Cell culture

HCC1806, MDA-MB-231, T47D and HEK293T cells were obtained from ATCC. MCF10-DCIS cells were provided by Kornelia Polyak (Harvard Medical School, USA). HCC1806 cells were maintained in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (FBS; Clontech). MDA-MB-231, T47D and HEK293T cells were cultured in Dulbecco’s Modified Eagle medium (DMEM; Gibco) containing 10% FBS. MCF10-DCIS was maintained in DMEM/F-12 (Gibco) supplemented with 5% horse serum, 20 ng/ml epidermal growth factor (EGF), 10 µg/ml insulin, 100 ng/ml final cholera toxin and 500 ng/ml hydrocortisone. All cell lines are regularly tested for mycoplasma contamination. They have been tested for authentication using short tandem repeat profiling and passaged for < 6 months.

Lentiviral vector infection

The shRNAs (sequences are listed in Supplementary Table 1) specific targeting FOSL1 mRNA were ligated to the lentiviral vector pLKO.1 digested by AgeI (NEB, R3552S) and EcoRI (NEB, R3101s) restriction enzyme to generate plasmids (pLKO.1-sh1/2-FOSL1). For overexpression of exogenous FOSL1 and TCOF1, CDS of FOSL1 and TCOF1 was synthesized and cloned into vector CD532A-1 by GENEWIZ. To prepare lentiviral supernatants, 10 µg lentiviral vectors (pLKO.1-shRNA CTL, pLKO.1-sh1-FOSL1, pLKO.1-sh2-FOSL1, CD532A-1, CD532A-1-FOSL1, CD532A-1-HA-TCOF1) were co-transfected with 7 µg psPAX2 and 2.4 µg VSV-G vectors to HEK293T cells in a 10-cm plate, using polyethylenimine as transfection reagent. Sixty hours post transfection, the supernatants containing lentiviruses were collected and filtered by 0.45 μm syringe filters (Thermo fisher, 7232545). For infection of lentivirus to TNBC cells, 0.15–0.2 ml lentivirus with 5 µg/ml polybrene was added to cells for 12–24 h in a well of 6-well plate. After around 48–60 h of infection with lentivirus, cells were used for protein and RNA extraction or next CellTiter-Glo® 3D and 2D Cell Viability Assay.

Overall survival analysis

The Kaplan-Meier plotter database (https://kmplot.com/analysis/) was used to analyze the prognosis of FOSL1 and FOSL2. The patients were split by auto-selected best cutoff with a follow-up time included. The TNBC (basal) subtype was chosen in the StGallen. All datasets were included for the analysis. The Kaplan-Meier survival plots, the hazard ratio, 95% confidence interval, and log rank P-value were displayed.

Quantitative reverse transcription-PCR (RT-qPCR)

Total RNA from HCC1806 and MCF10-DCIS cells in 6-well plates was extracted using the RNeasy Plus Mini Kit (Qiagen, #74134) according to the manual. Reverse transcription (RT) was performed using TaqMan Reverse Transcription Reagents (Applied Biosystems, N8080234). Quantitative RT-PCR was performed using a QuantStudio 12 K Flex Real-Time PCR System (Applied Biosystems). Relative expression levels of target genes were normalized to GAPDH. All primers used in the RT-qPCR are listed in Supplementary Table 1.

Chromatin immunoprecipitation (ChIP)-qPCR

ChIP-qPCR was performed following the previously described methods [8]. Briefly, HCC1806, MCF10-DCIS or MDA-MB-231 cells were cross-linked with 1% PFA at room temperature for 5 min, followed by washing twice with PBS. Cells were scraped into 1 ml ChIP lysis buffer (1% Triton, 0.1% Na-deoxycholate, 50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1× proteinase inhibitor cocktail) and incubated on ice for 15 min. The lysate of cells was sheared using Bioruptor Plus (Diagenode, UCD-300 TM) for 30 cycles (30 s ON and 30 s OFF at high power) to shear the chromatin, followed by two sequential centrifugations (10,000 × g for 5 and 15 min at 4 °C) to collect the soluble chromatin. The lysate was then incubated with the FOSL1 antibody, BRD4 antibody, H3K27ac antibody or isotype IgG at 4 °C overnight, followed by incubating with Protein G Sepharose (GE Healthcare, 17061802) pre-washed with ChIP lysis buffer for 1 h at 4 °C. After washing, samples were treated with 10% chelex (Bio-Rad, 142–1253) and then with 20 mg/ml Proteinase K (NEB, P8107S), followed by centrifugation. The precipitated DNA samples were measured by qPCR using Applied Biosystems QuantStudio 3 Real-Time PCR System. Primers for ChIP-qPCR are listed in Supplementary Table 1.

DNA pull-down assay

The TCOF1-E1 SE was amplified by PCR using 5′-biotinylated forward primer. PCR Primers of biotinylated-DNA of TCOF1-E1 SE are listed in Supplementary Table 1. PCR product was purified by QIAquick Gel extraction kit (Qiagen #28706) and immobilized on Streptavidin-Agarose beads (Thermo Scientific) in binding buffer (20 mM HEPES (pH 7.5), 2.5 mM KCl, 20% glycerol, 1 mM DTT, 0.02% NP40) for 30 min at room temperature, followed by wash once. Cells lysate in TNTE buffer (50 mM Tris/HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, protease and phosphatase inhibitors) was centrifuged at 13,000 g for 10 min at 4 °C. The lysate was incubated with the beads-biotinylated DNA complex, with or without 10 µg poly-dIdC (Sigma) used as a competitor for nonspecific DNA binding proteins, in a total volume of 1.4 ml, and incubated for 6 h at 4 °C. For samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), complexes were washed 3 times with binding buffer. For samples used for mass spectrometry for protein identification, complexes were washed twice with binding buffer and three times with 50 mM ammonium bicarbonate.

CellTiter-Glo® three-dimensional (3D) and two-dimensional (2D) cell viability assays

3D and 2D cell viability were determined as previously described [8]. Briefly, for 3D cultures, growth factor-reduced Matrigel (Corning) was used to coat 96-well plates (Corning # 3610). Cells were seeded to the Matrigel-precoated 96-well plates in assay medium (relevant complete medium with 2% Matrigel supplement), with a density of 2000 cells per well, and allowed to grow in 5% CO2 humidified incubator at 37℃ for 7 days to form spheroids. To quantify spheroid viability, CellTiter-Glo® 3D Cell Viability Assay (Promega #G9682) was performed under the instruction of the Kit manual. For assessment of 2D cell viability, cells were directly seeded to 96-well plate, with a density of 4000 cells per well, and cultured for 3 days. CellTiter-Glo® Luminescent Cell Viability Assay (Promega #G7571) was used to quantify 2D cell viability according to the manufacturer’s instructions. The bioluminescence signal was detected by a Synergy™ H1 Microplate Reader (BioTek).

Western blot

Total protein extracts were collected by lysing cells in EBN buffer (0.5% NP-40, 120 mM NaCl, 50 mM Tris-HCl (pH 7.4), proteinase inhibitor cocktail, 50 nM calyculin, 2 mM EDTA, 2 mM EGTA, 1 mM sodium pyrophosphate, 20 mM sodium fluoride) on ice for 30 min, followed by centrifugation at 13,000 × g at 4 °C for 10 min. Bio-Rad protein assay reagents (Bio-Rad, 5000114 and 5000113) were applied for the detection of protein concentration and the absorbance was read by a Synergy™ H1 Microplate Reader (BioTek). Then, 10 µg of protein was resolved by 10% SDS-PAGE followed by transfer to a nitrocellulose membrane. After blocking with 5% fat-free milk in TBST for 1 h, membranes were probed with indicated primary and secondary antibodies followed by enhanced chemiluminescence substrate (Pierce). The primary antibodies used were anti-FOSL1 (CST, # 5281T), anti-TCOF1 (sigma, # HPA038237), and anti-β-actin (CST, # 3700 S).

Dual-luciferase reporter assay

TCOF1-E1 was amplified from genomic DNA by PCR and cloned to firefly luciferase reporter pGL3-Promoter vector (Promega, #E1761). Plasmid pGL3-Promoter-TCOF1 E1 was co-transfected with pRL-TK vector (Promega, #E2241) into HCC1806 or MCF10-DCIS cells using FuGENE 6 transfection reagent (Promega, #2693). A pRL-TK vector plasmid expressing renilla luciferase was used as an internal transfection control. Two days after transfection, the Dual-Glo® Luciferase Assay System Kit (Promega, #E2920) was used to quantify luminescence signal. The firefly luciferase signal was first normalised to the renilla luciferase signal and then normalised to the empty pGL3-promoter plasmid signal.

Mammosphere formation assay

HCC1806 cells were seed to ultra-low attachment 6-well plate with cell density of 2000 cells per well and cultured in mammosphere assay medium (DMEM/F12 added with 2% B27 (Gibco, 12587010), 20 ng/mL FGF (PeproTech, 100-18B), and 20 ng/ml EGF (R&D, 236-EG)). Seven days after culturing, images of mammospheres were captured by Nikon Eclipse Tis2 microscope. Number of mammospheres with diameter ≥ 50 μm were counted using Nikon NIS-Elements D software.

AldeRed ALDH detection assay

The ALDH activity of cells was assessed using AldeRed ALDH Detection Assay kit (Merk, SCR150) following the manufacturer’s instructions. Briefly, cells cultured in 2D were trysinized and collected. After washing, 2 × 105 cells were incubated with AldeRed reagent and verapamil for 35 min at 37℃ in the dark. Subsequently, the cells were centrifuged, and the pellets were resuspended in 500 µl ice-cold AldeRed buffer while kept on ice. The signal of AldeRed was then measured using Beckman Coulter CytoFLEX S Flow cytometer analyser with the ECM detector (610/20 BP). Diethylaminobenzaldehyde (DEAB), the ALDH inhibitor, was used as negative control testing to assess the background fluorescence.

CD44/CD24 Flow cytometry

One million cells in stain buffer (PBS with 2% serum, 0.2 mM EDTA, and 0.015% NaN3) were incubated with CD44-PE (BD, 555479) and CD24-647 (BD, 561644) or isotype control IgG-PE (eBioscience, 12-4732-42) and IgG-647 (BD, 557715) at 4℃ for 30 min, protected from light. The cells were then washed and analyzed using a Beckman Coulter CytoFLEX S Flow cytometer. The threshold for positive signals is set according to the negative control of the isotype control IgG.

Fabrication of single-cell-laden microcapsules with droplet-based microfluidic device

A droplet-based microfluidic chip with a 50 μm thickness was fabricated by conventional photolithography for preparing single cell-laden microgels. After the fabrication of the device, a hydrophobic treatment was made on the surface of microchannel by 0.1% (v/v) of 1 H,1 H,2 H,2 H-perfluorododecyltrichloro (sigma) in Novec™ 7500 (3 M). To fabricate alginate microgels,1% (wt) alginate solution containing 50 mM Ca-EDTA was used as the internal phase, whereas the external phase was 1% (v/v) pico-surf in Novec™ 7500. Flow rates for internal and external phases were 0.15 and 0.45 ml per hour, respectively. After the formation of droplets containing single cell in microfluidic channels, the emulsions were collected in Eppendorf tubes and crosslinked with 0.05% (v/v) acetic acid for 2 min before demulsification by 20% (v/v) of perfluorooctanol solution (Sigma). The crosslinked alginate microgels were collected and transferred into mammosphere assay medium (DMEM/F-12 media supplemented with 2% B27 (Gibco, 12587010), 20 ng/mL FGF (PeproTech, 100-18B), 20 ng/mL EGF (R&D, 236-EG), 4 µg/mL Heparin, and 0.4% BSA (sigma, A6003-5G)) for long-term culture. To encapsulate single cell in each microgel, HCC1806 cells were suspended in the aforementioned alginate precursor with 16% (v/v) of Optiprep density gradient medium (Sigma). The mammosphere assay medium was refreshed every other day. After cultured for indicated days, the cell viability was determined by Calcein-AM/PI live/dead assay (Beyotime) according to the manufacturer’s instructions. The size of spheroids was observed with microscope and measured using ImageJ software.

Live/dead co-staining by Calcein-AM and PI

Spheroids formed in Single-cell-laden microcapsules were washed with PBS three times and then co-stained with a Calcein-AM/PI Cell Viability Assay Kit (Beyotime) with the concentration of Calcein-AM at 2.5 µM and PI 4.5 µM, respectively, at 37℃ for 30 min in the dark. The photographs were captured by an inverted fluorescence microscope, Nikon A1HD25 confocal microscope.

Statistical analysis

Statistical significance between conditions was assessed by Student’s t-tests. In all the figures, data are presented as mean ± standard error of the mean (SEM). Significance between conditions is denoted as *p < 0.05, **p < 0.01 and ***p < 0.001. At least three independent experiments were performed for each condition for verification of the emphasized trends in in vitro studies.