Cas5 regulates cell wall stability under basal and cell wall stress conditions. a WSC1 and WSC2, transmembrane sensors that respond to cell wall perturbation, require Cas5 for upregulation in response to caspofungin. The transcript level of WSC1 and WSC2 were monitored by qRT-PCR and normalized to TEF1. Plotted are the log2 fold-changes in transcript levels upon caspofungin treatment relative to untreated conditions in both the wildtype (black) and cas5∆/cas5∆ mutant (gray). Error bars represent standard deviation (s.d.) from the mean of triplicate samples. Significance was measured with an unpaired t test in GraphPad Prism (**P < 0.01). b Cas5 is required for tolerance and resistance to cell wall stress. Caspofungin or calcofluor white susceptibility assays were conducted in YPD medium. Growth was measured by absorbance at 600 nm after 48 h at 30 °C. Optical densities were averaged for duplicate measurements. Data was quantitatively displayed with color using Treeview (see color bar). c Deletion of CAS5 leads to the activation of the cell wall integrity pathway in the absence of cell wall stress. A SN95 wild-type strain and a cas5Δ/cas5Δ mutant were left untreated (−) or treated for 1 h with 125 ng/ml of caspofungin (+), as indicated. Phosphorylated Mkc1 was monitored by western blot and detected with α-p44/42 antibody. Full blots are shown in Supplementary Fig. 2a. Cdc28 was detected with α-PSTAIRE antibody and used as a loading control. d RLM1, a transcription factor downstream of Mkc1 in the cell wall integrity pathway, is induced in a cas5Δ/cas5Δ mutant in the absence of cell wall tress. The transcript level of RLM1 was monitored by qRT-PCR and normalized to TEF1. Differences in transcript level upon caspofungin treatment relative to untreated conditions are plotted. Error bars represent s.d. from the mean of triplicate samples. Significance was measured with an unpaired t test in GraphPad Prism (***P < 0.001)