Journal: Antioxidants (Basel, Switzerland)

Article Title: Delta-9-Tetrahydrocannabinol Blocks Bone Marrow-Derived Macrophage Differentiation through Elimination of Reactive Oxygen Species.

doi: 10.3390/antiox13080887

Figure Lengend Snippet: Figure 2. Identifying potential receptors through which THC blocks macrophage differentiation. (A–H) Representative flow and graphical representation of macrophage populations at 120 h between VEH- and THC-treated groups. Y-axis on flow plot is CD11b; X-axis is F4/80. (A–D) Preliminary flow gating shown in Figure S1A. (E–H) Preliminary flow gating shown in Figure S1B. (A) Wild-type mice; (B) CB1 knockout mice; (C) CB2 knockout mice; (D) CB1 and CB2 knockout mice; (E) GPR18 antagonist PSB CB5; (F) GPR55 antagonist ML 193; (G) A2aR antagonist SCH 442416; (H) G-PCR inhibitor PTX; (I) List of receptor inhibition methods and related citation. (A–H) n = 3, unpaired t-test was performed, levels of statistical significance were assigned according to the following cutoffs: *** p < 0.001, and **** p < 0.0001.

Article Snippet: THC was obtained from Cayman Chemical (Ann Arbor, MI, USA); GPCR inhibitors ML193, PSB CB5, SET 2, SCH442416, and Deferoxamine from Tocris (Minneaoikus, MN, USA); Pertussis Toxin (PTX) from List Biological Laboratories (Campbell, CA, USA); Recombinant Murine M-CSF and Annexin V Apoptosis Detection Kit from Biolegend (San Diego, CA, USA); PhenGreen SK (PGSK) and CellROX Deep Red and Sytox Blue from Thermo Fisher (Waltham, MA, USA); Penicillin/Streptomycin, FBS, phosphate-buffered saline (PBS) from VWR (West Chester, PA, USA).

Techniques: Knock-Out, Inhibition

Journal: bioRxiv

Article Title: A multi-subunit autophagic capture complex facilitates degradation of ER stalled MHC-I in pancreatic cancer

doi: 10.1101/2024.10.27.620516

Figure Lengend Snippet: A. Western blot showing levels of the indicated proteins in non-PDAC (HEK293T and Hela) and PDAC (KP4, PaTu8902, Panc10.05, PaTu8988T) cells. B. Immunoblot for total levels of FLAG and Tapasin (input) and following immunoprecipitation (IP) of stably expressed HLA-A-FLAG in the indicated cell lines. Cells were pre-treated with 400 nM Bafilomycin A1 (BafA1) for 6 h to block degradation. C. Time course measurement of amino acid starvation (EBSS) induced ER-phagy flux in the indicated cell lines stably expressing Keima-cb5 relative to DMEM growth media. N = 6 independent experiments. P values were determined using a two-way analysis of variance. D. Flow cytometry-based quantification of plasma membrane levels of MHC-I (HLA-A, - B, -C) in KP4 cells upon amino acid starvation in EBSS for 9 or 22 h compared to DMEM. N = 3 independent experiments. Data are the mean ± S.D. P values were determined using a one-way analysis of variance. E. Immunoblot for the indicated total protein (input) and following immunoprecipitation (IP) of stably expressed HLA-A-FLAG in KP4 cells under full nutrient conditions (DMEM), treatment with 400 nM BafA1 for 6 h, amino acid (AA) starvation for 6 h (EBSS), or combined AA starvation and 400nM BafA1 treatment. Blots are representative of N = 3 independent experiments. F. Flow cytometry-based quantification of plasma membrane levels of MHC-I (HLA-A, -B, -C) after siRNA-mediated knockdown of TEX264, FAM134B, and CCPG1 in KP4 cells. N = 3 independent experiment. Data are the mean ± S.D. P values were determined using a one-way analysis of variance. G. Immunoblot of the indicated proteins in KP4 cells following siRNA-mediated knockdown of TEX264, FAM134B, and CCPG1. Blots are representative of N = 3 independent experiments. Quantification is relative to siCTRL. H. Immunofluorescence staining of KP4 cells for TEX264 (green), MHC-I (red), and DAPI (blue). Inset shows the magnified view of the region indicated by the white box. Scale bar: 10 µm.

Article Snippet: pLJM1-HLA-A-FLAG was generated by subcloning the pcDNA of HLA-A (Addgene #85162) together with 2× FLAG into the EcoRI and NheI sites of the pLJM1 lentiviral vector. pMXs-GFP-NBR1, pMXs-GFP-NBR1 ΔUBA, and pMXs-GFP-NBR1 ΔLIR were provided by J. Debnath. pMRX-INU-TEX264-FLAG and pMRX-INU-TEX264 LIR4A- FLAG were gifts from Noboru Mizushima (Addgene #128258 and #128259). pLJM1- Keima-cb5 was generated by cloning the Keima-cb5 fragment from pRK5-GST-Keima- cb5 (a gift from Carol Mercer; Addgene plasmid #137755) into the AgeI and EcoRI sites of the pLJM1 lentiviral vector. pHAGE-CMV-ENV-SAV10-GFP and pHAGE-CMV-ENV- GFP were developed by Peter M. Bruno.

Techniques: Western Blot, Immunoprecipitation, Stable Transfection, Blocking Assay, Expressing, Flow Cytometry, Membrane, Knockdown, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: A multi-subunit autophagic capture complex facilitates degradation of ER stalled MHC-I in pancreatic cancer

doi: 10.1101/2024.10.27.620516

Figure Lengend Snippet: A. Flow cytometry gating strategy for Keima-cb5 experiments shown in . B,C . Flow cytometry-based quantification of plasma membrane levels of MHC-I (HLA-A, -B, -C) in PaTu8988T ( B ) or Panc1 ( C ) cells following siRNA-mediated knockdown of TEX264, FAM138B, or CCPG1. N = 3 independent experiments. Data are the mean ± S.D. P values were determined using a one-way analysis of variance.

Article Snippet: pLJM1-HLA-A-FLAG was generated by subcloning the pcDNA of HLA-A (Addgene #85162) together with 2× FLAG into the EcoRI and NheI sites of the pLJM1 lentiviral vector. pMXs-GFP-NBR1, pMXs-GFP-NBR1 ΔUBA, and pMXs-GFP-NBR1 ΔLIR were provided by J. Debnath. pMRX-INU-TEX264-FLAG and pMRX-INU-TEX264 LIR4A- FLAG were gifts from Noboru Mizushima (Addgene #128258 and #128259). pLJM1- Keima-cb5 was generated by cloning the Keima-cb5 fragment from pRK5-GST-Keima- cb5 (a gift from Carol Mercer; Addgene plasmid #137755) into the AgeI and EcoRI sites of the pLJM1 lentiviral vector. pHAGE-CMV-ENV-SAV10-GFP and pHAGE-CMV-ENV- GFP were developed by Peter M. Bruno.

Techniques: Flow Cytometry, Membrane, Knockdown

Journal: Autophagy

Article Title: Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy

doi: 10.1080/15548627.2020.1719746

Figure Lengend Snippet: CALCOCO1 has a role in reticulophagy. (A) GSTLSCSGFP-cb5 reticulophagy assay in HEK293 control and 2 sgCALCOCO1 KO cell lines, treated with Veh, 100 nM MLN, or glucose (Glc) starved. GST-tagged proteins/peptides in cell lysates were captured by glutathione agarose affinity purification. Ratio of the bottom fragment to the full-length fusion protein is shown, normalized to MLN in control cells. CALCOCO1 in cell lysates is shown. “B2” and “C3” represent different targets for guide RNA. *nonspecific fragment (B) GSTLSCSGFP-cb5 reticulophagy assay in HEK293 control and sgCALCOCO1 KO cells, treated with Veh, MLN, 10 ug/ml tunicamycin (Tun), or with galactose (Gal). Top panel. GST-tagged proteins/peptides in the LMF. Ratio of the bottom fragment to the full-length fusion protein is shown, normalized to MLN in control cells. Bottom panel. Immunoblot analyses of CALCOCO1 in LMF and high-speed supernatant (HSS) fractions. *nonspecific fragment (C) Immunoblot analysis of GST-Keima-cb5 assay in HEK293 cells, treated with Veh, MLN, and 5 ug/ml Tun as labeled. Ratio of 25 kD fragment to the full-length fusion protein is shown, normalized to MLN in control cells. (D) Relative protein abundances in sgCALCOCO1 KO HEK293 cells compared to sgGFP (GFP) control WT cells (mean, n = 5). Cells were treated for 24 h with 100 nM MLN0128. Proteins were quantified by label-free mass spectrometry. P-values for the protein abundance changes were determined by a two-sided Student’s t-test. (E) GST-BHMT autophagy assay in control and sgCALCOCO1 KO clones, with treatments as in Figure 6A. Ratio of BHMT fragment to GFP, normalized to MLN in control cells, is shown. Immunoblots were probed with antibodies as described. (F) Immunoblot analysis of WT MB231 and 3 sgCALCOCO1 KO cell lines, with antibodies, as shown. Cells were treated for 48 h with Veh, 100 nM MLN, and 20 nM bafilomycin A1, as shown

Article Snippet: GST - RFP-cb5 and GST-Keima-cb5 (Addgene, 137754 and 137755; deposited by Carol Mercer) were made by PCR of RFP or Keima from existing plasmids, using sequence-specific primers with recognition sites for NotI and XbaI restriction enzymes (RE).

Techniques: Affinity Purification, Western Blot, Labeling, Mass Spectrometry, Clone Assay

Journal: bioRxiv

Article Title: A functional map of phosphoprotein phosphatase regulation identifies an evolutionary conserved reductase for the catalytic metal ions

doi: 10.1101/2025.02.12.637884

Figure Lengend Snippet: A) Holoenzyme composition and regulators of PP2A-like phosphatases. B) Schematic of genome-wide CRISPR-Cas9 screen for genes whose knockout are synthetic lethal with okadaic acid (OA). NGS, next-generation sequencing. C) DrugZ analysis of CRISPR-Cas9 synthetic lethality screen performed in RPE1 p53-/-cells comparing treatment with a low dose of OA (2 nM) and untreated conditions. The top genes are indicated with established PP2A-like holoenzyme components and regulators in black and candidate regulators in grey. Significance threshold ( p -value of 0.05) is indicated with a dotted line at a DrugZ score of −3.3. PPP1CA is indicated as highest scoring non PP2A-like phosphatase. D) Human CYB5R4 and yeast Irc21 domain organization. Cytb5, cytochrome B5 domain. CS, CHORD/SGT domain. Cytb5-R, Cytochrome b5 reductase domain. E) Profiling of PPP composition by phosphatase inhibitor beads and mass spectrometry (PIB-MS). Volcano plot comparing the phosphatase components captured on phosphatase inhibitor beads from U2OS CYB5R4 knockout (KO) and parental (PAR) cells. PP2A holoenzyme components are indicated in green, PP4 in blue and PP6 in pink. ‘C’ indicates catalytic subunit. F) Peptide dephosphorylation assays measuring the activity of 3xFLAG-tagged PP2A C, PP4 C, and PP6 C, immunopurified from U2OS parental or CYB5R4 knockout stable cell lines. The data is representative of three independent experiments. G) Peptide dephosphorylation assays measuring the activity of HA-Pph21, myc-Pph3, and HA-Sit4 immunopurified from endogenously tagged wildtype ( wt ) and irc21 deletion strains. Data is shown for three independent experiments, and error bars represent standard deviations.

Article Snippet: Lysates were cleared by centrifugation for 30 minutes at 14,000 g whereafter supernatants were incubated with Fab-trap beads (Proteintech) for immunoprecipitation of 3xFLAG control and 3xFLAG-conjugated catalytic subunits or with GFP-trap beads (Proteintech) for immunoprecipitation of venus-tagged CYB5R4 wild-type and mutant.

Techniques: Genome Wide, CRISPR, Knock-Out, Next-Generation Sequencing, Mass Spectrometry, De-Phosphorylation Assay, Activity Assay, Stable Transfection

Journal: bioRxiv

Article Title: A functional map of phosphoprotein phosphatase regulation identifies an evolutionary conserved reductase for the catalytic metal ions

doi: 10.1101/2025.02.12.637884

Figure Lengend Snippet: A) Residue map of CYB5R4 showing synthetic lethality in OA. X-axis depicts the amino acid residue targeted for mutation and the y-axis the average log2 fold changes of gRNAs targeting the indicated residue in OA vs untreated conditions. The domains of CYB5R4 as well as H89R, H112R, and W114R are indicated. B) AlphaFold3 model of CYB5R4 with heme (salmon) and H89, H112, and W114 (yellow) indicated. C) Deep sequencing of the endogenous CYB5R4locus after transduction with a single gRNA targeting H112, followed by CRISPResso2 analysis shows the frequency of mutated alleles. Reference: the genomic sequence. The gRNA, PAM, and editing window are indicated as well as the amino acid translation. D) A colony formation assay was conducted in presence or absence of 2 nM OA with U2OS parental or CYB5R4 knockout cells. Cells were stably complemented with full length CYB5R4-venus either wildtype (WT) or with the specified mutations. The survival is calculated as the relative number of colonies in OA to untreated and represents three independent experiments. Error bares depict standard deviations and the shown p -values are based on one-way ANOVA analysis with Tukey’s multiple comparisons test.

Article Snippet: Lysates were cleared by centrifugation for 30 minutes at 14,000 g whereafter supernatants were incubated with Fab-trap beads (Proteintech) for immunoprecipitation of 3xFLAG control and 3xFLAG-conjugated catalytic subunits or with GFP-trap beads (Proteintech) for immunoprecipitation of venus-tagged CYB5R4 wild-type and mutant.

Techniques: Residue, Mutagenesis, Sequencing, Transduction, Colony Assay, Knock-Out, Stable Transfection

Journal: bioRxiv

Article Title: A functional map of phosphoprotein phosphatase regulation identifies an evolutionary conserved reductase for the catalytic metal ions

doi: 10.1101/2025.02.12.637884

Figure Lengend Snippet: A) Heatmap comparing the interactomes of venus-tagged CYB5R4 1-153 wildtype (WT), H89A/H112A, and W114A to venus control, which were immunoprecipitated from HeLa cells and analysed by mass spectrometry. PP4/6 holoenzyme components are shown. Colors represent log2 fold changes with red being increased and blue depleted compared to venus control. B) AlphaFold3 model of CYB5R4 1-153 and PP6 C with heme and residues H89, H112 and W114 indicated. The N-terminal tail of CYB5R4 is not shown in the close-up views for clarity. C) AlphaFold3 model of PP6 with annotation of CYB5R4 1-153 contacts (left) and a color gradient (right) that represents the average log2 fold changes of gRNAs targeting the indicated residue in OA vs untreated conditions. Blue values specify that mutation of the target residue causes depletion (synthetic lethality) in OA and red enrichment. Grey specifies residues not targeted. D) DiFMUP dephosphorylation assay measuring the activity of purified PP6 holoenzyme in presence or absence of pre-reduced purified CYB5R4 1-153 WT or H89A/H112A. Data is from three independent experiments, and error bars represent standard deviations. E) DiFMUP dephosphorylation assay measuring the activity of purified PP6 holoenzyme in presence or absence of the indicated reducing agents at 1 mM. Data is from three independent experiments, and error bars represent standard deviations. F) DiFMUP dephosphorylation assay measuring the activity of purified PP6 holoenzyme, which was first pre-incubated with or without EDTA to extract metal ions. Next, the enzyme was incubated in presence or absence of Mn 2+ and finally in the presence or absence of pre-reduced purified CYB5R4 1-153 WT. Activity after 30 minutes is shown (also see Supplementary Figure S7F). Data is from three independent experiments, and error bars represent standard deviations. G) Model of PP4/6 regulation by CYB5R4.

Article Snippet: Lysates were cleared by centrifugation for 30 minutes at 14,000 g whereafter supernatants were incubated with Fab-trap beads (Proteintech) for immunoprecipitation of 3xFLAG control and 3xFLAG-conjugated catalytic subunits or with GFP-trap beads (Proteintech) for immunoprecipitation of venus-tagged CYB5R4 wild-type and mutant.

Techniques: Control, Immunoprecipitation, Mass Spectrometry, Residue, Mutagenesis, De-Phosphorylation Assay, Activity Assay, Purification, Incubation

Journal: Autophagy

Article Title: Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy

doi: 10.1080/15548627.2020.1719746

Figure Lengend Snippet: CALCOCO1 has a role in reticulophagy. (A) GSTLSCSGFP-cb5 reticulophagy assay in HEK293 control and 2 sgCALCOCO1 KO cell lines, treated with Veh, 100 nM MLN, or glucose (Glc) starved. GST-tagged proteins/peptides in cell lysates were captured by glutathione agarose affinity purification. Ratio of the bottom fragment to the full-length fusion protein is shown, normalized to MLN in control cells. CALCOCO1 in cell lysates is shown. “B2” and “C3” represent different targets for guide RNA. *nonspecific fragment (B) GSTLSCSGFP-cb5 reticulophagy assay in HEK293 control and sgCALCOCO1 KO cells, treated with Veh, MLN, 10 ug/ml tunicamycin (Tun), or with galactose (Gal). Top panel. GST-tagged proteins/peptides in the LMF. Ratio of the bottom fragment to the full-length fusion protein is shown, normalized to MLN in control cells. Bottom panel. Immunoblot analyses of CALCOCO1 in LMF and high-speed supernatant (HSS) fractions. *nonspecific fragment (C) Immunoblot analysis of GST-Keima-cb5 assay in HEK293 cells, treated with Veh, MLN, and 5 ug/ml Tun as labeled. Ratio of 25 kD fragment to the full-length fusion protein is shown, normalized to MLN in control cells. (D) Relative protein abundances in sgCALCOCO1 KO HEK293 cells compared to sgGFP (GFP) control WT cells (mean, n = 5). Cells were treated for 24 h with 100 nM MLN0128. Proteins were quantified by label-free mass spectrometry. P-values for the protein abundance changes were determined by a two-sided Student’s t-test. (E) GST-BHMT autophagy assay in control and sgCALCOCO1 KO clones, with treatments as in Figure 6A. Ratio of BHMT fragment to GFP, normalized to MLN in control cells, is shown. Immunoblots were probed with antibodies as described. (F) Immunoblot analysis of WT MB231 and 3 sgCALCOCO1 KO cell lines, with antibodies, as shown. Cells were treated for 48 h with Veh, 100 nM MLN, and 20 nM bafilomycin A1, as shown

Article Snippet: GST-BHMT-IRES-GFP and GST LSCS GFP-cb5 have been previously described [ 39 , 59 ] (Addgene, 104442 and 104453; deposited by Carol Mercer).

Techniques: Affinity Purification, Western Blot, Labeling, Mass Spectrometry, Clone Assay