determine the levels of p38 α , JNK1/2, CYP7A1, PGC-1 α , PPAR α , CPT1A and ACOX1 mRNAs in the IF patients. ( f ) The relationships between p38 α mRNA and mRNA levels of CYP7A1, PGC-1 α , PPAR α , and between PGC-1 α mRNA and CYP7A1 mRNA, PPAR α mRNA in the liver tissues of the IF patients with Pearson’s correlations. Scale bar=25 μm * P <0.05, ** P <0.01

Article Snippet: Primary antibodies were applied in an optimal concentration overnight in a wet chamber (Phosphorylated p38 (Cell Signaling, Danvers, MA, USA, 4511), dilution 1:100; Phosphorylated JNK (Cell Signaling, 4668), dilution 1:100; CYP7A1 (Millipore, Darmstadt, Germany, MABD42), dilution 1:100; PPAR α (Abcam, Bristol, UK, ab8934) dilution 1:100; FXR (Invitrogen,Camarillo, CA, USA, 417200), dilution 1:500; CPT1A (Proteintech, Rosemont, IL, USA, 15184-1-AP), dilution 1:100; ACOX1 (Proteintech, 10957-1-AP), dilution 1:100 PGC-1 α (Abcam, ab54481), dilution 1:200).

Techniques: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: p38 α MAPK antagonizing JNK to control the hepatic fat accumulation in pediatric patients onset intestinal failure

doi: 10.1038/cddis.2017.523

Figure Lengend Snippet: p38 α MAPK and JNK have opposite effects on PN-associated hepatic steatosis. ( a ) H&E and Oil Red O staining showed that p38 α MAPK inhibition with SB203580 treatment increased the fat accumulated in the liver of PN-fed rats. In contrast, JNK inhibition with SP600125 treatment suppressed the PN-associated steatosis. ( b–e ) The alteration of levels of the triglyceride (TG) and phospholipids (PL) in liver and serum from groups of Sham, PN, PN+SB203580 and PN+SP600125. ( f , g ) The levels of total BA in the liver and serum from groups of Sham, PN, PN+SB203580 and PN+SP600125. ( h ) The measurement of primary BA including cholic acid (CA), chenodeoxycholic acid (CDCA) and α , β , ω -muricholic acid ( α -, β -, ω MCA) in livers from groups of Sham, PN, PN+SB203580 and PN+SP600125. Scale bar=25 μm * P <0.05, ** P <0.01

Article Snippet: Primary antibodies were applied in an optimal concentration overnight in a wet chamber (Phosphorylated p38 (Cell Signaling, Danvers, MA, USA, 4511), dilution 1:100; Phosphorylated JNK (Cell Signaling, 4668), dilution 1:100; CYP7A1 (Millipore, Darmstadt, Germany, MABD42), dilution 1:100; PPAR α (Abcam, Bristol, UK, ab8934) dilution 1:100; FXR (Invitrogen,Camarillo, CA, USA, 417200), dilution 1:500; CPT1A (Proteintech, Rosemont, IL, USA, 15184-1-AP), dilution 1:100; ACOX1 (Proteintech, 10957-1-AP), dilution 1:100 PGC-1 α (Abcam, ab54481), dilution 1:200).

Techniques: Staining, Inhibition

Journal: Cell Death & Disease

Article Title: p38 α MAPK antagonizing JNK to control the hepatic fat accumulation in pediatric patients onset intestinal failure

doi: 10.1038/cddis.2017.523

Figure Lengend Snippet: p38 α MAPK opposing JNK to suppress the expression of CYP7A1, PGC-1 α , PPAR α , CPT1A and ACOX1 in vivo . ( a ) Representative images of p-p38, p-JNK, CYP7A1, PGC-1 α , PPAR α , CPT1A and ACOX1 stainings in the liver tissues of Sham, PN, PN+SB203580 and PN+SP600125 groups. ( b ) Quantification of the results in panel A. ( c ) Western blot analyses of the p-p38, p-JNK,CYP7A1, PGC-1 α , PPAR α , CPT1A and ACOX1 protein levels in the livers of Sham, PN, PN+SB203580 and PN+SP600125 groups. ( d ) Quantification of the protein results in panel C. ( e ) qRT-PCR analyses determine the levels of p38 α , JNK1/2, CYP7A1, PGC-1 α , PPAR α , CPT1A and ACOX1 mRNAs in the livers of Sham, PN, PN+SB203580 and PN+SP600125 groups. Scale bar=25 μm * P <0.05, ** P <0.01

Article Snippet: Primary antibodies were applied in an optimal concentration overnight in a wet chamber (Phosphorylated p38 (Cell Signaling, Danvers, MA, USA, 4511), dilution 1:100; Phosphorylated JNK (Cell Signaling, 4668), dilution 1:100; CYP7A1 (Millipore, Darmstadt, Germany, MABD42), dilution 1:100; PPAR α (Abcam, Bristol, UK, ab8934) dilution 1:100; FXR (Invitrogen,Camarillo, CA, USA, 417200), dilution 1:500; CPT1A (Proteintech, Rosemont, IL, USA, 15184-1-AP), dilution 1:100; ACOX1 (Proteintech, 10957-1-AP), dilution 1:100 PGC-1 α (Abcam, ab54481), dilution 1:200).

Techniques: Expressing, In Vivo, Western Blot, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: p38 α MAPK antagonizing JNK to control the hepatic fat accumulation in pediatric patients onset intestinal failure

doi: 10.1038/cddis.2017.523

Figure Lengend Snippet: p38 α MAPK antagonistically JNK to control the palmitate (PA)-mediated lipogenesis. ( a ) Representative images of neutral lipids staining in the indicated cells. ( b , c ) Quantification of the contents of hepatic triglyceride (TG) and phospholipids (PL) in indicated treatments. ( d ) Western blot analyses for the p-p38, p-JNK, CYP7A1, PGC-1 α , PPAR α , CPT1A and ACOX1 protein levels in the human primary hepatic cell with indicated treatments. ( e–g ) Relative promoter activity of CYP7A1, PGC-1 α and PPAR α with indicated treatments. ( h ) The scheme illustrates a potential mechanism by which p38 α MAPK controls the fatty acid metabolism in the livers of IF patients. Scale bar=25 μm * P <0.05, ** P <0.01

Article Snippet: Primary antibodies were applied in an optimal concentration overnight in a wet chamber (Phosphorylated p38 (Cell Signaling, Danvers, MA, USA, 4511), dilution 1:100; Phosphorylated JNK (Cell Signaling, 4668), dilution 1:100; CYP7A1 (Millipore, Darmstadt, Germany, MABD42), dilution 1:100; PPAR α (Abcam, Bristol, UK, ab8934) dilution 1:100; FXR (Invitrogen,Camarillo, CA, USA, 417200), dilution 1:500; CPT1A (Proteintech, Rosemont, IL, USA, 15184-1-AP), dilution 1:100; ACOX1 (Proteintech, 10957-1-AP), dilution 1:100 PGC-1 α (Abcam, ab54481), dilution 1:200).

Techniques: Staining, Western Blot, Activity Assay

Journal: Nature Communications

Article Title: HuR controls glutaminase RNA metabolism

doi: 10.1038/s41467-024-49874-x

Figure Lengend Snippet: MIA PaCa-2 expression levels of ELAVL1 and GLS from RNA-Seq data ( a , mean and S.E.M.) and heatmap ( b ) with bidirectional clustering of singscores from KEGG Metabolic Pathways and scaled ELAVL1 expression. The letter ‘Q’ marks pathways that involve glutamine from (Control n = 4; Knockout n = 3). The dendrograms represent full bidirectional complete clustering based on correlation values. c Chromosome 2 indicating GLS loci and exon structure for KGA and GAC isoforms. Bellow, exon differential expression after ELAVL1 knockdown in MIA PaCa-2 or HeLa cells. Asterisks denote FDR < 0.05. d , above, Western blot of doxycycline-inducible ELAVL1 -silenced breast cancer cell lines (MDA-MB-231 and BT549) using two shRNAs, displaying a marked decrease in KGA protein level, accompanied by an increase in GAC. d , below, Western blot of constitutively silenced ELAVL1 breast cancer cell lines, grouped by telaglenastat resistance, which was defined elsewhere . Arrows indicate KGA and GAC isoforms and HuR-specific bands and western blots repeated at least two times with reproducible results. e Scheme for CRISPR-mediated knock-in of the mKO2-P2A-BleoR cassette into GLS exon 19 to produce the KGA-mKO2 fusion protein. f Increase and decrease in cellular fluorescence after ELAVL1 ectopic expression (bottom) or sh ELAVL1 -mediated transient silencing (top), respectively, in HEK293T knock-in cells, number independent cells evaluated per condition >1433. Representative images on the right. The scale bar is 50 μm. g Cell cycle analysis of BT549 knock-in cells KGA levels (evaluated by mKO2 fluorescence) are enhanced in S-G2/M phases, as expected , . Representative images in bottom part, with HiLo lookup table for DNA staining, one cell with higher DNA content and higher KGA content and the opposite example below, confirming the functionality of the system. Box plots represent the interquartile range; the vertical curve is the kernel density of the distribution, and the dark horizontal line denotes the median. Each dot represents an individual cell. Statistical significance derived from bootstrapped DEXSeq ( c ) or Two-sided Welch’s t -test ( f ). * p < 0.05, ** p < 0.01, *** p < 0.0001.

Article Snippet: BT549 (ATCC HTB-122), Hs578t (ATCC HTB-126), SKBR-3 (ATCC HCB-30), MDA-MB-157 (ATCC HTB-24), MDA-MB-231 (ATCC HTB-26), MDA-MB-436 (ATCC HTB-130), MDA-MB-453 (ATCC HTB-131), MDA-MB-468 (ATCC HTB-132), HCC38 (ATCC CRL-2314), HCC70 (ATCC CRL-2315), HCC1806 (ATCC CRL-2335), HCC1937 (ATCC CRL-2336), and PC-3 cell lines were cultivated in RPMI 1640 media (Sigma) supplemented with 10% fetal bovine serum (Vitrocell).

Techniques: Expressing, RNA Sequencing, Control, Knock-Out, Quantitative Proteomics, Knockdown, Western Blot, CRISPR, Knock-In, Fluorescence, Cell Cycle Assay, Staining, Derivative Assay

Journal: Nature Communications

Article Title: HuR controls glutaminase RNA metabolism

doi: 10.1038/s41467-024-49874-x

Figure Lengend Snippet: a Relative quantification of BT549 mRNA levels following doxycycline-inducible silencing of ELAVL1 , using qPCR for two distinct shRNA sequences. b HuR was immunoprecipitated (western blot above, arrows indicate HuR and IgG heavy chain). The control IgG were used from rabbit, in opposing to the mouse anti-HuR antibody. Quantification of mRNA derived from 3′UTR KGA regions immunoprecipitated bound in HuR from ( c ) PC-3 cells using agarose gel and ( d ) from BT549 using qRT-PCR. Quantification of mRNA derived from 3′UTR GAC regions immunoprecipitated bound in HuR from ( e ) PC-3 cells using agarose gel and ( f ) from BT549 using qRT-PCR. Agarose gel from PC-3 ( g ) and q-RT-PCR from BT549 ( h ) revealed that HuR binds to its mRNA (as already published elsewhere ) in addition to GLS intron 14. i In vitro FRAP analysis of recombinant mKO2-HuR incubated with in vitro transcribed control RNA or intron 14. The bar plot (right) denotes T 1/2 recovery times. Statistical significance derived from Two-sided Welch’s t-test ( a , d , f , h , and i ), error bars are SEM; qRT-PCR assays were evaluated in triplicate; otherwise, each point represents a replicate. * p < 0.05, ** p < 0.01, *** p < 0.0001.

Article Snippet: BT549 (ATCC HTB-122), Hs578t (ATCC HTB-126), SKBR-3 (ATCC HCB-30), MDA-MB-157 (ATCC HTB-24), MDA-MB-231 (ATCC HTB-26), MDA-MB-436 (ATCC HTB-130), MDA-MB-453 (ATCC HTB-131), MDA-MB-468 (ATCC HTB-132), HCC38 (ATCC CRL-2314), HCC70 (ATCC CRL-2315), HCC1806 (ATCC CRL-2335), HCC1937 (ATCC CRL-2336), and PC-3 cell lines were cultivated in RPMI 1640 media (Sigma) supplemented with 10% fetal bovine serum (Vitrocell).

Techniques: Quantitative Proteomics, shRNA, Immunoprecipitation, Western Blot, Control, Derivative Assay, Agarose Gel Electrophoresis, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, In Vitro, Recombinant, Incubation

Journal: Nature Communications

Article Title: HuR controls glutaminase RNA metabolism

doi: 10.1038/s41467-024-49874-x

Figure Lengend Snippet: a , top, Scheme of the RG6 splicing reporter for GLS intron 14 8 . HuR binding to intron 14 protects it from being spliced out and promotes an alternative splicing event that removes the reporter’s exon 2 and generates DsRed in frame (producing DsRed protein, a surrogate for KGA’s mRNA choice); HuR depletion promotes intron 14 splicing, leading to the retention of reporter’s exon 2, which places the eGFP coding sequence in the correct frame (producing eGFP protein, a surrogate for GAC expression). Doxycycline-induced silencing of the ELAVL1 cell line using sh ELAVL1 I and sh ELAVL1 II sequences in BT549 ( a , middle; representative images are shown on the right) and MDA-MB-231 ( b , bottom; representative images are shown on the right) cells led to a decrease in the DsRed/eGFP fluorescence ratio, indicating decreased KGA mRNA choice under this condition. At least 1158 independent cells evaluated for each group. b , top, Scheme of the psiCHECK2 reporter used to evaluate luciferase expression mediated by the GAC 3′-UTR or KGA 3′-UTR. ELAVL1 doxycycline-induced silencing decreased luciferase activity levels driven by both constructs but not the control ( b , bottom left); HuR ectopic expression promoted the inverse effect ( b , bottom right). At least 5 cell replicates for silencing and 3 for overexpression. c KGA (left) and GAC (right) mRNA decay after transcription blockage induced by actinomycin D incubation with PC-3 cancer cells (control cells or cells ectopically expressing HuR). d KGA (left) and GAC (right) mRNA decay after transcription blockage induced by actinomycin D incubation with BT549 cancer cells (control cells or ELAVL1 silenced using doxycicline) using two distinct shRNAs. e RT-qPCR data for polisomal enrichment analysis following ultracentrifugation in sucrose gradient ( n = 3). Each dot represents an individual cell or sample. Box plots represent the interquartile range; the vertical curve is the kernel density of the distribution, and the dark horizontal line denotes the median. Each dot represents an individual sample. Statistical significance was derived from Two-sided Welch’s t -test, and error bars are SEM. * p < 0.05, ** p < 0.01, *** p < 0.0001.

Article Snippet: BT549 (ATCC HTB-122), Hs578t (ATCC HTB-126), SKBR-3 (ATCC HCB-30), MDA-MB-157 (ATCC HTB-24), MDA-MB-231 (ATCC HTB-26), MDA-MB-436 (ATCC HTB-130), MDA-MB-453 (ATCC HTB-131), MDA-MB-468 (ATCC HTB-132), HCC38 (ATCC CRL-2314), HCC70 (ATCC CRL-2315), HCC1806 (ATCC CRL-2335), HCC1937 (ATCC CRL-2336), and PC-3 cell lines were cultivated in RPMI 1640 media (Sigma) supplemented with 10% fetal bovine serum (Vitrocell).

Techniques: Binding Assay, Alternative Splicing, Sequencing, Expressing, Fluorescence, Luciferase, Activity Assay, Construct, Control, Over Expression, Incubation, Quantitative RT-PCR, Derivative Assay

Journal: Nature Communications

Article Title: HuR controls glutaminase RNA metabolism

doi: 10.1038/s41467-024-49874-x

Figure Lengend Snippet: a PAR-CLIP-Seq experiments performed with HuR indicated a region proximal to the acceptor splicing site of intron 14 as the HuR binding region; the thick green bar indicates the region of PAR-CLIP in intron 14 with the maximum clustering of reads; ( b ) sequence conservation of the intronic region in Mus musculus , Ratus norvegicus and Bos taurus ; a value of 1 on the y-axis represents the maximum similarity to the Homo sapiens sequence. ( c ) Scheme illustrating the distance between GLS intron 14 elements related to the splicing event (AG sites, AG-exclusion zone, and putative branchpoint) and the region identified by PAR-CLIP as a HuR binding site (HuR binding region, thick green bar); ( c , bottom) multiple sequence alignment of the aforementioned sequence, indicating the mutation and deletion included in the RG6 splicing vector. Vertical dashed lines represent the identified region. d Mutation or deletion of the HuR binding site decreased the DsRed/eGFP fluorescence ratio compared to that in the control, indicating that HuR binding is important for the preference for KGA during splicing. Consistent with HuR being a key element in the process, ELAVL1 knockdown cells had a smaller variation in this ratio than control cells. The control is the wild-type sequence of intron 14. A total of 32,837 cells evaluated. Doxycycline-induced ELAVL1 silencing in BT549, MDA-MB-231, and Hs578t cells increased glutamine uptake ( e ), glutaminase activity ( f ), and cell growth ( g ), and glutamine dependence on oxygen consumption ( h ), with representative blot for ELAVL1 silencing. i , j OCR of BT549 cells silenced for ELAVL1 using distinct shRNA target sequences on mitochondrial stress test (injections of oligomycin, FCCP, antimycin, and rotenone). Representation of basal OCR ( k ), ECAR ( l ), and dot-plot OCR vs ECAR ( m ) from i and j . Box plots represent the interquartile range; the vertical curve is the kernel density of the distribution, and the dark horizontal line denotes the mean. Each dot represents an individual cell, n = 5 for ( f ), n > 2 for ( h ), n > 5 for ( i – m ). Statistical significance was derived from ANOVA followed by Tukey’s test ( d , g ) or Two-sided Welch’s t -test ( e , f , h , k , and l ), and error bars are SEM. * p < 0.05, ** p < 0.01, *** p < 0.0001. When indicated, ‘sh ELAVL1 ’ and ‘sh ELAVL1 II’ refer to two distinct shRNA sequences.

Article Snippet: BT549 (ATCC HTB-122), Hs578t (ATCC HTB-126), SKBR-3 (ATCC HCB-30), MDA-MB-157 (ATCC HTB-24), MDA-MB-231 (ATCC HTB-26), MDA-MB-436 (ATCC HTB-130), MDA-MB-453 (ATCC HTB-131), MDA-MB-468 (ATCC HTB-132), HCC38 (ATCC CRL-2314), HCC70 (ATCC CRL-2315), HCC1806 (ATCC CRL-2335), HCC1937 (ATCC CRL-2336), and PC-3 cell lines were cultivated in RPMI 1640 media (Sigma) supplemented with 10% fetal bovine serum (Vitrocell).

Techniques: Binding Assay, Sequencing, Mutagenesis, Plasmid Preparation, Fluorescence, Control, Knockdown, Activity Assay, shRNA, Derivative Assay

Journal: Nature Communications

Article Title: HuR controls glutaminase RNA metabolism

doi: 10.1038/s41467-024-49874-x

Figure Lengend Snippet: a Heatmap (above) from GC-MS metabolomics from Doxycycline-induced ELAVL1 silencing in BT549 cells cluster evaluated compounds in (blue) telaglenastat responsive and (green) HuR and Telaglenastat responsive. (Below) Principal component analysis from same data, with 80% probability circles. b Network-based pathway enrichment using FELLA for blue and green clusters. The effect of doxycycline-induced ELAVL1 silencing on intracellular glutamine ( c ), glutamate ( d ), and α-ketoglutarate ( e ). f Scheme denotes glutamine reductive carboxylation (above) and oxidation (below) metabolic pathways and the related isotopologues. Thick arrows identify reductive pathway-related (purple) and oxidative pathway-related (brown) isotopologues. The effect of doxycycline-induced ELAVL1 silencing on intracellular citrate ( g ), fumarate ( h ), aspartate ( i ), and malate ( j ), and succinate ( k ). For all mentioned letters, the overall pool is shown on the left and the isotopologue levels are shown on the right. Purple, brown and grays colors indicate isotopologues generated during reductive carboxylation and oxidative glutaminolysis and isotopologues common to both pathways, respectively. n = 4 for all experiments. Statistical significance was derived from Two-sided Welch’s t -test; each dot represents an individual replicate, and error bars are SEM. * p < 0.05, ** p < 0.01, *** p < 0.0001.

Article Snippet: BT549 (ATCC HTB-122), Hs578t (ATCC HTB-126), SKBR-3 (ATCC HCB-30), MDA-MB-157 (ATCC HTB-24), MDA-MB-231 (ATCC HTB-26), MDA-MB-436 (ATCC HTB-130), MDA-MB-453 (ATCC HTB-131), MDA-MB-468 (ATCC HTB-132), HCC38 (ATCC CRL-2314), HCC70 (ATCC CRL-2315), HCC1806 (ATCC CRL-2335), HCC1937 (ATCC CRL-2336), and PC-3 cell lines were cultivated in RPMI 1640 media (Sigma) supplemented with 10% fetal bovine serum (Vitrocell).

Techniques: Gas Chromatography-Mass Spectrometry, Generated, Derivative Assay

Journal: Nature Communications

Article Title: HuR controls glutaminase RNA metabolism

doi: 10.1038/s41467-024-49874-x

Figure Lengend Snippet: Compared to the control, doxycycline-induced ELAVL1 silencing decreased the proliferation of BT549 ( a , left), MDA-MB-231 ( a , middle), and Hs578t ( a , right) cells; the migration, as measured by scratch healing rate, of BT549 ( b , left), and MDA-MB-231 ( b , right) cells was also decreased, representative images in c ; cell invasion, as assayed by Matrigel-covered Boyden chamber assay, was also decreased in BT549, MDA-MB-231, and Hs578t ( d , left), representative images in ( d , right); in c , representative images below (scale bar represents 100 µm). In d , representative images of DAPI-stained nuclei located at the membrane. Statistical significance was derived from Two-sided Welch’s t -test; each dot represents an individual replicate; otherwise, n = 4 for proliferation assays and n = 8 for migration/invasion assays; error bars are SEM. * p < 0.05, ** p < 0.01, *** p < 0.0001. When indicated, ‘sh ELAVL1 ’ and ‘sh ELAVL1 II’ refer to two distinct shRNA sequences.

Article Snippet: BT549 (ATCC HTB-122), Hs578t (ATCC HTB-126), SKBR-3 (ATCC HCB-30), MDA-MB-157 (ATCC HTB-24), MDA-MB-231 (ATCC HTB-26), MDA-MB-436 (ATCC HTB-130), MDA-MB-453 (ATCC HTB-131), MDA-MB-468 (ATCC HTB-132), HCC38 (ATCC CRL-2314), HCC70 (ATCC CRL-2315), HCC1806 (ATCC CRL-2335), HCC1937 (ATCC CRL-2336), and PC-3 cell lines were cultivated in RPMI 1640 media (Sigma) supplemented with 10% fetal bovine serum (Vitrocell).

Techniques: Control, Migration, Boyden Chamber Assay, Staining, Membrane, Derivative Assay, shRNA

Journal: Nature Communications

Article Title: HuR controls glutaminase RNA metabolism

doi: 10.1038/s41467-024-49874-x

Figure Lengend Snippet: a Doxycycline-induced (left, sh ELAVL1 and sh ELAVL1 II) and stable (right) ELAVL1 silencing in BT549 cells combined with GLS inhibition with telaglenastat (CB-839) decreased cell proliferatio, compared to that in control cells (DMSO or shGFP). b Growth response of BT549 to increasing amounts of telaglenastat (4-day treatment) with ELAVL1 knockdown induced (+Dox, left) or not (−Dox, right). The IC50 value and 95% confidence interval (CI) are presented above the graphs. The gray dashed line indicates that there was a reduction in the final cell number compared to the number of seeded cells, denoting cell death. c Doxycycline-induced ELAVL1 silencing of BT549 cells combined with GLS inhibition and telaglenastat decreased cell migration (left) and invasion (right) by 37 and 42%, respectively, compared to control cells (DMSO - Dox). Statistical significance was derived from Two-sided Welch’s t-test; each dot represents an individual replicate; otherwise, n = 3 for proliferation assays and n = 8 for migration/invasion assays; error bars are SEM. * p < 0.05, ** p < 0.01, *** p < 0.0001.

Article Snippet: BT549 (ATCC HTB-122), Hs578t (ATCC HTB-126), SKBR-3 (ATCC HCB-30), MDA-MB-157 (ATCC HTB-24), MDA-MB-231 (ATCC HTB-26), MDA-MB-436 (ATCC HTB-130), MDA-MB-453 (ATCC HTB-131), MDA-MB-468 (ATCC HTB-132), HCC38 (ATCC CRL-2314), HCC70 (ATCC CRL-2315), HCC1806 (ATCC CRL-2335), HCC1937 (ATCC CRL-2336), and PC-3 cell lines were cultivated in RPMI 1640 media (Sigma) supplemented with 10% fetal bovine serum (Vitrocell).

Techniques: Inhibition, Control, Knockdown, Migration, Derivative Assay