Fig 2: D-lactate/pyruvate transition mediated by CDK7-YAP-LDHD axis regulates ESCC CSCs properties. a Diagram of D-lactate catabolism in mitochondria. b Untargeted metabolomic mass spectrometry analysis showing methylglyoxal pathway in KYSE410 cells. c D-lactate and pyruvate assays for their changed content in adherent and spheroid-forming KYSE410 cells. d D-lactate and pyruvate analysis to measure their levels in CDK7-overexpressed KYSE150, YAP-overexpressed and LDHD-overexpressed KYSE410 cells. e Cellular D-lactate and pyruvate content were detected with corresponding kits in YAP-knocked out HEK293A cells. f D-lactate and pyruvate analysis in CDK7, YAP or LDHD knocked-down KYSE410 cells. g Western blot analysis for the expression of indicted molecules in KYSE410 and KYSE450 cells supplementing 8 mM D-lactate for 72 h. h Representative micrographs suggesting the spheres formed by KYSE410 and KYSE450 cells treated with D-lactate (D) at indicated concentration. i Histogram showing the numbers of spheres in KYSE410 and KYSE450 cells supplementing indicated concentration of D-lactate (D). j Western blotting analysis for the expression of indicated molecules in KYSE150 and KYSE410 cells treated with indicated pyruvate for 72 h. k Representative phase contrast micrographs displaying the effect of 2 mM pyruvate (Py) on spheroids-forming capacity in KYSE410 and KYSE450 cells. l Histogram illustrating their changed spheroids formation efficiency in (k). β-actin was used as an internal reference in (g) and (j). Scale bars in (h) and (k) represent 100 μm. Error bars represent mean ± SD, (n = 3) in (c–f), (n = 6) in (i) and (l)

Fig 3: CDK7 phosphorylates YAP at S127 and S397 in the nucleus to enhance its transcriptional activity. a Western blot analysis of YAP protein expression in KYSE410 (left) and KYSE450 (right) cells cultured in the RPMI-1640 medium containing indicated concentration of THZ1 for 36 h. b Western blot analysis of YAP protein expression in shCDK7-transfected (shCDK7-1, shCDK7-2) and control (shCDK7-NC) KYSE410 and KYSE450 cells. c Interaction of endogenous YAP and CDK7 in KYSE410 cells. d Proximity ligation assay (PLA) showing the direct interaction between CDK7 and YAP. The nuclei were stained with DAPI. Scale bar, 10 μm. e Kinase assay in vitro suggesting the phosphorylated site and intensity of YAP mediated by CDK7. f Mass spectrometry peak photographs showing the phosphorylation of YAP S127 and S397 sites. g Immunofluorescent images representing the nuclear/cytoplasmic localization of CDK7 (Alexa Fluor 488, green) and phosphorylated YAP (S127 YAP, S397 YAP) (Alexa Fluor 543, red) in CDK7 overexpressed KYSE140 and KYSE150 cells, DAPI (blue) was used for the nuclear stain. Scale bar, 5 μm. h Phosphorylated YAP protein (S127, S397) levels in shCDK7-transfected (shCDK7-1, shCDK7-2) and control (shCDK7-NC) KYSE410 and KYSE450 cells were detected by Western blot. i Phosphorylated YAP protein (S127 YAP, S397 YAP) levels in KYSE410 and KYSE450 cells were detected by Western blot after treatment with the indicated concentrations of THZ1 for 4 h. j Phosphorylated YAP protein (S127 YAP, S397 YAP) levels in KYSE410 and KYSE450 cells were detected by Western blot after treatment with 200 nM THZ1 for indicated time. k Immunoblotting analysis of stemness-associated genes expression when TEAD1, TEAD3, TEAD4 (TEAD1/3/4) were knocked down by siRNA in KYSE410 and KYSE450 cells. l Immunoblotting analysis of stemness-associated genes expression when TEAD2 was knocked down by siRNA in KYSE410 and KYSE450 cells. m Western blot analysis of YAP protein status in wild type and transfected with lentivirus containing an empty vector or YAPS127A-S397A mutant. n Real-time PCR analysis of SOX9 mRNA levels in CDK7-overexpressed and control KYSE140 cells when both S127 and S397 sites were mutated into alanine. GAPDH was used as an internal reference for all above-mentioned Western blot assays. Error bars represent mean ± SD (n = 3)

Fig 4: D-lactate catabolism prevents ESCC CSCs ferroptosis to sustain their stem-like functions. a Cell viability analysis showing the IC50 value of D-lactate in KYSE410 and KYSE450 cells. b MTS measurement for cell viability of KYSE410 and KYSE450 cells treated with 8 mM and 16 mM D-lactate (D) alone or supplementing ZVAD, CQ, FER and NEC simultaneously. c Immunoblotting results suggesting the expression of ferroptosis markers in KYSE410 and KYSE450 cells with 8 mM or 16 mM D-lactate for 72 h. β-actin was used as an internal reference. d Flow cytometry analysis for lipid reactive oxygen species generation in KYSE410 and KYSE450 cells treated with D-lactate (D). e Histogram showing the relative reactive oxygen species production under the action of 8 mM or 16 mM D-lactate in KYSE410 and KYSE450 cells, respectively. f Flow cytometry results indicating the alternative mitochondrial membrane potential induced by 8 mM or 16 mM D-lactate. JC-1 monomers and JC-1 aggregates represent low and high MMP, respectively. g Histogram demonstrating relative mitochondrial membrane potential in KYSE410 and KYSE450 cells treated with D-lactate. h Histogram indicating changed Fe2+ concentration induced by D-lactate in KYSE410 and KYSE450 cells. i Histograms suggesting the changes of intracellular total GSH, reduced GSH and GSSG content in KYSE410 and KYSE450 cells treated with D-lactate. j MTS measurement showing the sensitivity of ESCC cells on Erastin treatment after CDK7-YAP-LDHD axis was up-regulated. k Working model of D-lactate catabolism driven by CDK7-YAP-LDHD axis promotes pyruvate generation and helps ESCC cells escape from ferroptosis induced by D-lactate, then support ESCC-CSCs’ properties. Error bars represent mean ± SD (n = 3)

Fig 5: CDK7-YAP-LDHD axis drives stemness-associated hallmarks of ESCC CSCs. a Western blotting analysis for detecting the expression of stemness-associated indicators in LDHD-overexpressed KYSE150, KYSE410 and KYSE450 cells. b Representative images demonstrating the spheres formed by KYSE150, KYSE410 and KYSE450 cells infected with empty vector and LDHD-overexpressed lentivirus. c Histogram displaying the impact of forced LDHD on sphere formation efficiency of these cells. d Photographs showing the dissected tumors formed by KYSE150 cells after LDHD overexpression in NOD/SCID mice. e The tumor-initiating frequencies of LDHD-overexpressed and control KYSE150 cells. f Immunoblotting analysis for the expression of indicated molecules in KYSE150, KYSE410 and KYSE450 cells after knocking down the expression of LDHD with shRNAs. g Representative photographs demonstrating the spheroids formed by KYSE410 and KYSE450 cells following suppressing LDHD expression by specific shRNAs. h Histogram illustrating the effect of reduced LDHD protein status on spheroid formation ability. i Images suggesting the dissected tumors formed by KYSE150 cells after knocking down LDHD. j The tumor-initiating frequencies of LDHD-depleted and control KYSE150 cells. k Western blotting results exhibiting the alternative protein levels of indicted molecules after knocking down LDHD in CDK7-overexpressed KYSE150 (left) and YAP-overexpressed KYSE410 (right) cells. l Western blot analysis for the expression of indicated molecules in CDK7-knocked down (left) and YAP-knocked down (right) KYSE410 cells after overexpressing LDHD. m Histograms showing the changing sphere-forming efficiencies of CDK7-overexpressed KYSE150 (left) and YAP-overexpressed (right) KYSE410 cells following knocking-down LDHD. n Histograms indicating the impact of forced LDHD expression on the spheroids formation efficiencies in CDK7-knocked down (left) and YAP-knocked down (right) KYSE410 cells. β-actin was used as an internal reference in Western blot experiments. Data were presented as mean ± SD of three independent experiments (n = 6). Scale bars in (b) and (g) represent 100 μm, 1 cm for (d) and (i)