Fig 2: p38 MAPK priming by anisomycin treatment leads to increased mitochondrial fragmentation and PDGF-BB-induced proliferation in VSMCs in a PGC1a-dependent manner. (A) Representative western blot of PGC1a, phospho-p38, p38 and Tubulin in MT4-MMP+/+ and MT4-MMP-/- VSMCs treated or not with PDGF-BB. (B) Correlation graphs between phospho-p38/p38 and PGC1a/Tubulin in MT4-MMP+/+ and MT4-MMP-/- VSMCs treated or not with PDGF-BB. n = 6 VSMC cultures in 6 independent experiments. Correlation was analysed by a simple linear regression test. (C) Western blot of phospho-p38, p38 and Tubulin in two independent batches of PGC1a+/+ and PGC1a-/- VSMCs pre-treated with vehicle (DMSO) or anisomycin. (D) Representative confocal microscopy images showing immunostaining for TOMM20 and its thresholded binary image of PGC1a+/+ and PGC1a-/- VSMCs pre-treated or not with vehicle or anisomycin. Cells were also treated or not afterwards with PDGF-BB (images not shown). Scale bar: 10 µm. (E) Quantification of number of individuals, inverse aspect ratio, mean area and number of branch junctions. n = 5 VSMC cultures in 5 independent experiments. (F) Quantification of the percentage of proliferative VSMCs (DAPI+/SMA+/Ki67+) in PGC1a+/+ and PGC1a-/- VSMCs pre-treated with vehicle (DMSO) or anisomycin and in presence or absence of PDGF-BB. n = 5 VSMC cultures in five 5 independent experiments. In (E,F) data are means ± s.e.m. analysed by two-way ANOVA with Benjamini and Hochberg post-test; *, # p < 0.05, ** p < 0.01, ***, ### p < 0.001, ****, #### p < 0.0001.

Fig 3: Sin3B Is Not Required for Entry into Quiescence(A) Western blot for two independent Sin3B wild-type (left) and two independent Sin3B knockout (right) T98G cell lines. Immunoblotting was performed using 40 µg of whole-cell lysates probed using the indicated antibodies.(B) Representative flow plots of KI67 staining in Sin3B+/+ and Sin3B-/- T98G cells. Cells were stained for KI67. Cycling, asynchronous, cycling cells; Sync, serumstarved cells for 72 hr; Isotype, isotype control antibody.(C) Quantification of KI67 mean fluorescence intensity (MFI) from (A). n = 4 (2 independent cell lines per genotype with 2 experimental replicates each).(D) Representative flow plots of BrdU staining in Sin3B+/+ and Sin3B-/- T98G cells. Cells were starved for 72 hr, pulsed for 2 hr with BrdU, and stained.(E) Propidium iodide flow plots from (D).(F) Quantification of (C). n = 4 (2 independent cell lines per genotype with 2 experimental replicates each).(G) Representative flow plots of PI staining for Sin3B+/+ and Sin3B-/- T98G cells, synchronized in G0 by serum deprivation for 72 hr (T0) or release upon addition of serum for 10 hr (T10) or 18 hr (T18).(H) Sin3B+/+ and Sin3B-/- T98G cells expressing FUCCI probes (RFP, pFUCCI-G1; GFP, pFUCCI-S/G2/M) were serum deprived for 72 hr and released into 10% serum for the indicated time, and the amount of RFP and GFP fluorescence was quantified. n = 6 (2 independent cell lines per genotype with 3 technical replicates each).

Fig 4: Acetylcholine/muscarinic signaling maintains SOX2 and the acinar lineage in human SG AHuman salivary gland obtained from healthy individuals (no IR; submandibular) or patients who received radiation therapy for head and neck cancer (IR) were subjected to qPCR.B–DHuman SMG explants cultured for 7 days with either murine embryonic day (E) 13 parasympathetic ganglia (nerves) or E13 mesenchyme (MES). Explants were analyzed by immunostaining for markers of nerves (B, TUBB3) or cell proliferation (C, Ki67) or by qPCR (D). Scale bars = 50 µm (B, C).EqPCR analysis of adult human salivary gland (SMG or PG) explants of four different individuals cultured for 4 h ± CCh (200 nM, n = 4). Individual datasets are shown in Fig EV5D.Data information: Data in (A) n = 11 for no IR and n = 7 for IR; 30–85 years. Data were normalized to GAPDH with individual values plotted and analyzed using a Student's t-test with a false discovery rate set to 0.05. Error bars show mean ± SD. AQP3 P = 0.017, MIST1 P = 0.086, AMY1 P = 0.005, SOX2 P = 0.079, CHRM1 P = 0.040, CHRM3 P = 0.011, EGFR P = 0.931, KRT19 P = 0.618. Data in (D) are means + SD of n = 2 individuals where solid line columns represent individual #1 and broken line columns represent individual #2. Data were normalized to salivary glands from the same individuals cultured with murine mesenchyme (Control, black dashed line). Data in (E) are box and whisker plots of n = 4 different individuals, showing means (horizontal line), upper and lower quartiles (box) and upper and lower values (whiskers). Data were normalized to the untreated control (black dashed line). n = 5–8 explants per individual. Data were analyzed using a one-way analysis of variance with post hoc Dunnett's test. SOX2 **P = 0.00834, CHRM3 *P = 0.0449, CHRM1 **P = 0.0093, AQP5 **P = 0.0069, AQP3 *P = 0.0375, MIST1 *P = 0.0379, CD44 *P = 0.0485, KRT19 *P = 0.0461.

Fig 5: SOX2 marks a subset of acinar cells that replenish acini Wild-type murine PG stained for SOX2, ECAD, and nuclei. Scale bar is 50 µm.The percentage of acinar SOX2+ and SOX2- cells that are Ki67+ were counted using FACS and shown as a percentage of total AQP5+SOX2+ or AQP5+SOX2- cells.The % of SOX2+ acinar cells that are either CyclinD1+ or CyclinD1-.Schematic of Rosa26 mTmG Cre-mediated gene excision (adapted from Muzumdar et al, 2007).Representative image of Sox2 lineage-traced SLG. Cre-mediated recombination was induced in Sox2 CreERT2 ;Rosa26 mTmG mice and SLG analyzed 14 or 30 days later by immunostaining for SOX2. Scale bar = 25 µm.Representative images of Kit lineage-traced SLG and SMG. Cre-mediated recombination was induced in Kit CreERT2 ;Rosa26 mTmG mice and SMG/SLG analyzed 14 days and 6 months later. Tissue was stained with AQP5 to mark acinar cells and KRT8 to mark intercalated duct cells. Scale bar = 25 µm. mT = membrane-bound Tomato.Data information: Data in (B), SLG were pooled from n = 2 mice (85,000 events). Data in (C) were calculated from three non-consecutive fluorescent sections of each SLG from n = 3 mice with individual values plotted. Error bars show mean ± SD.