Figure 3: hCINAP directly binds to LDHA and promotes LDHA activity.

(a) Immunoprecipitation analysis of the interaction between endogenous hCINAP and LDHA protein in SW480 cells. Rabbit IgG served as a negative control. See also the hCINAP-interacting partners screening in Supplementary Fig. 2. (b) In vitro pull-down analysis of the interaction between GST-tagged hCINAP and His-tagged LDHA protein purified from E. coli, with GST protein as a negative control. See also the details within the FGFR1–LDHA–hCINAP interaction in Supplementary Fig. 3. (c) Lactate production levels in SW480 cells transfected with the indicated shRNAs and plasmids were determined by a spectrophotometer and normalized to the cell number. (d) LDHA enzyme activity was determined in SW480 cellls as indicated. (e) Endogenous LDHA protein was immunoprecipitated and subjected to enzymatic activity assay. Western blot analysis showed immunoprecipitated LDHA protein in lysates of SW480 cells treated with control shRNA and hCINAP shRNA 2. The experiments were performed three times. (f,g) Measurements of ECAR and OCR levels of SW480 cells in e. (h) Limiting dilution assay in CRCSCs as indicated under normaxia or hypoxia (3% O₂). CRCSC frequency is calculated by extreme limiting dilution analysis. **P<0.01. Also see LDHA Y10 phosphorylation and LDHA activity in Supplementary Fig. 6a,b. Data in c–g are determined by Student's t-test and presented as mean±s.e.m. (n=3). **P<0.01, ***P<0.001, NS, not significant.