Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 1. ICAM-1 cross-linking in- duced time-dependent activation of SRC tyrosine kinases. TNF--pre- treated ECs were incubated for 30 min with 10 g/ml mouse anti-human ICAM-1 antibody and washed. A cross-linking sec- ondary antibody was added for 0–15 min, and the activity of SRC was evaluated by an in vitro kinase assay using Sam68- (331–443) as a substrate after immuno- precipitating SRC. Tyrosine phosphoryla- tion of Sam68-(331–443) was detected by immunoblot using an anti-phosphoty- rosine antibody as described under “Ex- perimental Procedures.” A, representa- tive immunoblot showing activation of SRC tyrosine kinases in response to ICAM-1 cross-linking. The amount of im- munoprecipitated SRC was also deter- mined for each sample. Lanes 1–5, SRC activity in ECs prior to (lane 1) or 0.25–15 min after ICAM-1 cross-linking (lanes 2–5). B, densitometric analysis of immu- noblots. The activity of SRC tyrosine ki- nases was normalized by the amount of immunoprecipitated SRC. Data are pre- sented as fold changes over the non-cross- linked controls and expressed as means S.E. (n 4). *, p 0.05 when compared with controls.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Incubation, Activity Assay, In Vitro, Kinase Assay, Western Blot, Immunoprecipitation

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 2. Activation of SRC tyrosine ki- nases was inhibited by allopurinol, a xanthine oxidase inhibitor (A), as well as Me2SO, a hydroxyl radical scav- enger, and deferoxamine, an iron che- lator (B). ECs were treated with 10 g/ml anti-ICAM-1 along with 0.3 mg/ml allo- purinol, 1% Me2SO, or 1.5 mM deferoxa- mine, or their respective control vehicle for 30 min and washed. A cross-linking sec- ondary antibody was added for 0–6 min, and SRC activity was evaluated as de- scribed under “Experimental Procedures.” Open bars, no cross-linking; closed bars, cross-linking for 6 min. Data are expressed as fold changes from the non-cross-linked controls in vehicle-pretreated samples and presented as mean S.E. (n 4). *, p 0.05 when compared with the non-cross- linked controls; #, p 0.05 when compared with the vehicle-pretreated samples.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Control, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 3. Modulation of SRC activity by PAO, a tyrosine phosphatase in- hibitor. ECs were treated with 10 g/ml anti-ICAM-1 along with control vehicle or 20 M PAO for 30 min and washed. A cross-linking secondary antibody was added for 0–6 min, and SRC activity was evaluated as described under “Experi- mental Procedures.” Data are expressed as fold changes from the non-cross-linked controls in vehicle-pretreated samples, and presented as mean S.E. (n 4). *, p 0.05 when compared with the non- cross-linked controls; #, p 0.05 when compared with the vehicle-pretreated samples.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activity Assay, Control

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 4. Activation of SRC tyrosine kinases required SHP-2. ECs were treated with 10 nM control or SHP-2 an- tisense oligonucleotides as described un- der “Experimental Procedures.” A, the ef- fect of SHP-2 antisense on the protein expression of SHP-2 or SHP-1 in ECs as examined by immunoblot. B, the effect of SHP-2 antisense on SRC activity before or after ICAM-1 cross-linking for 6 min. Data are expressed as fold changes from the non-cross-linked controls and pre- sented as means S.E. (n 8). *, p 0.05 when compared with the non-cross- linked controls; #, p 0.05 when com- pared with the control antisense-treated samples.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Control, Expressing, Western Blot, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 5. Immunoprecipitated SHP-2 from ECs can dephosphorylate the phospho-SRC peptide at residue Tyr-530. SHP-2 was immunoprecipitated (IP) from ECs, and dephosphorylation of the phospho-SRC peptide at residue Tyr-530 was examined as described under “Experimental Procedures.” A, examples of two independent samples showing that immunoprecipitated SHP-2 can decrease the phosphorylation levels of the phospho-SRC peptide. Top gel, SHP-2 was specifically immunoprecipitated using a SHP-2 antibody. Bottom gel, incubation with the immunoprecipitated SHP-2 resulted in a decrease in the phosphorylation levels of the phospho-SRC peptide. B, densitometric analysis of the decrease in the phosphorylation levels of the phospho-SRC peptide as shown in A. Data are expressed relative to the control samples and presented as means S.E. (n 5). *, p 0.05 when compared with control samples.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunoprecipitation, Residue, De-Phosphorylation Assay, Phospho-proteomics, Incubation, Control

Journal: The Journal of biological chemistry

Article Title: Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells.

doi: 10.1074/jbc.M308466200

Figure Lengend Snippet: FIG. 6. Activation of p38 MAPK in- duced by ICAM-1 cross-linking was inhibited by PP2, an inhibitor of SRC tyrosine kinases. ECs were incubated with 10 g/ml anti-ICAM-1 antibody along with vehicle or 20 M PP2 for 30 min and washed. The cells were either left untreated or treated with cross-linking secondary an- tibody for 6 min. The activity of p38 MAPK was evaluated by an in vitro kinase assay using ATF-2 as a substrate. A, activity of p38 MAPK as evaluated by phosphorylation of ATF-2. As a loading control, the amount of ezrin in the samples used for immunopre- cipitation was also examined. B, densitomet- ric analysis of ATF-2 phosphorylation as in A. Open bars, no cross-linking; closed bars, cross-linking ICAM-1 for 6 min. The data are expressed as fold changes from the non- cross-linked controls in vehicle-pretreated samples and are presented as means S.E. (n 6 or 7). *, p 0.05 when compared with the non-cross-linked controls.

Article Snippet: Goat anti-tyrosine-phosphorylated ezrin at residue Tyr-146 (pY146), SRC-associated during mitosis, 68-kDa Sam-(68–331-443) fusion protein, rabbit anti-human SHP-1 and SHP-2 antibody, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Incubation, Activity Assay, In Vitro, Kinase Assay, Phospho-proteomics, Control

Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa depletion significantly inhibits spreading-induced signaling. (A) siRNA-transfected cells held in suspension for 1 h (left panel) or plated on collagen IV-coated dishes for the indicated times (minutes, right panel) were lysed and subjected to western blotting with anti-active FAK [pFAK(Y397)], active Src [pSrc(Y416)] and anti-phosphorylated paxillin [pPax(Y118)] antibodies. (B) Protein phosphorylation in control cells at 30 min after plating was set as 100%. The results represent a summary from five to seven experiments. *P<0.05; **P<0.01. (C) Lysates from plated cells treated as in A were analyzed by western blotting with antibodies against Src-dependent FAK phosphorylation sites (Y576 and Y925). The blots shown represent one of three independent experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Transfection, Western Blot

Journal: RNA

Article Title: ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5′ splice site motifs within nuclear speckles

doi: 10.1261/rna.079104.122

Figure Lengend Snippet: ZFC3H1 depletion leads to cytoplasmic accumulation of endogenous 5′SS motif containing mRNAs (or intronic polyadenylated transcripts). ( A ) Workflow for RNA Frac-seq. ZFC3H1- or control-depleted U2OS cells were fractionated into nuclear and cytoplasmic/ER fractions (see Materials and Methods for more details). RNA was purified from these fractions and from total cell lysates, and then analyzed by Illumina sequencing. ( B ) Nuclear “N” and cytoplasmic “C” fractions were collected from ZFC3H1- or control-depleted U2OS cells, then separated by SDS-PAGE and analyzed by immunoblot for nuclear (Aly), ER (Trap-α), and cytoplasmic (tubulin) protein markers. ( C ) Lysates collected from ZFC3H1- or control-depleted U2OS cells (96 h post-transduction with lentiviral-delivered ZFC3H1-2 shRNA) were analyzed by immunoblot for ZFC3H1 and tubulin. ( D ) Fold change in total levels of intronic polyadenylated (IPA) transcripts (ZFC3H1 depletion vs. control depletion) ( x -axis), plotted against the change in the total levels of fully processed mRNA (using cUTR reads, y -axis). Each dot corresponds to reads from one gene that is known to produce IPA transcripts (listed in Supplemental Table 1 ). Note that ZFC3H1 depletion leads to the up-regulation of IPA transcripts, but not fully processed mRNAs. ( E ) ( Top ) Schematic of a fully processed mRNA and IPA transcript generated from the same gene. Note that the IPA transcript is generated from a 3′ cleavage/polyadenylation signal in the first intron and contains a 5′SS motif. ( Bottom ) genome browser tracks of the PCF11 gene in control- “shCon” or ZFC3H1-depleted “shZFC” cells at 500× and 50× resolution. Note the large peak for the IPA transcript (intronic cleavage/polyadenylation site is denoted with a red arrow), which is up-regulated in ZFC3H1-depleted cells. Also note that the reads corresponding to the full-length transcript are unaffected by ZFC3H1 depletion. ( F ) Similar to D , except that the fold change of IPA transcript levels in the cytoplasmic fraction (ZFC3H1 depletion vs. control depletion, x -axis) is plotted against the fold change in the nuclear fraction (ZFC3H1 depletion vs. control depletion, y -axis). Note that ZFC3H1 depletion leads to cytoplasmic accumulation of many IPA transcripts (compare blue dots to red). To account for reads from fully processed mRNAs, the IPA transcript levels are normalized to the cUTR transcript levels of the same gene. ( G ) Similar to E , except the IPA peaks in the nuclear and cytoplasmic fractions are shown for PCF11 . In control-depleted cells, reads from the PCF11 IPA are enriched in the nuclear, but not the cytoplasmic fractions. In ZFC3H1-depleted cells, reads from the PCF11 IPA are at comparable levels in the nuclear and cytoplasmic fractions. ( H – J ) Genome browser tracks of three IPA transcript-producing genes, “ CCDC71 ,” “ BRD3 ,” and “ ZFPM1 .” Note the accumulation of IPA transcripts in the cytoplasmic fractions upon ZFC3H1 depletion. The intronic 3′ cleavage/polyadenylation sites are denoted by red arrows.

Article Snippet: Antibodies used in this study include rabbit polyclonals against ZFC3H1 (also known as CCDC131) (Bethyl Laboratories, A301-457A), MTR4 (also known as SKIV2L2) (Bethyl Laboratories, A300-614A), PABPN1 (Bethyl Laboratories, A303-523A), U1-70K (Abcam, ab83306), Aly , and TRAPα ( ) or mouse monoclonals against U1-70K (Sigma-Aldrich, clone 9C4.1), mAb414 (Sigma), SC35 (Clone SC35, Sigma), and α-tubulin (DM1A, Sigma).

Techniques: Control, Purification, Illumina Sequencing, SDS Page, Western Blot, Transduction, shRNA, Generated

Journal: RNA

Article Title: ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5′ splice site motifs within nuclear speckles

doi: 10.1261/rna.079104.122

Figure Lengend Snippet: ZFC3H1 is required for the nuclear retention of 5′SS motif containing mRNAs. ( A ) U2OS cells were treated with different lentivirus shRNAs against ZFC3H1 (“ZFC3H1-1” and “ZFC3H1-2”), MTR4 (“MTR4-1 + 2”) or control shRNA. Lysates were collected 96 h post-transduction, separated by SDS-PAGE and immunoprobed for ZFC3H1, MTR4, or tubulin. Note that to effectively deplete MTR4, cells were treated with lentivirus containing two shRNA plasmids. ( B ) Schematic of the intronless ( Δi ) ftz reporter ( ftz-Δi ) construct used in this study, with and without the V5-His element in the 3′UTR ( ftz-Δi-5 ′ SS ). Note that the V5-His element contains a consensus 5′SS motif, which promotes nuclear retention. ( C , D ) Control-, MTR4-, and ZFC3H1-depleted cells were transfected with the intronless ftz reporter plasmid (± 5 ′ SS ). Eighteen to twenty-four hours later, the cells were fixed and the mRNA was visualized by FISH. Note that depletion of ZFC3H1, but not MTR4, caused the cytoplasmic accumulation of the ftz-Δi-5′SS mRNA. Representative images are shown in C (scale bar, 10 µm) and quantification is shown in D . Each bar represents the average and standard error of at least three independent experiments, each experiment consisting of at least 30 to 60 cells. Student's t -test was performed for D. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. ( E ) Schematic of CCDC71 -IPA reporter used in this study (see also H). The position of the FISH probe used to visualize the IPA RNA is marked in gray and the position of the 3′ cleavage site in the intron is as indicated. ( F ) U2OS cells were transfected with the CCDC71-IPA reporter and, 18 to 24 hours later, the cells were fixed. The IPA transcript was visualized by FISH and nuclear speckles were visualized by immunofluorescence against SC35. Representative images are shown with a merged overlay showing the CCDC71-IPA mRNA in red and SC35 in green. Scale bar, 10 µM. Examples of CCDC71-IPA /SC35 colocalization are indicated with arrows. ( G , H ) Control- and ZFC3H1-depleted cells were transfected with the CCDC71-IPA reporter and the IPA transcript was visualized by FISH. ZFC3H1 depletion increased the cytoplasmic accumulation of the CCDC71-IPA . Representative images are shown in G (scale bar, 10 µm) and quantification is shown in H . Each bar represents the average and standard error of at least three independent experiments, each experiment consisting of at least 30 to 60 cells. Student's t -test was performed for H. (**) P < 0.01. ( I – K ) ePAT assay and 3′RACE were used to examine 3′ end processing. ( I ) Schematic of the ePAT assay as described in . The ftz -specific ( F′ ) and universal ( R′ ) primers used to amplify the ePAT amplicon are indicated. The sequence of the ePAT amplicon before the cleavage site is shown in J and is 117 nt long. ( J ) The sequence of the end of the 3′UTR is shown. Indicated in bold are the ftz -specific F ′ primer annealing site (used in the ePAT and 3 ′ RACE experiments), the hexanucleotide motif, and the cleavage site (as determined by 3 ′ RACE experiments on mRNAs derived from U2OS cells transfected with either ftz-Δi or ftz-Δi-5′SS ). ( K ) PCR products from the ePAT assay were separated on a 1% agarose gel and stained with ethidium bromide. Lane 1 : Molecular weight markers with sizes in bp indicated on the left ; lanes 3–6 : ePAT amplicons from U2OS cells that were transfected with plasmids containing the indicated versions of the ftz reporter (without [ Δi ] or with [ i ] an intron, without or with the 5′SS motif). Note that the amplicons generated from all four reactions are the same length (∼230 nt). Since the amplified region in the 3 ′ UTR is 117 bp long (see J ), and the universal primer has a 14 nt extension (see J ), the poly(A)-tail is estimated to be ∼100 nt long.

Article Snippet: Antibodies used in this study include rabbit polyclonals against ZFC3H1 (also known as CCDC131) (Bethyl Laboratories, A301-457A), MTR4 (also known as SKIV2L2) (Bethyl Laboratories, A300-614A), PABPN1 (Bethyl Laboratories, A303-523A), U1-70K (Abcam, ab83306), Aly , and TRAPα ( ) or mouse monoclonals against U1-70K (Sigma-Aldrich, clone 9C4.1), mAb414 (Sigma), SC35 (Clone SC35, Sigma), and α-tubulin (DM1A, Sigma).

Techniques: Control, shRNA, Transduction, SDS Page, Construct, Transfection, Plasmid Preparation, Immunofluorescence, Amplification, Sequencing, Derivative Assay, Agarose Gel Electrophoresis, Staining, Molecular Weight, Generated

Journal: RNA

Article Title: ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5′ splice site motifs within nuclear speckles

doi: 10.1261/rna.079104.122

Figure Lengend Snippet: ZFC3H1 and U1-70K function in the same pathway for the nuclear retention of 5′SS motif containing mRNAs. ( A ) U2OS cells were treated with lentivirus shRNA against either U1-70K, ZFC3H1, or a mixture of the two. Lysates were collected 96 h post-transduction, separated by SDS-PAGE and immunoprobed for U1-70K, ZFC3H1, and mAb414. Note that to effectively deplete U1-70K, cells were treated with lentivirus containing four shRNA plasmids. Also note that the asterisk (*) denotes a non-specific band. ( B , C ) Control-, U1-70K-, ZFC3H1-, or codepleted cells were transfected with the intronless ftz reporter ± 5′SS as described in . Note that the cytoplasmic/nuclear distribution of ftz-Δi-5′SS mRNA in cells codepleted of U1-70K and ZFC3H1 resembles the distribution in single depletion cells, suggesting that both proteins function in the same pathway. Representative images are shown in C (scale bar, 10 µm) and quantification is shown in D . Each bar represents the average and standard error of at least three independent experiments, each experiment consisting of at least 30 to 60 cells. Student's t -test was performed for C . (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. ( D ) HEK cells expressing carboxy-terminally tagged ZFC3H1 (ZFC3H1-FLAG) were lysed and subjected to immunoprecipitation reactions with FLAG M2 beads or mouse IgG (“Control IP”). Immunoprecipitates were separated by SDS-PAGE and immunoprobed for FLAG and U1-70K. For comparison, 1% of the input lysate was also analyzed. The full-length ZFC3H1-FLAG protein is denoted by the asterisk (*) and a shorter band, likely a degradation product, is denoted by the pound (#) sign.

Article Snippet: Antibodies used in this study include rabbit polyclonals against ZFC3H1 (also known as CCDC131) (Bethyl Laboratories, A301-457A), MTR4 (also known as SKIV2L2) (Bethyl Laboratories, A300-614A), PABPN1 (Bethyl Laboratories, A303-523A), U1-70K (Abcam, ab83306), Aly , and TRAPα ( ) or mouse monoclonals against U1-70K (Sigma-Aldrich, clone 9C4.1), mAb414 (Sigma), SC35 (Clone SC35, Sigma), and α-tubulin (DM1A, Sigma).

Techniques: shRNA, Transduction, SDS Page, Control, Transfection, Expressing, Immunoprecipitation, Comparison

Journal: RNA

Article Title: ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5′ splice site motifs within nuclear speckles

doi: 10.1261/rna.079104.122

Figure Lengend Snippet: ZFC3H1 and U1-70K are required for nuclear retention of 5′SS motif containing mRNAs in speckles. ( A , B ) Control or ZFC3H1-depleted U2OS cells were microinjected with plasmids containing the βG-Δi reporter ± the 5′SS motif. After the indicated times, the cells were fixed and stained for βG mRNA by FISH and for the nuclear speckle marker SC35 by immunofluorescence. ( A ) Example images of U2OS cells fixed 2 h post-injection with βG reporter mRNA ± the 5′SS motif, with each row representing a single field of view with white arrows pointing to examples of βG mRNA/SC35 colocalization. The merged overlayed image shows βG mRNA in red, SC35 in green. ( B ) Quantification of the degree of βG mRNA/SC35 colocalization in cells depleted of ZFC3H1 or control shRNA treatment by Pearson correlation coefficient analysis as previously described . Each bar represents the average and standard error of three independent experiments, each experiment consisting of 150 to 200 nuclear speckles from 15 to 20 cells. Note that ZFC3H1 is not required for the targeting of 5′SS motif containing mRNAs to nuclear speckles. ( C – E ) Control, U1-70K- or ZFC3H1-depleted U2OS cells were transfected with ftz-Δi ± 5 ′ SS motif. Eighteen to twenty-four hours post-transfection, the cells were fixed and stained for ftz mRNA by FISH and for the nuclear speckle marker SC35 by immunofluorescence. Representative images, with each row depicting a single field of view, is shown in C with merged overlays showing ftz mRNA in red and SC35 in green. Scale bar, 10 µM. Examples of ftz /SC35 colocalization are indicated with arrows. ( D ) The degree of ftz /SC35 colocalization by Pearson correlation coefficient analysis was quantified as above, except that values <0.25 were counted as “not colocalized.” Note that U1-70K or ZFC3H1 depletion leads to decreased level of colocalization between ftz mRNA and SC35. Each bar represents the average and standard error of three independent experiments, each experiment consisting of 100–200 nuclear speckles from 10 to 20 cells. ( E ) The amount of ftz reporter mRNA present in nuclear speckles as a percentage of either the nuclear (“Spec/Nuc”) or total cellular (“Spec/Total”) mRNA levels in transfected cells. Each data point represents the average and standard error of the mean of at least three independent experiments, each experiment consisting of 10–20 cells. Student's t -test was performed, (**) P < 0.01, (***) P < 0.001.

Article Snippet: Antibodies used in this study include rabbit polyclonals against ZFC3H1 (also known as CCDC131) (Bethyl Laboratories, A301-457A), MTR4 (also known as SKIV2L2) (Bethyl Laboratories, A300-614A), PABPN1 (Bethyl Laboratories, A303-523A), U1-70K (Abcam, ab83306), Aly , and TRAPα ( ) or mouse monoclonals against U1-70K (Sigma-Aldrich, clone 9C4.1), mAb414 (Sigma), SC35 (Clone SC35, Sigma), and α-tubulin (DM1A, Sigma).

Techniques: Control, Staining, Marker, Immunofluorescence, Injection, shRNA, Transfection

Journal: bioRxiv

Article Title: Druggable genome CRISPRi screen in 3D hydrogels reveals regulators of cortactin-driven actin remodeling in invading glioblastoma cells

doi: 10.1101/2025.01.20.633978

Figure Lengend Snippet: Proximity labeling identifies cortactin as a binding partner for AURKB and ACP1, and a key regulator of GBM invasion, whose expression increases in invasive GBM cells. (a) Workflow for Turbo-ID AURKB and ACP1. Plasmids were created in which AURKB, ACP1, and control sequence encoding amino acids 1-29 of cytochrome P450 were fused to the TurboID sequence, an engineered biotin ligase that uses ATP to convert biotin into biotin–AMP, a reactive intermediate that covalently labels proximal proteins. (b) Shown are KEGG pathways shared amongst proteins bound to both AURKB and ACP1 plotted based on their fold enrichment (x-axis) and -log 10 (FDR) (y-axis) with pathways related to invasion highlighted. Three of these pathways are relevant to cancer invasion and are highlighted along with the correlation between their gene sets: proteoglycans in cancer, regulation of the actin cytoskeleton, and focal adhesion. (c) Proteins bound to AURKB or ACP1 listed based on the log 2 Fold Change of their binding to AURKB relative to cytochrome P450 (x-axis) and the log 2 Fold Change of their binding to ACP1 relative to cytochrome P450 (y-axis). Proteins shaded gray did not exhibit significant binding to either protein relative to control. Proteins in cyan exhibited significant binding to ACP1 relative to control. Proteins in pink exhibited significant binding to AURKB relative to control. Proteins in light purple exhibited significant binding to AURKB and ACP1 relative to control. The eight proteins shaded dark purple and identified are part of the three invasion-related KEGG pathways highlighted in ( b ). (d) Genes for proteins that significantly bound AURKB and ACP1 are stratified according to their gene expression in the edge vs. core fractions of 3D hydrogels into which GBM43 cells invaded. (e) CellSCAPE analysis of how cortactin interacts with proteins known to interact with AURKB ( left ) and ACP1 ( right )

Article Snippet: Primary antibodies for ACP1 (R&D Systems, MAB5075-SP), AURKB (EnCor Biotechnology, MCA-6G2), cortactin (Proteintech, 11381-1-AP), phospho-Serine (Santa Cruz Biotechnology, sc-81514), phospho-Threonine (Santa Cruz Biotechnology, sc-5267) and p-Tyrosine (Santa Cruz Biotechnology, sc-7020) were used to characterize their distribution and quantity.

Techniques: Labeling, Binding Assay, Expressing, Control, Sequencing

Journal: bioRxiv

Article Title: Druggable genome CRISPRi screen in 3D hydrogels reveals regulators of cortactin-driven actin remodeling in invading glioblastoma cells

doi: 10.1101/2025.01.20.633978

Figure Lengend Snippet: Serine phosphorylation of cortactin by AURKB and tyrosine dephosphorylation of cortactin by ACP1. (a) Immunoprecipitation of cortactin from GBM43 cell lysates followed by blotting for AURKB (upper row) and ACP1 (middle row), along with immunoprecipitation of ACP1 followed by blotting for cortactin (lower row) confirms cortactin binding to AURKB and ACP1. (b) Targeting AURKB with CRISPRi in GBM43/sgAURKB cells or via the drug AZD1152-HQPA leads to reduced cortactin serine phosphorylation, as determined by immunoprecipitation of cortactin followed by blotting for phosphorylated serine/threonine (first row) and blotting for cortactin (second row) or blotting the lysates without precipitation for cortactin (third row) or β-actin (fourth row). Quantification shown in the graph to the right was derived from band intensities in 3 technical replicates. (c) Targeting ACP1 with CRISPRi in GBM43/sgACP1 cells or via the drug LMW-PTP Inhibitor I leads to increased cortactin tyrosine phosphorylation, as determined by immunoprecipitation for phosphorylated tyrosine followed by blotting for cortactin (first row) and blotting for heavy chain antibody (second row), as well as blotting the non-precipitated lysates for cortactin (third row) and beta-actin (fourth row). Quantification shown in the graph to the right was derived from band intensities in 3 technical replicates. (d) Western blot for total cortactin levels confirms CTTN knockdown in GBM43/sgCTTN cells relative to GBM43/sgGAL4 control cells and also reveals decreased cortactin levels in GBM43/sgAURKB cells relative to GBM43/sgGAL4 control cells, as determined by quantification of band intensities in 3 technical replicates to generate the graph to the right. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Scatter dot plots show the mean (horizontal bar) with standard deviation (vertical bar).

Article Snippet: Primary antibodies for ACP1 (R&D Systems, MAB5075-SP), AURKB (EnCor Biotechnology, MCA-6G2), cortactin (Proteintech, 11381-1-AP), phospho-Serine (Santa Cruz Biotechnology, sc-81514), phospho-Threonine (Santa Cruz Biotechnology, sc-5267) and p-Tyrosine (Santa Cruz Biotechnology, sc-7020) were used to characterize their distribution and quantity.

Techniques: De-Phosphorylation Assay, Immunoprecipitation, Binding Assay, Derivative Assay, Western Blot, Knockdown, Control, Standard Deviation

Journal: bioRxiv

Article Title: Druggable genome CRISPRi screen in 3D hydrogels reveals regulators of cortactin-driven actin remodeling in invading glioblastoma cells

doi: 10.1101/2025.01.20.633978

Figure Lengend Snippet: AURKB and ACP1 affect cortactin phosphorylation, altering actin polymerization, cortactin levels, and actin-cortactin overlap. (a) Lysates from GBM43/sgGAL4 cells (black), GBM43/sgGAL4 cells treated with AURKB inhibitor AZD1152-HQPA (green), and GBM43/sgAURKB cells (red) were used in actin polymerization assays, with lysates from GBM43/sgGAL4 cells treated with AURKB inhibitor AZD1152-HQPA (P=0.003) and GBM43/sgAURKB cells (P<0.0001) slowing the rate of actin polymerization relative to GBM43/sgGAL4 lysates (assessed by comparing the slope of the polymerization curves derived from linear regression). Differences in polymerization levels at individual timepoints occurred for GBM43/sgAURKB lysates vs. GBM43/sgGAL4 lysates as shown by red asterisks below the x-axis, differences in polymerization between lysates of GBM43/sgGAL4 cells treated with AZD1152-HQPA vs. GBM43/sgGAL4 lysates were not significant during the range of times assessed, although the differences in actin polymerization rates between the two conditions suggested they would eventually differ. n=3/group. Points represent means, error bars represent standard errors. (b) Lysates from GBM43/sgGAL4 cells (black), GBM43/sgGAL4 cells treated with LMW-PTP Inhibitor I to target ACP1 (green), and GBM43/sgACP1 cells (red) were used in actin polymerization assays, with lysates from GBM43/sgGAL4 cells treated with LMW-PTP Inhibitor I to target ACP1 (P<0.0001) and GBM43/sgACP1 cells (P<0.0001) slowing the rate of actin polymerization relative to GBM43/sgGAL4 lysates (assessed by comparing the slope of the polymerization curves derived from linear regression). Differences in polymerization levels at individual timepoints occurred for GBM43/sgACP1 lysates vs. GBM43/sgGAL4 lysates as shown by red asterisks below the x-axis, differences in polymerization between lysates of GBM43/sgGAL4 cells treated with LMW-PTP Inhibitor I vs. GBM43/sgGAL4 lysates at individual timepoints are shown by green asterisks below the x-axis. n=3/group. Points represent means, error bars represent standard errors. (c) Targeting AURKB with CRISPRi or drug (AZD1152-HQPA) in cultured GBM43 cells (n=5/group) leads to reduced cortactin levels (P<0.0001, upper graph) and reduced cortactin-actin overlap (P<0.0001; lower graph) normalized to cell area at a total intracellular level as well as when separating the cell core from cell edge. 60x magnification, scale bar: 2 μm. 60x magnification, scale bar: 2 μm. Scatter dot plots show the mean (horizontal bar) with standard deviation (vertical bar). (d) Targeting ACP1 with CRISPRi or drug (LMW-PTP Inhibitor I) in cultured GBM43 cells (n=5/group) leads to reduced cortactin immunostaining at a total intracellular level (P=0.042 CRISPRi and P=0.045 inhibitor) and in the cell core (P=0.006 CRISPRi and P=0.02 inhibitor), but not at the cell edge (P=0.6-0.8) where background levels of cortactin started out lower (upper graph) and reduced cortactin-actin overlap throughout the cell (P<0.0001 total, core, and edge; lower graph) normalized to cell area. 60x magnification, scale bar: 2 μm. Scatter dot plots show the mean (horizontal bar) with standard deviation (vertical bar). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Article Snippet: Primary antibodies for ACP1 (R&D Systems, MAB5075-SP), AURKB (EnCor Biotechnology, MCA-6G2), cortactin (Proteintech, 11381-1-AP), phospho-Serine (Santa Cruz Biotechnology, sc-81514), phospho-Threonine (Santa Cruz Biotechnology, sc-5267) and p-Tyrosine (Santa Cruz Biotechnology, sc-7020) were used to characterize their distribution and quantity.

Techniques: Derivative Assay, Cell Culture, Standard Deviation, Immunostaining

Journal: bioRxiv

Article Title: Druggable genome CRISPRi screen in 3D hydrogels reveals regulators of cortactin-driven actin remodeling in invading glioblastoma cells

doi: 10.1101/2025.01.20.633978

Figure Lengend Snippet: Targeting AURKB and ACP1 in orthotopic GBM xenografts in vivo slows invasion and uncouples cortactin from actin in tumor cells without affecting survival. GBM43/sgGAL4, GBM43/sgAURKB, GBM43/sgACP1, and GBM43/sgCTTN cells were implanted into the right frontal lobes of athymic mice and allowed to grow until mice reached endpoint, after which tumors were explanted and analyzed by immunofluorescence. (a) GBM43/sgAURKB (P=0.02; n=7) and GBM43/sgCTTN (P=0.02; n=4) xenografts were less invasive than GBM43/sgGAL4 (n=6) based on fractal analysis of images of tumors and their surrounding brain, which yields fractal dimension, a measure of invasive tumor growth pattern as a continuous number between 1 and 2, with higher numbers representing greater invasiveness. GBM43/sgACP1 (n=6) xenografts exhibited no change in fractal dimension compared to GBM43/sgGAL4 xenografts (P=0.8). Composite of images taken at 20x magnification, scale bar: 500 mm. (b) Knockdowns did not impact survival, with no difference noted in the survival of mice with GBM43/sgGAL4, GBM43/sgAURKB, GBM43/sgACP1, and GBM43/sgCTTN xenografts (P=0.6, n=6-10/group). (c) Maximal cross-sectional area of tumors at endpoint was noted to be smaller in GBM43/sgACP1 (n=9, P=0.007) and GBM43/sgCTTN (n=6, P=0.03) xenografts compared to GBM43/sgGAL4 (n=6) xenografts, but unchanged in GBM43/sgAURKB (n=7) xenografts compared to GBM43/sgGAL4 (P=0.08). (d) Immunofluorescence of xenografts at endpoint revealed decreased immunopositive cortactin area normalized to total area of cellular tumor in GBM43/sgAURKB (P=0.003) and GBM43/sgACP1 (P=0.005) xenografts relative to GBM43/sgGAL4 xenografts (upper graph) and increased decoupling of cortactin from actin in GBM43/sgAURKB (P=0.004) xenografts (lower graph). n=4/group, 60x magnification, scale bar: 20 μm. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Scatter dot plots show the mean (horizontal bar) with standard deviation (vertical bar).

Article Snippet: Primary antibodies for ACP1 (R&D Systems, MAB5075-SP), AURKB (EnCor Biotechnology, MCA-6G2), cortactin (Proteintech, 11381-1-AP), phospho-Serine (Santa Cruz Biotechnology, sc-81514), phospho-Threonine (Santa Cruz Biotechnology, sc-5267) and p-Tyrosine (Santa Cruz Biotechnology, sc-7020) were used to characterize their distribution and quantity.

Techniques: In Vivo, Immunofluorescence, Standard Deviation

Journal: Scientific Reports

Article Title: Overexpression of JMJD6 drives immune evasion via the BRD4–IRF1–PD-L1 axis and promotes malignancy in gastric cancer

doi: 10.1038/s41598-025-30705-y

Figure Lengend Snippet: Graphical abstract. Graphical abstract illustrating the hypothetical mechanism by which JMJD6 promotes tumor progression and immune evasion in GC. JMJD6 is overexpressed in gastric cancer cells and promotes BRD4 expression, which upregulates IRF1 and consequently increases PD-L1 expression. Elevated PD-L1 expression on tumor cells inhibits T cell–mediated antitumor immunity, thereby facilitating immune escape.

Article Snippet: Anti-JMJD6 mouse monoclonal antibody (sc-28348; Santa Cruz Biotechnology, TX, USA), anti-PD-L1 rabbit monoclonal antibody (13684; Cell Signaling Technology, MA, USA), anti-ACTB rabbit monoclonal antibody (3700; Cell Signaling Technology), anti-BRD4 rabbit polyclonal antibody (A301-985A50; Bethyl Laboratories, TX, USA), and anti-IRF1 rabbit monoclonal antibody (8478; Cell Signaling Technology) were used.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Overexpression of JMJD6 drives immune evasion via the BRD4–IRF1–PD-L1 axis and promotes malignancy in gastric cancer

doi: 10.1038/s41598-025-30705-y

Figure Lengend Snippet: Overexpression of JMJD6 in gastric cancer. ( a ) The expression of JMJD6 in six GC cell lines compared with the fibroblast cell strain WI-38. The level of JMJD6 mRNA was determined by quantitative RT-PCR in a panel of GC cell lines. The results shown are means ± SD (bars). Black bars represent cell lines where up-regulation of JMJD6 mRNA expression was observed, compared with that in WI-38. The status of a TP53 mutation was the reported status of a TP53 mutation in the database ( http://p53.free.fr/index.html , W: wild-type TP 53, M: mutant TP53 ). Note that among TP53 -mutated GC cell lines, KATO-III had a p53 gene deletion and a frameshift mutation. ( b ) Specific JMJD6 immunostaining in a representative primary tumor sample. Based on this result, the intensity scores for JMJD6 staining were determined as follows: 0 = negative, 1 = weak, 2 = moderate, 3 = strong. Kaplan–Meier plots depending on the intensity ( c and proportion ( d ) scores of specific immunostainings of JMJD6. The log-rank test was used for statistical analysis; P < 0.05 was considered to be statistically significant. e. For scoring JMJD6 expression by immunohistochemistry (IHC), the intensity and the percentage of the total cell population (0% ≤ 0 ≤ 20%, 20% < 1 ≤ 40%, 40% < 2 ≤ 60%, 60% < 3 ≤ 100%) that expressed JMJD6 were evaluated for each case. Expression of JMJD6 was graded as high expression (intensity plus proportion scores ≥ 4 for tumor cells showing immunopositivity) or low expression (intensity plus proportion scores ≤ 3 for tumor cells showing immunopositivity). f. Cancer-specific survival rates of patients with GC (as determined by Kaplan–Meier plots), depending on JMJD6 expression.

Article Snippet: Anti-JMJD6 mouse monoclonal antibody (sc-28348; Santa Cruz Biotechnology, TX, USA), anti-PD-L1 rabbit monoclonal antibody (13684; Cell Signaling Technology, MA, USA), anti-ACTB rabbit monoclonal antibody (3700; Cell Signaling Technology), anti-BRD4 rabbit polyclonal antibody (A301-985A50; Bethyl Laboratories, TX, USA), and anti-IRF1 rabbit monoclonal antibody (8478; Cell Signaling Technology) were used.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Mutagenesis, Immunostaining, Staining, Immunohistochemistry

Journal: Scientific Reports

Article Title: Overexpression of JMJD6 drives immune evasion via the BRD4–IRF1–PD-L1 axis and promotes malignancy in gastric cancer

doi: 10.1038/s41598-025-30705-y

Figure Lengend Snippet: Suppression of malignant activities in GC cells by JMJD6 knockdown. ( a ) The effects of JMJD6 knockdown on cell proliferation at the indicated times by siRNA (siRNA-JMJD6) compared with control siRNA in MKN45 (wild-type TP53 ) and MKN74 (mutant TP53 ) cell lines. ( b ) Representative population results in each phase of the cell cycle in each cell, as assessed by FACS at 72 h post-treatment with siRNA. ( c ) Suppression of migration, invasion, and epithelial-mesenchymal transition in GC cells by JMJD6 knockdown. Knocking down JMJD6 by siRNA-JMJD6 suppressed migration and invasion in MKN45 (wild-type TP53 ) (A) and MKN74 (mutant TP53 ) cell lines. The bar graphs show the means ± SD ( n = 4). The Mann–Whitney U-test was used for statistical analysis. P < 0.05 was considered statistically significant.

Article Snippet: Anti-JMJD6 mouse monoclonal antibody (sc-28348; Santa Cruz Biotechnology, TX, USA), anti-PD-L1 rabbit monoclonal antibody (13684; Cell Signaling Technology, MA, USA), anti-ACTB rabbit monoclonal antibody (3700; Cell Signaling Technology), anti-BRD4 rabbit polyclonal antibody (A301-985A50; Bethyl Laboratories, TX, USA), and anti-IRF1 rabbit monoclonal antibody (8478; Cell Signaling Technology) were used.

Techniques: Knockdown, Control, Mutagenesis, Migration, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Overexpression of JMJD6 drives immune evasion via the BRD4–IRF1–PD-L1 axis and promotes malignancy in gastric cancer

doi: 10.1038/s41598-025-30705-y

Figure Lengend Snippet: JMJD6 regulates BRD4, IRF1 and PD-L1 expression. ( a ) The knockdown of JMJD6 by transfection with siRNA-JMJD6 suppressed BRD4, IRF1 and PD-L1 in MKN74. In addition, the knockdown of BRD4 by transfection with siRNA-BRD4 suppressed IRF1 and PD-L1 in MKN74. In contrast, the knockdown of BRD4 did not suppress JMJD6 in MKN74. ( b ) Knockdown of JMJD6 suppressed PD-L1 and BRD4 expression in MKN74 gastric cancer (GC) cells. White dotted lines indicate nuclear boundaries. ( c ) Co-culture assay of GC cells and T cells. Under JMJD6 knockdown, T cells had more potent anti-tumor activity against GC cells compared with NC, and the proliferation ratio of GC cells was significantly decreased (mean ± SD, n = 3; error bars indicate SD, n = 3). ( d ) An impedance-based tumor-cell killing assay. The knockdown of JMJD6 increased the anti-tumor activity of T cells and inhibited the proliferation of GC cells. ( e ) JMJD6 overexpression using plasmid transfection promotes BRD4, IRF1 and PD-L1 expression. ( f ) A hypothetical model of the overexpression or activation of JMJD6 in GC cells.

Article Snippet: Anti-JMJD6 mouse monoclonal antibody (sc-28348; Santa Cruz Biotechnology, TX, USA), anti-PD-L1 rabbit monoclonal antibody (13684; Cell Signaling Technology, MA, USA), anti-ACTB rabbit monoclonal antibody (3700; Cell Signaling Technology), anti-BRD4 rabbit polyclonal antibody (A301-985A50; Bethyl Laboratories, TX, USA), and anti-IRF1 rabbit monoclonal antibody (8478; Cell Signaling Technology) were used.

Techniques: Expressing, Knockdown, Transfection, Co-culture Assay, Activity Assay, Over Expression, Plasmid Preparation, Activation Assay

Journal: Scientific Reports

Article Title: Overexpression of JMJD6 drives immune evasion via the BRD4–IRF1–PD-L1 axis and promotes malignancy in gastric cancer

doi: 10.1038/s41598-025-30705-y

Figure Lengend Snippet: JMJD6 expression is positively correlated with PD-L1 in GC tissues. Immunohistochemical staining of JMJD6 and PD-L1 was performed on GC tissues from 174 patients. Twenty high JMJD6-expressing cases and twenty low JMJD6-expressing cases were selected for PD-L1 staining and CPS evaluation. Representative images from four cases are shown. High JMJD6 expression was significantly associated with CPS ≥ 1 ( P = 0.0281, Fisher’s exact test).

Article Snippet: Anti-JMJD6 mouse monoclonal antibody (sc-28348; Santa Cruz Biotechnology, TX, USA), anti-PD-L1 rabbit monoclonal antibody (13684; Cell Signaling Technology, MA, USA), anti-ACTB rabbit monoclonal antibody (3700; Cell Signaling Technology), anti-BRD4 rabbit polyclonal antibody (A301-985A50; Bethyl Laboratories, TX, USA), and anti-IRF1 rabbit monoclonal antibody (8478; Cell Signaling Technology) were used.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , b , Unsupervised hierarchical clustering of the phospho-catalytic activity signatures of WiDr cells treated with vemurafenib (VEM; n = 13 independent experiments) ± gefitinib (GEF; n = 5 independent experiments) or cetuximab (CET; n = 5 independent experiments) as compared to their untreated control counterparts ( n = 23 independent experiments). a , ATP consumption in cell extracts using 228 peptide sensors. b , Kinase signatures deconvoluted from the peptide phosphorylation profiles in a . Bar graphs next to the heatmaps show the P values (two-sided Student’s t test) for each of the peptides ( a ) or kinases ( b ) comparing all treated samples to controls. c , Volcano plot of the data in b displaying the change in kinase activity versus P value for each treatment arm (same as b : VEM, n = 13; VEM + GEF, n = 5; VEM + CET, n = 5; as compared to their untreated control counterparts ( n = 23), where n is the number of independent experiments). d , Bar graphs of the data in b representing the shift in activity of SRC, SFK, EGFR and HER family kinases when cells were treated with vemurafenib alone or in combination with gefitinib or cetuximab. Kinase activity is compared to that in untreated control cells, and data are displayed as the average ± standard error in nM of ATP. Same as in b , c : VEM, n = 13; VEM + GEF, n = 5; VEM + CET, n = 5; as compared to their untreated control counterparts ( n = 23), where n is the number of independent experiments. e , Representative IHC images showing staining intensity for active SFK (phosphorylated Y419 epitope in the SRC activation site) following treatment of a BRAF V600E CRC PDX model with vehicle control, dabrafenib (DAB) and/or trametinib (TRA) for 3 or 21 d. The color-coded bottom panel highlights differences in bin intensities from automated image analysis (see for details). IHC images and intensity quantifications are representative of n = 2 independent PDX tumors per treatment condition and n = 20 independent tissue areas per tumor and per condition. f , Quantification of IHC staining intensity for total and activated SFK in two PDX models treated for 3 or 21 d with dabrafenib ± trametinib versus vehicle control (two-sided Student’s t test, P < 1 × 10 –15 ). Using batch processing and automated analysis of IHC images, protein expression was measured at the single-cell level (that is, n ≥ 10,000 individual cancer cells per treatment condition and tumor). g , Proposed parallel mechanism of SRC activation in response to BRAF/MEK/EGFR therapies in BRAF V600E CRC. BRAF*, BRAF V600E .

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Activity Assay, Control, Phospho-proteomics, Staining, Activation Assay, Immunohistochemistry, Expressing

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , BRAF V600E CRC cell lines were treated with vemurafenib (VEM) for 7 to 8 hours. Vemurafenib was used at 1.75 uM in HT29, 2 uM in KM20, 0.15 uM in LIM2405, 2.25 uM in SNUC5, and 1.5 uM in WiDr (details of treatment conditions (concentration and time) are available in spreadsheets Supplementary Table and of the Supplementary Tables document). Cell lysates were assayed by western blot with the indicated antibodies. Upper panels: SFK activation is reflected by increased phosphorylation of the SRC activation site, Y419 (pY419). HSP90 is used as loading control. Bottom panel: reduction in ERK 1/2 phosphorylation as control of BRAF inhibition. Molecular weight/size markers are indicated on the right (kDa). The experiment was repeated ≥3 times with similar results. b , Representative IHC images showing total SRC staining intensity following treatment of a BRAF V600E CRC PDX model with vehicle control, dabrafenib (DAB) and/or trametinib (TRA) for 3 or 21 days. The color-coded bottom panel highlights differences in bin intensities resulting from automated image analysis (see Methods for details). A scale bar is provided (100 micrometers). As in main Fig. , IHC images and intensity quantifications are representative of n = 2 independent PDX tumors per treatment condition, and n = 20 independent tissue areas per tumor and per condition. c , Quantification of IHC staining intensity for total and activated SFK in two PDX models treated for 3 or 21 days with DAB ± TRA vs. vehicle control. As in main Fig. , we used batch processing and automated analysis of IHC images to quantify protein expression at the single cell level (that is, n ≥ 10,000 individual cancer cells per treatment condition and tumor). d , SRC staining score by IHC in untreated patient CRC tumor specimens with or without a BRAF V600E mutation, from primary (prim.) or metastatic (met.) sites.

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Concentration Assay, Western Blot, Activation Assay, Phospho-proteomics, Control, Inhibition, Molecular Weight, Staining, Immunohistochemistry, Expressing, Mutagenesis

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , BRAF V600E CRC cell lines treated with vemurafenib ± gefitinib were lysed and immunoblotted with the indicated antibodies. SFK activation is reflected by increased phosphorylation of the SRC activation site Y419 (pY419) and lack of phosphorylation of the inhibitory site Y530 (non-pY530). Active SRC can be deactivated by rephosphorylation of Y530 by CSK. HSP90 serves as a loading control. Molecular weight/size markers are indicated on the right (kDa). The experiment was repeated three times with similar results. b , Shift in vemurafenib sensitivity measured by cell viability assay (left) and calculation of the CI (right) upon treatment of BRAF V600E CRC or melanoma cell lines with vemurafenib + gefitinib ± a SRC inhibitor, dasatinib, for 3 d ( n = 4 independent experiments per cell line). c , Colony formation assays in which BRAF V600E CRC cells were treated with an increasing concentration of vemurafenib alone (control) or with a fixed dose of gefitinib ± dasatinib. Data are representative of n = 2 independent repeats. d , Treatment of cell line-derived xenograft mouse models with a vemurafenib progenitor, PLX4720 (PLX); dasatinib; saracatinib; and/or gefitinib for 21 d ( n = 7 mice per group). Plotted is the percent change in tumor volume relative to baseline (day 1). Data are displayed as the average for all mice in a specified treatment group ± standard error. e , Treatment of PDX models with vemurafenib ± gefitinib ± dasatinib for 21 d, with data plotted as in d ( n = 8 mice per group). All raw and relative tumor volumes and exact P values shown in d , e are available as Source Data; P values are from a two-sided Student’s t test. f , g , GLMs testing the association of change in tumor volume between treatment arms and vehicle over time shown in d , e . Effect size is measured as the GLM standard coefficient. A GLM was applied to each tumor model separately or combined. Results for cell line xenografts and PDXs are shown in f and g , respectively. GLM P values corrected for FDR are shown in g . NT, not tested. h , i , Comparison of the effect sizes and FDR-corrected P values of treatment arms with and without a SRC inhibitor. The same number of mice per group shown in d , e was used for the analyses in f – i (that is, n = 7 mice per treatment group for WiDr and KM20 cell line xenografts and n = 8 mice per treatment group for PDX models 1 and 2).

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Activation Assay, Phospho-proteomics, Control, Molecular Weight, Viability Assay, Concentration Assay, Derivative Assay, Comparison

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , Western blots to detect phospho-T202/Y204 ERK1/2 and total ERK1/2 in BRAF V600E CRC cell lines treated with vemurafenib (VEM) ± gefitinib (GEF) or dasatinib (DAS) collected after 8 h, 24 h, 48 h or 72 h. HSP90 is used as a loading control. The experiment was repeated 2 independent times with similar results. b , Quantification of western blots shown in panel ( a ). The bar plot (averages and standard deviations per treatment condition across cell lines) was overlaid with a dot plot displaying individual measurements per cell line and condition. Data are normalized to p-ERK levels after 8 h treatment with VEM alone. See table below for detailed values and color codes; n = 8 cell lines. c , Western blots to detect total and phospho-Y654 beta-catenin (CTNNB1) in BRAF V600E CRC cell lines treated with VEM, or GEF, or DAS, or combinations of VEM + GEF, or VEM + DAS, or VEM + GEF + DAS. The detection of phospho-Y419 and total SFK serves as a control for the effect of SFK-inhibition (with DAS). The experiment was repeated 3 independent times with similar results. In panels a , c , molecular weight/size markers are indicated on the right (kDa).

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Western Blot, Control, Inhibition, Molecular Weight

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , Levels of secreted PGE2 were measured by ELISA in the conditioned medium of BRAF V600E CRC cell lines treated with vemurafenib ± gefitinib. Data are displayed as the average PGE2 secretion in pg ml –1 per 100,000 cells ± s.d. ( n = 3 independent experiments per cell line). b , BRAF V600E CRC cell lines were treated with exogenous PGE2. Cell lysates were assayed by western blot as indicated. Y419 phosphorylation and lack of phosphorylation of Y530 (non-pY530) are used as readouts of SFK activation. HSP90 serves as a loading control. The experiment was repeated two independent times per cell line with similar results. c , Bar graphs representing fold change (log 2 scale) ± standard error for change in sensitivity to vemurafenib upon further treatment with PGE2 or untreated control in 3-day cell viability assays. Top, CI, Bliss model. Same methods as in Fig. ( n = 3 independent experiments per cell line). d , Western blots to detect pY654 of CTNNB1 in BRAF V600E CRC cell lines treated with exogenous PGE2. The experiment was repeated two independent times per cell line with similar results. e , Three BRAF V600E CRC cell lines engineered with a doxycycline-inducible constitutively active GNAS construct, iGNAS R201C , were treated with doxycycline. Cell lysates were assayed by western blot as indicated. The experiment was repeated three times with similar results. f , Bar graphs representing fold change (log 2 scale) ± standard error for change in sensitivity to vemurafenib or vemurafenib + gefitinib after iGNAS R201C induction in 3-day cell viability assays. Top, CI, as in c ( n = 3 independent experiments per cell line). g , GNAS was knocked out in BRAF V600E CRC cells using CRISPR (GNAS-KO). GNAS knockout was validated by western blot (top). GNAS-KO cells were treated with vemurafenib, and cell lysates were assayed by western blot with the indicated antibodies (bottom). The experiment was repeated ≥2 times with similar results. In b , d , e , g , molecular weight/size markers are indicated on the right (kDa). h , Bar graphs representing fold change (log 2 scale) ± standard error for change in sensitivity to vemurafenib or vemurafenib + gefitinib with GNAS knockout in 3-day cell viability assays. Top, CI, as in c ( n = 3 independent experiments per cell line). i , Representative IHC images showing COX2 staining intensity following treatment of a BRAF V600E CRC PDX model with vehicle control, dabrafenib and/or trametinib for 3 or 21 d (where n is the same as defined in Fig. ). The color-coded bottom panel highlights differences in bin intensities from automated image analysis (see for details). j , Quantification of COX2 staining intensity by IHC for two PDX models treated for 3 or 21 d with dabrafenib ± trametinib versus vehicle control (two-sided Student’s t test, P < 1 × 10 –15 ; n is the same as defined in Fig. ). k , Proposed mechanism of COX2–PGE2-mediated SRC-driven resistance to BRAF/MEK/EGFR therapies in BRAF V600E CRC.

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Activation Assay, Control, Construct, CRISPR, Knock-Out, Molecular Weight, Staining