(A) TCGA data analysis showing SLC8B1 mRNA levels in tumor tissues and adjacent normal tissues of COADREAD (colon and rectal adenocarcinoma) patients. Each data point represents an individual …
Source data Figure 1.
(A) TCGA data analysis showing SLC8B1 mRNA levels in tumors of male and female COADREAD patients. (B, C) TCGA data analysis showing a comparison of SLC8B1 mRNA levels in COAD patients based on age (B…
(A) Schematic representation of CRISPR-generated HCT116 NCLX KO #33 cells. Luciferase-tagged control HCT116 and HCT116 NCLX KO #33 cells were injected at 5 × 105 cells/mice into spleens of male …
Source data Figure 2.
Bioluminescence images of male NOD/SCID mice injected with luciferase-tagged HCT116 cells or HCT116 NCLX KO #33 cells.
(A, B) CyQUANT proliferation assays of HCT116 cells (A), DLD1 cells (B), and their respective NCLX KO clones. (C, D) Representative bright-field images of in vitro wound healing migration assay (C), …
Source data Figure 3.
(A–D) CRISPR/Cas9 knockout strategy of the SLC8B1 gene in NCLX KO clones #33 (A), #37 (B), and #59 (C) with g1 and g2 representing the cut site and red box non-translated region. Location of primers …
Source data Figure 3—figure supplement 1.
(A, B) Flow cytometric analysis of mitochondrial membrane potential using the dye TMRE in HCT116 cells (A) and DLD1 cells (B) and their respective NCLX KO clones. Each data point represents one …
Source data Figure 4.
(A) Representative images of HCT116 cells and HCT116 NCLX KO cells loaded with the mitochondrial Ca2+ dye Rhod-2 AM. Cells were stimulated with 300 µM ATP in 5 mM extracellular Ca2+ at time 0, and …
Source Data Figure 4—figure supplement 1.
Source Data Figure 4—figure supplement 2.
(A–C) Representative transmission electron micrographs (TEM) of mitochondria in HCT116, HCT116 NCLX KO #37 cells, HCT116 NCLX KO #59 cells (A), DLD1 cells, DLD1 NCLX KO#06 cells (B) with red arrows …
(A) Volcano plot of differentially expressed genes between HCT116 cells and HCT116 NCLX KO #33 cells showing Log2 fold change vs. false discovery rate. The thresholds in the figure correspond to a …
Source data Figure 5.
(A) Heat map of expression of glycolytic genes that are significantly different between HCT116 cells and HCT116 NCLX KO #33 cells. (B, C) Gene set enrichment analysis (GSEA), and of HCT116 cells, …
Source Data Figure 5—figure supplement 1.
(A–F) Western blot of HCT116 cells and clones of HCT116 NCLX KO cells (A), DLD1 cells and clones of DLD1 NCLX KO cells (C) and HCT116 cells infected with either scramble shRNA or three combinations …
Source data Figure 6.
(A) The proliferation of HCT116 cells and HCT116 NCLX KO #33 cells in the presence of 0 µM, 1 µM, 10 µM, and 25 µM 5-FU (n = 3). (B) Measurement of IC50 of 5-FU for HCT116 and HCT116 NCLX KO #33 …
Source Data Figure 6—figure supplement 1.
(A) RT-qPCR analysis of glycolytic gene expression in HCT116 cells and three clones of HCT116 NCLX KO cells. RT-qPCR results are plotted as 2-ΔCt relative to tubulin and normalized to control. (B, C)…
Source data Figure 7.
(A–D) Western blots for HK2, ALDOA, LDHA, with GAPDH as a loading control from HCT116, HCT116 NCLX KO #37, #59 (A), DLD1, clones of DLD1 NCLX KO cells (C), and quantification of band intensity …
Source Data Figure 7—figure supplement 1.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Homo sapiens) | SLC8B1/NCLX | GenBank | Gene ID: 80024 | |
Gene Mus musculus | Slc8b1/NCLX | GenBank | Gene ID: 170756 | |
Genetic reagent Mus musculus | NOD.CB17-Prkdcscid/J | Jackson Laboratory | Stock No: 010636, RRID:IMSR_JAX:010636 | Male |
Cell line (Homo-sapiens) | HCT116 (colon, epithelial) | ATCC | ATCC# CCL-247 | Male |
Cell line (Homo-sapiens) | HT29 (colon, epithelial) | ATCC | ATCC# HTB-38 | Female |
Cell line (Homo-sapiens) | DLD1 (colon, epithelial) | ATCC | ATCC# CCL-221 | Male |
Cell line (Homo-sapiens) | HCT116 NCLX KO (colon, epithelial) | This paper | NCLX Knockout clones of HCT116 cells were generated by the Trebak lab using CRISPR/Cas9 and are available upon request | |
Cell line (Homo-sapiens) | DLD1 NCLX KO (colon, epithelial) | This paper | NCLX Knockout clones of DLD1 cells were generated in the Trebak lab using CRISPR/Cas9 and are available upon request | |
Cell line (Homo-sapiens) | HCT116 shNCLX (colon, epithelial) | This paper | HCT116 cells with stable shRNA-mediated knockdown of NCLX (shNCLX) were generated by the Trebak Lab using shRNA sequences (listed below in this table) cloned in the lentiviral vector pLKO. These plasmids are available upon request | |
Cell line (Homo-sapiens) | DLD1 shNCLX (colon, epithelial) | This paper | DLD1 cells with stable shRNA-mediated knockdown of NCLX (shNCLX) were generated by the Trebak Lab using shRNA sequences (listed below in this table) cloned in the lentiviral vector pLKO. These plasmids are available upon request | |
Antibody | anti-Hif1α (Rabbit polyclonal) | Cell Signaling Technology | Cat# 14179s, RRID:AB_2622225 | WB (1:500) |
Antibody | anti- ALDOA (Rabbit polyclonal) | Cell Signaling Technology | Cat# 8060S, RRID:AB_2797635 | WB (1:1000) |
Antibody | anti- HK2 (Rabbit polyclonal) | Cell Signaling Technology | Cat# 2867S, RRID:AB_2232946 | WB (1:1000) |
Antibody | anti- LDHA (Rabbit polyclonal) | Cell Signaling Technology | Cat# 2012S, RRID:AB_2137173 | WB (1:1000) |
Antibody | anti- MMP1 (Rabbit polyclonal) | Abcam | Cat# ab38929, RRID:AB_776395 | WB (1:1000) |
Antibody | anti- MMP2 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-13594, RRID:AB_627956 | WB (1:500) |
Antibody | anti- MMP9 (Rabbit polyclonal) | Abcam | Cat# ab73734, RRID:AB_1860201 | WB (1:1000) |
Antibody | anti- LC3B (Rabbit polyclonal) | Abcam | Cat# ab51520, RRID:AB_881429 | WB (1:1000) |
Antibody | anti- OXPHOS (Mouse monoclonal) | Abcam | Cat# ab110413, RRID:AB_2629281 | WB (1:5000) |
Antibody | anti- GAPDH (Mouse monoclonal) | Millipore Sigma | Cat# MAB374, RRID:AB_2107445 | WB (1:10000) |
Antibody | anti- HSC70 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-24, RRID:AB_627760 | WB (1:5000) |
Antibody | anti- p62 (Rabbit polyclonal) | Abcam | Cat# ab109012, RRID:AB_2810880 | WB (1:1000) |
Antibody | anti- cleaved caspase-3 (Rabbit polyclonal) | Cell Signaling Technology | Cat# 9661S, RRID:AB_2341188 | WB (1:1000) IF (1:500) |
Antibody | anti- pAMPK (Rabbit polyclonal) | Cell Signaling Technology | Cat# 2535S, RRID:AB_331250 | WB (1:1000) |
Antibody | anti- AMPK (Rabbit polyclonal) | Cell Signaling Technology | Cat# 5831S, RRID:AB_10622186 | WB (1:1000) |
Antibody | anti- pS6K (Rabbit polyclonal) | Cell Signaling Technology | Cat# 9234S, RRID:AB_2269803 | WB (1:1000) |
Antibody | anti- S6K (Rabbit polyclonal) | Cell Signaling Technology | Cat# 2708S, RRID:AB_390722 | WB (1:1000) |
Sequence-based reagent | SLC7A11_F | This paper | RT-PCR primers | AGGGTCACCTTCCAGAAATC |
Sequence-based reagent | SLC7A11_R | This paper | RT-PCR primers | GAAGATAAATCAGCCCAGCA |
Sequence-based reagent | GCLM _F | This paper | RT-PCR primers | CATTTACAGCCTTACTGGGAGG |
Sequence-based reagent | GCLM _R | This paper | RT-PCR primers | ATGCAGTCAAATCTGGTGGCA |
Sequence-based reagent | FOXO3_F | This paper | RT-PCR primers | CGGACAAACGGCTCACTCT |
Sequence-based reagent | FOXO3_R | This paper | RT-PCR primers | GGACCCGCATGAATCGACTAT |
Sequence-based reagent | NANOG _F | This paper | RT-PCR primers | TTTGTGGGCCTGAAGAAAACT |
Sequence-based reagent | NANOG _R | This paper | RT-PCR primers | AGGGCTGTCCTGAATAAGCAG |
Sequence-based reagent | OCT4_F | This paper | RT-PCR primers | TTCAGCCAAACGACCATCTG |
Sequence-based reagent | OCT4_R | This paper | RT-PCR primers | CACGAGGGTTTCTGCTTTGC |
Sequence-based reagent | SOX2_F | This paper | RT-PCR primers | GCCGAGTGGAAACTTTTGTCG |
Sequence-based reagent | SOX2_R | This paper | RT-PCR primers | GGCAGCGTGTACTTATCCTTCT |
Sequence-based reagent | GLUT1_F | This paper | RT-PCR primers | TATCGTCAACACGGCCTTCACT |
Sequence-based reagent | GLUT1_R | This paper | RT-PCR primers | AACAGCTCCTCGGGTGTCTTAT |
Sequence-based reagent | HK2_F | This paper | RT-PCR primers | GCCATCCTGCAACACTTAGGG |
Sequence-based reagent | HK2_R | This paper | RT-PCR primers | GTGAGGATGTAGCTTGTAGAGGGT |
Sequence-based reagent | GPI_F | This paper | RT-PCR primers | TGTGTTCACCAAGCTCACAC |
Sequence-based reagent | GPI_R | This paper | RT-PCR primers | GTAGAAGCGTCGTGAGAGGT |
Sequence-based reagent | ALDOA_F | This paper | RT-PCR primers | AGGCCATGCTTGCACTCAG |
Sequence-based reagent | ALDOA_R | This paper | RT-PCR primers | AGGGCCCAGGGCTTCAG |
Sequence-based reagent | ENO1_F | This paper | RT-PCR primers | GACTTGGCTGGCAACTCTG |
Sequence-based reagent | ENO1_R | This paper | RT-PCR primers | GGTCATCGGGAGACTTGAAG |
Sequence-based reagent | LDHA_F | This paper | RT-PCR primers | GGTTGGTGCTGTTGGCATGG |
Sequence-based reagent | LDHA_R | This paper | RT-PCR primers | TGCCCCAGCCGTGATAATGA |
Sequence-based reagent | MMP1_F | This paper | RT-PCR primers | ATGCTGAAACCCTGAAGGTG |
Sequence-based reagent | MMP1_R | This paper | RT-PCR primers | GAGCATCCCCTCCAATACCT |
Sequence-based reagent | MMP2_F | This paper | RT-PCR primers | ACCAGCTGGCCTAGTGATGATG |
Sequence-based reagent | MMP2_R | This paper | RT-PCR primers | GGCTTCCGCATGGTCTCGATG |
Sequence-based reagent | MMP9_F | This paper | RT-PCR primers | ACGCACGACGTCTTCCAGTA |
Sequence-based reagent | MMP9_R | This paper | RT-PCR primers | CCACCTGGTTCAACTCACTCC |
Sequence-based reagent | qNCLX_1_F | This paper | RT-PCR primers | GCGTGCTGGTTACCACAGT |
Sequence-based reagent | qNCLX_1_R | This paper | RT-PCR primers | CCACGGAAGAGCATGAGGAA |
Sequence-based reagent | qNCLX_2_F | This paper | RT-PCR primers | CCGGCAGAAGGCTGAATCTG |
Sequence-based reagent | qNCLX_2_R | This paper | RT-PCR primers | ACCTTGCGGCAGTCTACCAC |
Sequence-based reagent | GAPDH_F | This paper | RT-PCR primers | CCCTTCATTGACCTCAACTACA |
Sequence-based reagent | GAPDH_R | This paper | RT-PCR primers | ATGACAAGCTTCCCGTTCTC |
Sequence-based reagent | NONO_F | This paper | RT-PCR primers | TCCGAGGAGATACCAGTCGG |
Sequence-based reagent | NONO_R | This paper | RT-PCR primers | CCTGGGCCTCTCAACTTCGAT |
Sequence-based reagent | Tubulin_F | This paper | RT-PCR primers | AGTCCAAGCTGGAGTTCTCTAT |
Sequence-based reagent | Tubulin_R | This paper | RT-PCR primers | CAATCAGAGTGCTCCAGGGT |
Sequence-based reagent | G6PD_F | This paper | RT-PCR primers | CGAGGCCGTCACCAAGAAC |
Sequence-based reagent | G6PD_R | This paper | RT-PCR primers | GTAGTGGTCGATGCGGTAGA |
Sequence-based reagent | PGD_F | This paper | RT-PCR primers | ATGGCCCAAGCTGACATCG |
Sequence-based reagent | PGD_R | This paper | RT-PCR primers | AAAGCCGTGGTCATTCATGTT |
Sequence-based reagent | TKT_F | This paper | RT-PCR primers | TCCACACCATGCGCTACAAG |
Sequence-based reagent | TKT_R | This paper | RT-PCR primers | CAAGTCGGAGCTGATCTTCCT |
Sequence-based reagent | g1 | This paper | Guide RNA sequences (Figure 2A and Figure 3—figure supplement 1A) | GCGCAGATTCAGCCTTCTGC |
Sequence-based reagent | g2 | This paper | Guide RNA sequences (Figure 3—figure supplement 1B and C) | GGGATACTCACGTCTACCAC |
Sequence-based reagent | g3 | This paper | Guide RNA sequences (Figure 3—figure supplement 1E) | GTAGACGTGAGTATCCCGGT |
Sequence-based reagent | g4 | This paper | Guide RNA sequences (Figure 3—figure supplement 1E) | ACCCACACCAGCAGTCCGTC |
Sequence-based reagent | shRNA (shNCLX#2) | This paper | Figure 3—figure supplement 1I | GCCTTCTTGCTGTCATGCAAT |
Sequence-based reagent | shRNA (shNCLX#3) | This paper | Figure 3—figure supplement 1I | GCTCCTCTTCTACCTGAACTT |
Sequence-based reagent | siRNA (siNCLX) | This paper | Figure 4—figure supplement 1M | AACGGCCCCUCAACUGUCUT |
Sequence-based reagent | NCLX_1 | This paper | PCR primers for screening genomic DNA of NCLX KO clones (Figure 3—figure supplement 1F) | GCCAGCATTTGTGTCCATTT |
Sequence-based reagent | NCLX_2 | This paper | PCR primers for screening genomic DNA of NCLX KO clones (Figure 3—figure supplement 1F) | AATTCGTCTCGGCCACTTAC |
Sequence-based reagent | NCLX_3 | This paper | PCR primers for screening genomic DNA of NCLX KO clones (Figure 3—figure supplement 1F) | ACTTAGCACATCGCCACCTG |
Sequence-based reagent | NCLX_4 | This paper | PCR primers for screening genomic DNA of NCLX KO clones (Figure 3—figure supplement 1F) | CTGATCTGCACGCTGAATGG |
Sequence-based reagent | NCLX_5 | This paper | PCR primers for screening genomic DNA of NCLX KO clones (Figure 3—figure supplement 1F) | GAGGTACACAGCAGTTCT CCC |
Sequence-based reagent | NCLX_6 | This paper | PCR primers for screening genomic DNA of NCLX KO clones (Figure 3—figure supplement 1F) | CAGCTGGTGCCCTCAAACAC |
Sequence-based reagent | PX75 NCLX test F | This paper | PCR primers for screening genomic DNA of NCLX KO HCT116 cells | GTTGTTGAGACAGAGTCTTGC TTC |
Sequence-based reagent | PX76 NCLX test R | This paper | PCR primers for screening genomic DNA of NCLX KO HCT116 cells | TCCAGCGAGACTGTGCAGAA |
Sequence-based reagent | px77 | This paper | PCR primers for genotyping NCLX -/- mice | TACAGTCTGGCTCGTTCC CT |
Sequence-based reagent | px78 | This paper | PCR primers for genotyping NCLX -/- mice | CGGTCCCAGACGCCG T |
Sequence-based reagent | px79 | This paper | PCR primers for genotyping NCLX -/- mice | CGCTGGGGTCCATCT TTG AT |
Sequence-based reagent | px80 | This paper | PCR primers for genotyping NCLX -/- mice | TGGGTCTCCGGTCCCAGT A |
Commercial assay or kit | cDNA Reverse Transcription Kit | Applied biosystems | Cat# 4368814 | |
Commercial assay or kit | Seahorse XFp Mito Fuel Flex Test Kit | Agilent Technologies | Cat# 103270-100 | |
Commercial assay or kit | Seahorse XFp Glycolysis Stress Test Kit | Agilent Technologies | Cat# 103017-100 | |
Commercial assay or kit | Seahorse XFp Cell Mito Stress Test Kit | Agilent Technologies | Cat# 103010-100 | |
Commercial assay or kit | BCA assay kit | Thermo Fisher Scientific | Cat# A53225 | |
Commercial assay or kit | TMRE | Thermo Fisher Scientific | Cat# T669 | |
Commercial assay or kit | CyQUANT | Thermo Fisher Scientific | Cat# C35006 | |
Chemical compound, drug | Antibiotic and Antimycotic | Thermo Fisher Scientific | Cat# 15240062 | |
Chemical compound, drug | McCoy’s 5A | Corning | Cat# 10-050CV | |
Chemical compound, drug | RPMI-1640 | Corning | Cat# 10-040CV | |
Chemical compound, drug | Lipofectamine 2000 | Thermo Fisher Scientific | Cat# 11668019 | |
Chemical compound, drug | TrypLE | Thermo Fisher Scientific | Cat# 12605028 | |
Chemical compound, drug | CoCl2 | Sigma-Aldrich | Cat# 15862 | |
Chemical compound, drug | 2-deoxy-D-glucose (2-DG) | Sigma-Aldrich | Cat# D8375 | |
Chemical compound, drug | 5- Fluorouracil | Sigma-Aldrich | Cat# F6627 | |
Chemical compound, drug | Glucose | Sigma-Aldrich | Cat# D9434 | |
Chemical compound, drug | Puromycin | MP Biomedical | Cat# 02100552 | |
Chemical compound, drug | RIPA buffer | Sigma | Cat# R0278 | |
Chemical compound, drug | ATP | Sigma | Cat# A9187 | |
Chemical compound, drug | Dextrose | Fisher Scientific | Cat# D14 | |
Chemical compound, drug | Tris Base | Fisher Scientific | Cat# BP152-5 | |
Chemical compound, drug | NaCl | Fisher Scientific | Cat# S671 | |
Chemical compound, drug | MOPS SDS running buffer | Thermo Fisher Scientific | Cat# NP0001 | |
Chemical compound, drug | Tris-Glycine transfer buffer | Bio-rad | Cat#161-0734 | |
Chemical compound, drug | KCl | Fisher Scientific | Cat# P217 | |
Chemical compound, drug | MgCl2 | Fisher Scientific | Cat# M33 | |
Chemical compound, drug | CaCl2 | Fisher Scientific | Cat# C614 | |
Chemical compound, drug | HEPES | Fisher Scientific | Cat# BP310 | |
Chemical compound, drug | LDS sample buffer | Thermo Fisher Scientific | Cat# NP0007 | |
Chemical compound, drug | NuPAGE Bis-Tris precast gels | Thermo Fisher Scientific | Cat# NP0321 | |
Chemical compound, drug | Polyvinylidene difluoride membrane | Li-Core Biosciences | Cat# 88518 | |
Chemical compound, drug | Odyssey Blocking Buffer (TBS) | Li-Core Biosciences | Cat# 937-50003 | |
Chemical compound, drug | Dextran sulfate sodium | MP Biomedical | Cat# 0216011080 | |
Chemical compound, drug | Azoxymethane | Sigma | Cat# A5486 | |
Chemical compound, drug | DNase I | Thermo Fisher Scientific | Cat# 18068-015 | |
Chemical compound, drug | TRIzol | Thermo Fisher Scientific | Cat# 15596018 | |
Chemical compound, drug | Seahorse XF DMEM Medium pH 7.4 | Agilent Technologies | Cat# 103575-100 | |
Chemical compound, drug | Seahorse XF 100 mM pyruvate solution | Agilent Technologies | Cat# 103578-100 | |
Chemical compound, drug | Seahorse XF 200 mM glutamine solution | Agilent Technologies | Cat# 103579-100 | |
Chemical compound, drug | Seahorse XF 1.0 M glucose solution | Agilent Technologies | Cat# 103577-100 | |
Chemical compound, drug | Zymogram Developing Buffer (10X) | Thermo Fisher Scientific | Cat# LC2671 | |
Chemical compound, drug | Zymogram Renaturing Buffer (10X) | Thermo Fisher Scientific | Cat# LC2670 | |
Chemical compound, drug | Tris-Glycine SDS Running Buffer (10X) | Thermo Fisher Scientific | Cat# LC2675 | |
Chemical compound, drug | Tween 20 | Fisher Scientific | Cat# BP337 | |
Software, algorithm | Image J | https://imagej.net/ | RRID:SCR_003070 | |
Other | DAPI | Sigma-Aldrich | Cat# D9542 | 1µg/ml |
Other | Hoechst | Thermo Fisher Scientific | Cat# H3570 | 1µg/ml |
Other | IRDye 800CW Goat anti-Mouse | Li-Core Biosciences | Cat# 925-32210 | 1:10000 |
Other | IRDye 800CW Donkey anti-Rabbit | Li-Core Biosciences | Cat# 925-32213 | 1:5000 |
Other | MitoSox Red | Thermo Fisher Scientific | Cat# M36008 | |
Other | Mito TEMPO | Thermo Fisher Scientific | Cat# SML0737 | |
Other | Mito Tracker Green FM | Thermo Fisher Scientific | Cat# M7514 | |
Other | Mito Tracker Deep red FM | Cell Signaling Technology | Cat# 8778S | |
Other | Fura-2 AM | Thermo Fisher Scientific | Cat# F1221 | |
Other | FluoroBlok | Corning | Cat# 351152 | |
Other | BioCoat Tumor Invasion Plate | Corning | Cat# 80774380 | |
Other | SYBER select master mix | Thermo Fisher Scientific | Cat# 4472920 | |
Other | Novex 10% Zymogram Plus (Gelatin) Protein Gels, 1.0 mm, 10-well | Thermo Fisher Scientific | Cat# ZY00100 | |
Other | SimplyBlue Safe Stain | Thermo Fisher Scientific | Cat# LC6060 |
Genomic sequencing of NCLX KO clones of HCT116 and DLD1 cells.
This file shows the genome sequencing of NCLX KO clones #33, #37, and #59 of HCT116 and #06, #24, and #32 of DLD1 cells. For genomic sequencing, PCR was performed to amplify specific parts of the NCLX gene using primers listed in the key resources table. The primers used for cloning genomic DNA from HCT116 cells were PX75 NCLX test F and PX76 NCLX test R. The primers used for cloning genomic DNA from DLD1 cells were NCLX_4 and NCLX_5. PCR products were cloned using StrataClone Blunt PCR Cloning Kit and sent to Genewiz for sanger sequencing. The genome sequencing data for the HCT116 NCLX KO clones confirm the introduction of STOP codons in coding sequences of HCT116 NCLX KO #33 at predicted positions 181-183 bp and 184-186 bp, in HCT116 NCLX KO #37 at 16-18 bp and 46-48 bp, and in HCT116 NCLX KO #59 at 31-33 bp, and 52-54 bp. Furthermore, the genomic sequencing of NCLX KO clones of DLD1 cells showed that the middle ~32 kb portion of the NCLX gene was deleted in all clones. Therefore, providing decisive evidence that these cells are indeed NCLX KO.
Source data RNAseq_HCT116_HCT116 NCLX KO.