Cell Culture: MDA-MB-231 human triple-negative breast cancer cell lines were all grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics. Cells are cultured with standard sterile tissue culture technique.

CRISPR Protocol: Cells used for the analysis of the first cell line (MDA-MB-231 human TNBC cells) were thawed from a previous graduate student at the Oudin lab who, along with one of us (Maia),  had performed the CRISPR gene knockdown. This involved creating MDA-MB-231-Cas9 cells, lentiviral transduction of gRNAs, and then the cells were expanded for use.

Cell Viability Assay: To test the effect of gene knockdowns of MAPT and TUBB3 in chemotherapy sensitivity, cell viability experiments were performed. 5000 cancer cells were seeded in each well of a 96 well plate, then treated with chemotherapy drugs at varying concentrations. Cell viability was measured on day 1 and day 3 using Presto Blue reagent (P50200) according to manufacturer recommendations. Viability was normalized with blank and control samples, then displayed as fractions of the no drug control.

siRNA Infection: For the initial viability evaluation of MAPT and TUBB3 knockouts, siRNA-TUBB3 and  siRNA-MAPT Silencer will be used from provider ThermoFisher with code 4392420 and 9854367. The protocol for infection was followed as instructed by the provider. Three experimental groups were tested to evaluate the best siRNA concentration for knockouts: 5μM, 2.5μM, and 1.25μM. MAPT gene expression was measured with qPCR and TUBB3 with immunostaining given the accessibility of the well-known antibody for this gene. 

Migration Assay: On day one, a glass-bottom 24 well plate was coated with 0.1mg/mL Collagen I for 1 hour at 37C. 300 μL cells were seeded at 12k/well in media and incubated for 2 hours at 37C. The migration chamber was set up with image acquisition settings using Brightfield and TXRed to image the cells. Multipoint settings were used to select 4-5 POVs for each well, and a 10-minute interval between image acquisitions for a total of 97 times.

Cell adhesion assay: 24h after culture was established, cells were fixed and stained with DAPI. Images were taken at 20x with approximately 30 field of views per condition. CellProfiler v3.1.8164 was used to identify cell  shape: DAPI was used to identify individual cells then mCherry was used to determine cell shape parameters.

Statistical analysis: GraphPad Prism v8.4.3 was used for generation of graphs and statistical analysis. To compare between  two  groups, unpaired two-tailed Student’s t-test was used and  a  p-value of ≤ 0.05  is considered  significant. To compare  between  multiple  groups,  one-way  ANOVA  with  Tukey’s multiple testing correction was used with a corrected p-value of ≤ 0.05 is considered significant. For  RNA-seq, adequately expressed genes passing  a  fold change  threshold  of 1.2 and with p value ≤ 0.05 in edgeR analysis were considered differentially expressed. Pathways with p value ≤ 0.05 and FDR ≤ 0.01 were considered differentially regulated.