Journal: Cell chemical biology

Article Title: The efflux pump ABCC1/MRP1 constitutively restricts PROTAC sensitivity in cancer cells.

doi: 10.1016/j.chembiol.2024.11.009

Figure Lengend Snippet: Figure 2. ABCC1 KO reduces resistance to MZ1-mediated cytotoxicity and BET degradation (A) MZ1, macroPROTAC-1, and SIM1 drug response curves for HAP1 WT and two independently generated ABCC1 KO clones with normalized mean ± SEM (n = 3) CellTiter-Glo (CTG) luminescent values. Half-maximal efficiency concentration (EC50) values for WT and KO (mean of two clones) cells are indicated by dotted lines. (B) Mean ± SEM (n = 3) EC50 values for MZ1 in HAP1 WT and ABCC1, ABCB1, or ABCF2 KO clones (two independent clones per gene). Significantly changed EC50 values in KO vs. WT (unpaired t test, p value < 0.05 for each KO clone) are marked with an asterisk. Underlying drug response curves are shown in Figures 2A and S2D.

Article Snippet: To generate individual cell lines re-expressing or overexpressing ABC transporters, KO or WT HAP1 cells were transduced with modified pLIX_401 (Addgene #41393) vectors encoding codon-optimized cDNAs from the cDNA library described above.

Techniques: Generated, Clone Assay, Concentration Assay

Journal: Cell chemical biology

Article Title: The efflux pump ABCC1/MRP1 constitutively restricts PROTAC sensitivity in cancer cells.

doi: 10.1016/j.chembiol.2024.11.009

Figure Lengend Snippet: Figure 3. Genetic depletion or chemical inhibition of ABCC1 reduces PROTAC resistance in cancer cells (A and B) Data are represented as mean ± SEM (n = 3) MZ1 EC50 values for MZ1 in HAP1 WT and KO cell lines (two independent clones per KO) in the presence of DMSO and (A) reversan or (B) tariquidar. Significantly changed EC50 values in DMSO vs. reversan/tariquidar co-treated cells (unpaired t test, p value < 0.05) are marked by an asterisk. (C) ABCC1 vs. ABCB1 mRNA expression in transcripts per million (TPMs). Cancer cell lines used in this study are marked in red; data for 1,406 other cancer cell lines were retrieved from the DepMap database (marked in gray). (D) ABCB1 and ABCC1 mRNA and protein expression levels as retrieved from public datasets deposited by the RESOLUTE consortium. (E) Mean ± SEM (n = 3) MZ1 EC50 values in WT and KO cell lines (two independent clones per gene) generated from HCT116, LS180, and 1321N1 cell lines. Significantly changed EC50 values in KO vs. WT (unpaired t test, p value < 0.05 for both KO clones) are marked by an asterisk. (F) Mean ± SEM (n = 3) EC50 values for MZ1 in cancer cell lines in the presence of reversan or tariquidar. Significantly changed EC50 values in DMSO vs. inhibitor co-treated cells (unpaired t test, p value <0.05) are marked by an asterisk. (G) Mean ± SEM (n = 3) MZ1 EC50 values in LS180 single and double KO cells. Significantly changed EC50 values in double KO vs. single KO control (+OR1A1) (unpaired t test, p value < 0.05) are marked by an asterisk. (H) Mean ± SEM (n = 3) MZ1 EC50 values in LS180 WT and ABCC1 KO cells lines in the presence of tariquidar. Significantly changed EC50 values in DMSO vs. inhibitor co-treated cells (unpaired t test, p value < 0.05) are marked by an asterisk.

Article Snippet: To generate individual cell lines re-expressing or overexpressing ABC transporters, KO or WT HAP1 cells were transduced with modified pLIX_401 (Addgene #41393) vectors encoding codon-optimized cDNAs from the cDNA library described above.

Techniques: Inhibition, Clone Assay, Expressing, Generated, Control

Journal: Cell chemical biology

Article Title: The efflux pump ABCC1/MRP1 constitutively restricts PROTAC sensitivity in cancer cells.

doi: 10.1016/j.chembiol.2024.11.009

Figure Lengend Snippet: Figure 4. ABCC1 confers resistance to structurally diverse PROTACs (A) Molecular structure of tested PROTACs. POI and E3 ligase ligands are marked by colored circles as indicated. (B) Mean ± SEM (n = 3) EC50 values for various PROTACs and glues in HAP1 WT and KO cells (two independent clones per gene). Significantly changed EC50 values in KO vs. WT (unpaired t test, p value <0.05 for both KO clones) are marked by an asterisk.

Article Snippet: To generate individual cell lines re-expressing or overexpressing ABC transporters, KO or WT HAP1 cells were transduced with modified pLIX_401 (Addgene #41393) vectors encoding codon-optimized cDNAs from the cDNA library described above.

Techniques: Clone Assay

Journal: The EMBO Journal

Article Title: qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging

doi: 10.1038/s44318-024-00337-5

Figure Lengend Snippet: ( A – C ) Left—Representative images of WT, HDR, and MMEJ-targeted H2BC11 -moxGFP-Puro tagging in HAP1, ARPE-19, and U-2 OS cells co-stained with DAPI. Scale bars: 10 µm. Right—Flow cytometry quantifications of GFP-positive cells, based on three distinct biological replicates with each measurement encompassing 200,000 cells (Right). Data were mean ± SEM. ( D ) Genomic PCR outside the homology arms probing for locus-specific integration of the qTAG cassette in HAP1, ARPE-19, and U-2 OS cells. ( E ) Left—Representative images of WT, selected H2BC11 -moxGFP-Blast, selected H2BC11 -moxGFP-Puro, and selected H2BC11 -moxGFP-Zeo HEK293T cells co-stained with DAPI (Left). Scale bars: 10 µm. Right—Flow cytometry quantifications of GFP-positive cells, based on three distinct biological replicates with each measurement encompassing 200,000 cells (Right). Data were mean ± SEM. ( F ) Genomic PCR outside the homology arms probing for locus-specific integration of the qTAG cassettes with alternative mammalian selectable markers in HEK293T cells. ( G ) Overview of an alternate strategy using electroporation and RNPs to edit the tubulin TUBA1B gene with a qTAG-Blast-mScarlet cassette in H9 stem cells. ( H ) Representative images of WT and Blast-mScarlet-TUBA1B H9 cells co-stained with DAPI and probed for pluripotency marker OCT4. Scale bar: 10 µm. .

Article Snippet: The human cell lines used in this study included: HAP1 cells (Horizon Genomics, cat. No. C631, RRID: CVCL_Y019), HEK293T cells (ATCC, cat. No. CRL-3216, RRID: CVCL_0063 ), ARPE-19 cells (ATCC, cat. No. CRL-3216, RRID: CVCL_0145 ), RPE-1 cells (ATCC, cat. No. CRL-4000, RRID: CVCL_4388 ), U-2 OS cells (ATCC, cat. No. HTB-96, RRID: CVCL_0042 ), H9 cells (WiCell, cat. WA09, RRID: CVCL_9773 ).

Techniques: Staining, Flow Cytometry, Electroporation, Marker

Journal: The EMBO Journal

Article Title: qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging

doi: 10.1038/s44318-024-00337-5

Figure Lengend Snippet: ( A ) Schematic gene design of LMNB1 displaying the binding of the sgRNA (dark green) and its PAM binding site (yellow), homology arms, and targeted insertion site. ( B ) Overview of the strategy to carry out tagging of LMNB1 with a variety of functional proteomic qTAG cassettes. ( C ) Representative images of WT, V5-miniTurbo, and V5-ultraID tagged LMNB1 in HAP1 cells. Cells were stained with DAPI and probed with streptavidin in the absence or presence of biotin for 1 h. Scale bars: 10 µm. ( D ) A representative immunoblot of WT, V5-miniTurbo, and V5-ultraID tagged LMNB1 in HAP1 cells probed with antibodies against streptavidin, V5, and LMNB1 in the absence or presence of biotinylation for 10 min, 1 h, and 12 h. ( E ) Representative images of WT and V5-dTAG tagged LMNB1 in HAP1 cells stained for DAPI and probed for V5 in the absence or presence of dTAG-13 for 24 h. Scale bars: 10 µm. ( F ) A representative immunoblot of dTAG-V5 tagged LMNB1 in HAP1 cells probed for V5 and LMNB1 in the absence or presence of dTAG-13 for 2, 7, and 24 h. ( G ) Representative images of WT, 3xFLAG tagged, 3xHA tagged, and V5 tagged LMNB1 in HAP1 cells stained for DAPI and probed for their respective epitopes. Scale bars: 10 µm. ( H ) Representative immunoblots of WT, 3xFLAG tagged, 3xHA tagged, and V5 tagged LMNB1 in HAP1 cells with their respective epitope-specific antibodies. .

Article Snippet: The human cell lines used in this study included: HAP1 cells (Horizon Genomics, cat. No. C631, RRID: CVCL_Y019), HEK293T cells (ATCC, cat. No. CRL-3216, RRID: CVCL_0063 ), ARPE-19 cells (ATCC, cat. No. CRL-3216, RRID: CVCL_0145 ), RPE-1 cells (ATCC, cat. No. CRL-4000, RRID: CVCL_4388 ), U-2 OS cells (ATCC, cat. No. HTB-96, RRID: CVCL_0042 ), H9 cells (WiCell, cat. WA09, RRID: CVCL_9773 ).

Techniques: Binding Assay, Functional Assay, Staining, Western Blot

Journal: The EMBO Journal

Article Title: qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging

doi: 10.1038/s44318-024-00337-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The human cell lines used in this study included: HAP1 cells (Horizon Genomics, cat. No. C631, RRID: CVCL_Y019), HEK293T cells (ATCC, cat. No. CRL-3216, RRID: CVCL_0063 ), ARPE-19 cells (ATCC, cat. No. CRL-3216, RRID: CVCL_0145 ), RPE-1 cells (ATCC, cat. No. CRL-4000, RRID: CVCL_4388 ), U-2 OS cells (ATCC, cat. No. HTB-96, RRID: CVCL_0042 ), H9 cells (WiCell, cat. WA09, RRID: CVCL_9773 ).

Techniques: Recombinant, Produced, Sequencing, Cloning, Gel Extraction, Protease Inhibitor, Plasmid Preparation, Purification, Ligation, Saline, Transfection, Bradford Protein Assay, Software