Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) GSEA plots showing pathway signatures that are enriched in MAP3K8 -high PDAC samples analyzed from the TCGA PDAC Firehose Legacy database. (B) Relative SRE reporter activity in HEK293T cells stably expressing EV or KRAS G12V and KRAS G12D -mutant HPAC and Pa01C cells treated with different concentrations of TPL2i for 48 h. Data were pooled from 2 to 3 independent experiments, each done in biological triplicates and presented as mean ± SEM (**** p < 0.0001, *** p < 0.0002, ** p < 0.0021 by two-way ANOVA). (C) IP western blots of HEK293T cells stably expressing EV or HA-TPL2 transfected with EV or FLAG-KRAS G12V . 48 h after transfection, cells were treated with DMSO or bortezomib (0.1 μM) for 6 h and harvested for western blots. (D) Western blots and t 1/2 of HA-TPL2 protein in HEK293T cells stably expressing EV or FLAG-KRAS G12V treated with 10 μg/mL CHX for the indicated durations. (E) Representative fluorescence images and quantification showing in situ TPL2-TRIM4 punctate dots by Duolink PLA in HEK293T cells expressing EV or FLAG-KRAS G12V . Data are presented as mean ± SEM. **** p < 0.0001 by two-tailed t test, six fields per condition were analyzed. Scale bars, 50 μm). (F) Western blots and t 1/2 of endogenous TRIM4 protein in control or KRAS -silenced Pa01C cells treated with CHX (10 μg/mL) for the indicated durations. (G) Western blots and t 1/2 of endogenous TRIM4 protein in MIA Paca-2 cells pre-treated overnight with AMG-510 (0.2 μM), followed by (CHX 10 μg/mL) for the indicated durations. For (D), (F), and (G), the t 1/2 of the indicated proteins were calculated by comparing band intensities at t 0 and performing one-phase exponential decay analysis. Data from three independent experiments are quantified, with blots from one representative experiment shown.

Article Snippet: The pBabe expression FLAG-KRAD G12D was obtained from Dr. Adhikari (Washington University, St. Louis, MO, USA). pLVX puro TRIM21-GFP (Cat# 116941) and pLVX puro TRIM21 C54Y-GFP (Cat# 116942), pXL010-Wnt dual (GFP-Fire) reporter (Cat# 40588), pCMV-Pam-FL (Cat# 42570), pcDNA5FRT/TO-RNF185-GFP (Cat #113498), HA-GSK3β pcDNA3 (Cat #14753) were purchased from Addgene.

Techniques: Activity Assay, Stable Transfection, Expressing, Mutagenesis, Western Blot, Transfection, Fluorescence, In Situ, Two Tailed Test, Control

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) Volcano plot of KRAS G12V BioID experiments, which identified RNF185 and MYCBP2 as the two E3 ligases enriched by >2-fold through proximal biotinylation by BirA-KRAS G12V . Identification of the known KRAS G12V effectors BRAF and RALA confirmed the robustness of the data. EFR3A was confirmed and published previously. (B) IP-western blots confirming the interaction of HA-KRAS G12V with endogenous RNF185 and TRIM21 in HEK293T cells. (C) IP-western blots of HEK293T cells stably expressing EV or FLAG-tagged KRAS G12D treated with DMSO or MRTX1133 at 0.1 and 0.5 μM overnight and harvested for western blots. (D and E) Representative Duolink PLA fluorescence images and quantification of fluorescent puncta showing in situ interaction between (D) endogenous RNF185 and KRAS or (E) TRIM21-GFP and endogenous RNF185 in Pa01C and HPAC cells treated overnight with MRTX1133. Data presented as mean ± SEM (****p < 0.0001 by one-way ANOVA; six fields per condition were analyzed; scale bars, 20 μm). (F) IP-western blot of HEK293T cells stably expressing EV or HA-KRAS G12V and transiently transfected with TRIM21-GFP or/and FLAG-RNF185. (G and H) Western blots and t 1/2 of endogenous TRIM21 protein in (G) HEK293T FLAG-KRAS G12D cells stably expressing EV or GFP-RNF185 or (H) Pa01C cell stably expressing a scramble or shRNF185, following treatment with CHX (10 μg/mL) for the indicated durations. (I) Western blots and densitometric quantification of the indicated markers in scramble and two different RNF186 -silenced Pa01C cells. (J) Western blots and t 1/2 of endogenous TRIM21 and TRIM4 proteins in HPAC cells pre-treated overnight with DMSO or MRTX1133, followed by CHX (10 μg/mL) for the indicated durations. (K) Western blots and densitometric quantification showing the impact of overnight MRTX1133 treatment on the indicated markers in Pa01C and HPAC cells. For (G), (H), and (J), data from three independent experiments were quantified, with blots from one representative experiment shown.

Article Snippet: The pBabe expression FLAG-KRAD G12D was obtained from Dr. Adhikari (Washington University, St. Louis, MO, USA). pLVX puro TRIM21-GFP (Cat# 116941) and pLVX puro TRIM21 C54Y-GFP (Cat# 116942), pXL010-Wnt dual (GFP-Fire) reporter (Cat# 40588), pCMV-Pam-FL (Cat# 42570), pcDNA5FRT/TO-RNF185-GFP (Cat #113498), HA-GSK3β pcDNA3 (Cat #14753) were purchased from Addgene.

Techniques: Western Blot, Stable Transfection, Expressing, Fluorescence, In Situ, Transfection

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet:

Article Snippet: The pBabe expression FLAG-KRAD G12D was obtained from Dr. Adhikari (Washington University, St. Louis, MO, USA). pLVX puro TRIM21-GFP (Cat# 116941) and pLVX puro TRIM21 C54Y-GFP (Cat# 116942), pXL010-Wnt dual (GFP-Fire) reporter (Cat# 40588), pCMV-Pam-FL (Cat# 42570), pcDNA5FRT/TO-RNF185-GFP (Cat #113498), HA-GSK3β pcDNA3 (Cat #14753) were purchased from Addgene.

Techniques: Ubiquitin Proteomics, Recombinant, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Ligation, Western Blot, Over Expression, Plasmid Preparation, Amplification, Clone Assay, Expressing, Software, Microscopy

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) Schematic workflow of IP-mass spectrometry with HEK293T cells stably expressing FLAG-KRAS G12V or FLAG-RALA G23V . Selected protein hits with more than 10 significant sequence matches and enriched in KRAS G12V lysates by ≥3-fold are shown. An asterisk denotes known KRAS interactors based on BioGRID 4.4 . (B) IP-western blots confirming the interaction of HA-KRAS G12V with endogenous TRIM21 in HEK293T cells. (C) IP-western blots of HEK293T cells stably expressing EV or HA-TPL2 transfected with EV (denoted as −), GFP-tagged WT, or enzymatically inactive (C54Y) TRIM21. 48 h after transfection, cells were treated with DMSO or bortezomib (0.1 μM) for 6 h and harvested for western blots. (D) Western blots and t 1/2 of HA-TPL2 proteins in HEK293T cells stably expressing EV or TRIM21-GFP treated with CHX (10 μg/mL) for the indicated durations. Data from three independent experiments is quantified with blots from one representative experiment shown. (E) Relative SRE-driven luciferase reporter activity in HEK293T cells transfected with EV or the indicated constructs. Data were pooled from three independent experiments, each done in triplicates, and are presented as mean ± SEM (****p < 0.0001). (F) Western blots and densitometric quantification of the indicated markers in Pa01C and HPAC cells stably expressing a scramble or two different shRNAs against TRIM21 . (G) GSEA plots showing KRAS and MEK oncogenic signatures in TRIM21 -silenced Pa01C cells.

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Mass Spectrometry, Stable Transfection, Expressing, Sequencing, Western Blot, Transfection, Luciferase, Activity Assay, Construct

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) IP-western blots of HEK293T cells stably expressing EV or FLAG-TRIM4 and transfected with EV or TRIM21-GFP. (B) IP-western blots of HEK293T cells stably expressing EV or HA-TPL2 cells transfected with a fixed amount of TRIM4 (6 μg in 10 cm 2 plates) and different amounts (1.5 μg, 3 μg, and 6 μg) of TRIM21. (C) Western blots and t 1/2 of endogenous TRIM4 protein in HEK293T cells transfected with the indicated cDNAs and treated with CHX (10 μg/mL) for the indicated durations. Data from three independent experiments were quantified, with blots from one representative experiment shown. (D) IP-western blots of HEK293T cells stably expressing EV or HA-TPL2 and transfected with EV, TRIM21-GFP, and FLAG-KRAS G12V . (E) IP-western blots showing TRIM21 polyubiquitination in HEK293T cells transfected with EV, TRIM21-GFP, and/or HA-KRAS G12V . For (A), (B), (D), and (E), 48 h after transfection, cells were treated with DMSO or bortezomib (0.1 μM) for 6 h and harvested for western blots.

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Western Blot, Stable Transfection, Expressing, Transfection

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) Volcano plot of KRAS G12V BioID experiments, which identified RNF185 and MYCBP2 as the two E3 ligases enriched by >2-fold through proximal biotinylation by BirA-KRAS G12V . Identification of the known KRAS G12V effectors BRAF and RALA confirmed the robustness of the data. EFR3A was confirmed and published previously. (B) IP-western blots confirming the interaction of HA-KRAS G12V with endogenous RNF185 and TRIM21 in HEK293T cells. (C) IP-western blots of HEK293T cells stably expressing EV or FLAG-tagged KRAS G12D treated with DMSO or MRTX1133 at 0.1 and 0.5 μM overnight and harvested for western blots. (D and E) Representative Duolink PLA fluorescence images and quantification of fluorescent puncta showing in situ interaction between (D) endogenous RNF185 and KRAS or (E) TRIM21-GFP and endogenous RNF185 in Pa01C and HPAC cells treated overnight with MRTX1133. Data presented as mean ± SEM (****p < 0.0001 by one-way ANOVA; six fields per condition were analyzed; scale bars, 20 μm). (F) IP-western blot of HEK293T cells stably expressing EV or HA-KRAS G12V and transiently transfected with TRIM21-GFP or/and FLAG-RNF185. (G and H) Western blots and t 1/2 of endogenous TRIM21 protein in (G) HEK293T FLAG-KRAS G12D cells stably expressing EV or GFP-RNF185 or (H) Pa01C cell stably expressing a scramble or shRNF185, following treatment with CHX (10 μg/mL) for the indicated durations. (I) Western blots and densitometric quantification of the indicated markers in scramble and two different RNF186 -silenced Pa01C cells. (J) Western blots and t 1/2 of endogenous TRIM21 and TRIM4 proteins in HPAC cells pre-treated overnight with DMSO or MRTX1133, followed by CHX (10 μg/mL) for the indicated durations. (K) Western blots and densitometric quantification showing the impact of overnight MRTX1133 treatment on the indicated markers in Pa01C and HPAC cells. For (G), (H), and (J), data from three independent experiments were quantified, with blots from one representative experiment shown.

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Western Blot, Stable Transfection, Expressing, Fluorescence, In Situ, Transfection

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) Workflow of IP-mass spectrometry and enrichment plot demonstrating GSK3β as one of the top hits enriched in HEK293T cells expressing HA-TPL2 plus TRIM21-GFP when compared to HA-TPL2 alone. (B) IP-western blots confirming the interaction of HA-TPL2 with endogenous GSK3β when TRIM21 is overexpressed. (C and D) Bar graphs showing relative luciferase reporter activity in (C) 293T TOPFlash reporter cells transfected with EV or HA-TPL2 and (D) Pa01C and HPAC cells stably expressing a Wnt luciferase reporter treated overnight with TPL2i. (E) Bar graphs showing selected up- and downregulated Molecular Signatures Database (MSigDB) oncogenic signatures with normalized enrichment score and false discovery rate q values. Shown are GSEA plots focusing on Wnt and MEK signatures in MAP3K8 -silenced Pa01C cells. (F and G) Western blots and densitometric quantification of the indicated markers in (F) HEK293T cells stably expressing EV or HA-TPL2 and (G) Pa01C and HPAC cells treated overnight with TPL2i. (H) Relative Wnt luciferase reporter activity in Pa01C and HPAC cells treated with the indicated agents overnight. (I and J) Western blots and densitometric quantification of p-GSK3β and p-β-catenin in (I) Pa01C and HPAC cells stably expressing EV or TRIM21-GFP and (J) scramble or TRIM21 -silenced Pa01C cells. (K) GSEA plot showing the upregulated Wnt pathway signature in TRIM21 -silenced Pa01C cells. (L) IP-western blots showing GSK3β polyubiquitination in HEK293T cells stably expressing the indicated cDNAs. (M) Western blots and t 1/2 of endogenous GSK3β in Pa01C cells stably expressing EV or TRIM21-GFP treated with CHX (10 μg/mL) for the indicated durations. Data from three independent experiments were quantified, with blots from one representative experiment shown. (N and O) Relative TOPFlash luciferase reporter activity in HEK293T cells stably expressing the indicated cDNAs (N) treated overnight with DMSO or GSK3β inhibitor and (O) treated overnight with TPL2i. (P) Relative Wnt luciferase reporter activity in Pa01C and HPAC cells treated overnight with the indicated agents. For (C), (D), (H), and (N)–(P), data were pooled from two or three independent experiments, each done in technical triplicates, and are presented as mean ± SEM. The p values were calculated from two-tailed t test or ANOVA for multiple groups. **** p < 0.0001, *** p < 0.0002, ** p < 0.0021, * p < 0.0332.

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Mass Spectrometry, Expressing, Western Blot, Luciferase, Activity Assay, Transfection, Stable Transfection, Two Tailed Test

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet:

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Ubiquitin Proteomics, Recombinant, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Ligation, Western Blot, Over Expression, Plasmid Preparation, Amplification, Clone Assay, Expressing, Software, Microscopy

Journal: bioRxiv

Article Title: Distinct molecular and functional properties of human induced-proprioceptor and low-threshold mechanoreceptor neurons

doi: 10.1101/2024.05.12.593730

Figure Lengend Snippet: (A) Schematic of the protocol to derive induced-proprioceptor neurons (iPN: NGN2+RUNX3) and induced-LTMR neurons (iLTMR: NGN2+SHOX2). Growth factors (GF) include BDNF, GDNF, NT-3, and ß-NGF. (B) Heatmap of key stem cell ( NANOG , POU5F1 ), neuron ( MAP2 , TUBB3 , RBFOX3 ), and sensory neuron-specific ( PRPH , NEFH , ISL1 , POU4F1 , LDHB , VSNL1 ) markers by bulk RNA sequencing (Log 2 TPM) of the iPN and iLTMR cultures, n = 3 biological replicates. (C) Representative immunocytochemistry images of the neuronal marker ß-III-TUBULIN ( red ) and the sensory neuronal markers NF200 ( red ), BRN3A ( red ), and ISLET1 ( red ). Nuclei are shown in blue. Scale bars = 20 μm. The percentage of iPN and iLTMR cells expressing (D) BRN3A and (E) ISLET1 compared to the number of nuclei. n = 3 biological replicates, >100 cells counted per biological replicate. (F) Representative immunocytochemistry images of the sensory neuron subtype markers TRKA ( red ), TRKB ( red ) and TRKC ( red ) in the iPN and iLTMRs. Nuclei are shown in blue. Scale bar = 20 μm. (G) Heatmap of key proprioceptor, LTMR and nociceptor markers by bulk RNA sequencing of the iPN and iLTMR cultures, presented as Log 2 TPM, n = 3 biological replicates.

Article Snippet: The lentiviral expression vectors used in this study include pLV-TetO-eGFP-PuroR (GFP control vector), pLV-TetO-hNGN2-hRUNX3-GFP-PuroR (NGN2 + RUNX3 expression vector) and pLV-TetO-hNGN2-hSHOX2-GFP-PuroR (NGN2 + SHOX2 expression vector) , which were designed in-house using the pLV-TetO-hNGN2-eGFP-PuroR (Addgene #79823) as a backbone.

Techniques: RNA Sequencing Assay, Immunocytochemistry, Marker, Expressing

Journal: bioRxiv

Article Title: Distinct molecular and functional properties of human induced-proprioceptor and low-threshold mechanoreceptor neurons

doi: 10.1101/2024.05.12.593730

Figure Lengend Snippet: hPSCs were transduced with lentiviral viral particles containing the reverse tetracycline transactivator (rTTA) and either pLV-TetO-eGFP-PuroR (GFP control), pLV-TetO-hNGN2-hRUNX3-GFP-PuroR (iNGN2 + iRUNX3) or pLV-TetO-hNGN2-hSHOX2-GFP-PuroR (iNGN2 + iSHOX2). Viral particles were removed and doxycycline was administered 24 h following transduction. hPSCs were harvested for Western blotting or immunocytochemistry after 96 h of doxycycline administration. (A) Representative images of transduced cultures that confirm the expression of GFP after 96 h of doxycycline incubation. Western blots of hPSC protein lysates probed for (B) NGN2 and RUNX3 or (C) NGN2 and SHOX2 protein expression. (D) Representative immunocytochemistry images showing the cellular co-localisation of GFP ( green ) with NGN2 ( magenta ) and RUNX3 ( red ) or (E) with NGN2 ( magenta ) and SHOX2 ( red ). Nuclei are shown in blue. Scale bars = 50 μm.

Article Snippet: The lentiviral expression vectors used in this study include pLV-TetO-eGFP-PuroR (GFP control vector), pLV-TetO-hNGN2-hRUNX3-GFP-PuroR (NGN2 + RUNX3 expression vector) and pLV-TetO-hNGN2-hSHOX2-GFP-PuroR (NGN2 + SHOX2 expression vector) , which were designed in-house using the pLV-TetO-hNGN2-eGFP-PuroR (Addgene #79823) as a backbone.

Techniques: Transduction, Western Blot, Immunocytochemistry, Expressing, Incubation

Journal: Cell Reports

Article Title: Caspase cleavage and nuclear retention of the energy sensor AMPK-α1 during apoptosis

doi: 10.1016/j.celrep.2022.110761

Figure Lengend Snippet: Identification of caspase cleavage site in AMPK-α1 (A) Sequences showing mutation of possible cleavage sites near C terminus of AMPK-α1. (B) Jurkat cells stably transfected with indicated constructs expressing GFP-tagged AMPK-α1 were treated with anti-Fas antibody and western blot analysis carried out. (C) In vitro caspase cleavage assay was performed using Jurkat cell lysates containing GFP-tagged wild type or D529A-mutant AMPK-α1. (D) Schematic diagram of AMPK-α1 cleavage by caspase. (E) Sequence alignment of AMPK-α1 in different species. Cleavage site is marked in red, and the region containing NES is marked in blue. (F) Sequence alignment of AMPK-α1 and α2 at the C-terminal tail. The cleavage site on α1 and corresponding site on α2 are marked in red and region containing NES is marked in blue.

Article Snippet: pLV[Exp]-Puro-EF1A>AMPK-α1(D529A)-EGFP with C-terminal GFP tag , VectorBuilder , VB180420-1016sdf.

Techniques: Mutagenesis, Stable Transfection, Transfection, Construct, Expressing, Western Blot, In Vitro, Cleavage Assay, Sequencing

Journal: Cell Reports

Article Title: Caspase cleavage and nuclear retention of the energy sensor AMPK-α1 during apoptosis

doi: 10.1016/j.celrep.2022.110761

Figure Lengend Snippet: Biological significance of AMPK-α1 cleavage (A) Human α1β2γ1 complexes (WT or D529A or STREP-stop mutants) were expressed in E. coli ; following purification on Ni 2+ -agarose (WT or D529A mutant) or Ni 2+ -agarose and Strep-Tactin columns (STREP-stop mutant), duplicate samples were analyzed by western blots probed with the indicated antibodies. (B–E) Bacterially expressed, unphosphorylated human α1β2γ1 complexes (WT, D529A, or STREP-stop) were incubated in duplicate with the indicated amounts of either (B and C) human LKB1:STRAD:MO25 complex or (D and E) CaMKK2. Western blots and AMPK activity were measured. (F) Purified rat liver AMPK was first depleted of α2-containing complexes by immunoprecipitation, and the remaining α1 complexes were incubated for 2 h with the amounts of bacterially expressed caspase-3 shown in Figure S4 C. Samples were assayed for AMPK activity in the presence or absence of AMP (200 μM) or A-769662 (10 μM). Results are mean ± SEM (n = 3); significant differences from controls without AMP or A-769662 are indicated ( ∗∗∗ p < 0.001). (G) Jurkat cells were treated with indicated concentrations of anti-Fas. Nuclear and cytoplasmic extractions were prepared and western blot analysis carried out. (H) HeLa cells were transiently transfected with the indicated GFP-tagged full-length (GFP-WT-AMPK-α1) or truncated (GFP-cl-AMPK-α1 and GFP-ACTP) proteins. Following transfection, cells were visualized by confocal microscopy with nuclei stained using Hoechst 33342 (scale bars, 10 μm). (I) mCherry-tagged full-length (mCherry-WT-AMPK-α1, red) and truncated (GFP-cl-AMPK-α1, green) AMPK-α1 were co-transfected into HeLa cells and cells visualized by confocal microscopy with nuclei stained with Hoechst 33342 (scale bars, 10 μm). (J–L) Jurkat cells expressing indicated constructs were exposed to etoposide (10 μM for 6 h). Cytotoxicity, caspase activity, and western blots were carried out; data shown are mean ± SD (n = 3; ∗∗ p < 0.01 and ∗ p < 0.05).

Article Snippet: pLV[Exp]-Puro-EF1A>AMPK-α1(D529A)-EGFP with C-terminal GFP tag , VectorBuilder , VB180420-1016sdf.

Techniques: Purification, Mutagenesis, Western Blot, Incubation, Activity Assay, Immunoprecipitation, Transfection, Confocal Microscopy, Staining, Expressing, Construct