Fig 2: Sox6 cumulative fate map(A) Sox6-FSF-Cre, CAG-Flpe, RC-Frepe brain at E11.5 (mCherry not shown).(B) Sox6-Cre, Th-2A-Flpo, RC-Frepe brain at E14.5.(C) Adult Sox6-Cre, Th-2A-Flpo, RC-Frepe brain (n = 9).(D) In Sox6-FSF-Cre, Th-2A-Flpo, RC-Frepe brains, DA neurons expressing Sox6 postmitotically (after the onset of Th expression) are labeled with GFP. In Sox6-Cre, Th-2A-Flpo, RC-Frepe brains, Cre activation no longer depends on Th; thus, TH+ DA neurons expressing Sox6 at any point in their history are labeled with GFP.(E) Comparison of cumulative versus postmitotic labeling. In VTA, there are more GFP+ neurons in the cumulative analysis (p = 0.06).(F and G) SNc of Sox6-Cre, Th-2A-Flpo, RC-Frepe brains labeled with ALDH1A1, or CALBINDIN-D28k+ (CB+) (GFP, triangles; mCherry arrowheads).(H) In the SNpl, the majority of Vglut2+ neurons are TH+,mCherry+ (arrowheads), while only a few are TH+,mCherry- (triangles). *Typically GFP+, lost during in situ hybridization.Scale bars: (A) 50 µm; (B) 100 µm; (C) 200 µm. Error bars are SEMs. (A, B, and E–G) n = 3.

Fig 3: GABA co-release from SNcDA axons requires Gat1-mediated GABA uptake(A) Confocal images of SNc in Gat1 cKODA mice expressing Cre-dependent HA-tagged Gat1 (left) and Gat1(R69K) (right) immunolabeled for TH (green), HA (red), and DAPI (blue). Inset: detailed view (scale, 50 µm).(B–F) oIPSCs recorded from SPNs (Vh = 0 mV) upon stimulation of SNcDA axons (blue bar) in Gat1 cKODA mice (B) exogenously expressing Gat1 (C), Gat1(R69K) (D), Gad67 (E), or Aldh1a1 (F).(G) Fluorescence in situ hybridization for Gad67 (red) and TH (green) in Gat1 cKODA mice with (right) or without (left) Gad67 in DA neurons. Inset: detail (scale, 50 µm).(H) oIPSC amplitude in SPNs of Gat1 cKODA mice virally expressing ChR2 in SNcDA neurons (n = 22, p = 1 × 10-15), or ChR2 + Gat1 (n = 14; p = 0.07), ChR2 + Gat1(R69K) (n = 18; p = 1 × 10-15), ChR2 + Gad67 (n = 13; p = 0.018), or ChR2 + Aldh1a1 (n = 17; p = 2 × 10-15) normalized to control. All p values versus control, Mann-Whitney. nd, not detected. Individual values shown along with population mean ± SEM.(I) Same as (H) for latency (Gat1DA: p = 0.32; Gad67DA: p = 0.0007 versus control, Mann-Whitney).(J) Same as (I) for rise time (Gat1DA: p = 0.18; Gad67DA: p = 0.39 versus control, Mann-Whitney).

Fig 4: Sox6 progenitor fate map(A) Schematic of PRISM alleles.(B) Sox6-Cre, Nestin-LSL-Flpo, RC-LacZ (from here on Sox6 PRISM) at E11.5. ßGal+ progenitors (Sox6+) are located medially in the Lmx1a+ floor plate.(C) Sox6 PRISM adult brains show TH+,ßGal+ neurons in the SNc and VTA (n = 8).(C’) High magnification of SNc TH+,ßGal+ cells (arrowheads) and TH+,ßGal- cells (triangles).(D) Average TH+,ßGal+/TH+ neurons in Sox6 PRISM adult brains, also corrected by PRISM efficiency; p = 0.1.(E) High magnification of Sox6 PRISM SNc neurons co-expressing TH, ALDH1A1, and ßGal.(F) Sox6 PRISM SNc cells co-expressing dopamine transporter (DAT), CALBINDIN-D28k (CB), and ßGal. Most CB+,DAT+ cells are ßGal- (triangles). For reference, arrowhead shows a ßGal+,DAT+,CB- cell.(G) Percentage of ßGal+ cells that express ALDH1A1 or CB in the SNc and VTA of Sox6 PRISM adult brains (p values: SNc p = 0.008, VTAp = 0.5).Scale bars: (C) 200 µm; all other scale bars, 50 µm. Error bars are SEMs. (B and D–G) n = 3.

Fig 5: Matrix stiffness is correlated with CSC abundance in human breast cancer.a Representative images of ultrasound B-mode, UE, HE, picrosirius red staining, AFM map (inserted in picrosirius red pictures), and ALDH1/CK staining of patients in the NAC cohort (n = 124 patients). White dashed lines represent tumor outlines. Green and yellow squares in the HE pictures indicate the area shown in the picrosirius red and immunofluorescence pictures, respectively. Asterisks (*) indicate location of higher magnification views in ALDH1/CK staining images. Bars in the pictures indicate the scale. b Quantitation of CK+ALDH1+ CSCs percentage in total cancer cells of patients in (a) (n = 124 patients). Mean ± SEM, ****p < 0.0001 by two-tailed Mann-Whitney U test. c The correlation between the strain ratio and the percentage of CK+ALDH1+ CSCs of patients in (a) was evaluated by Pearson’s correlation analysis (n = 124 patients). Correlation coefficient (r) and p value were acquired by Pearson correlation test and shown in the figure. For b and c, source data are provided as a Source Data file.