Fig 2: Fgfr2 signaling continuously prevents adult AT2 apoptosis.a (Left) Time course showing the proportion of AT2 (Muc1+) cells labeled with GFP in control (LyzM-Cre; Rosa26-mTmG Fgfr2fl/+) or Fgfr2 deleted (LyzM-Cre; Rosa26-mTmG; Fgfr2fl/fl) lungs in postnatal life (control, extension of Fig. 5p, mean values ± SD). No further accumulation of GFPpos AT2 occurs in Fgfr2 deleted lungs (dashed line) after PN20, despite continued LyzM-Cre activity (AT2 labeling) apparent in control (solid line). n = 3955 AT2 (3 animals/genotype). (Right) Time course showing identities of GFPpos cells following Fgfr2 loss (extension of Fig. 5r). After a rapid increase in AT1 due to AT2 reprogramming in the juvenile period (through PN20), no further accumulation of GFPpos AT1 or AT2 occurs. n = 300 GFPpos cells scored (3 animals/condition and timepoint, mean ± SD). b Close-up of the alveolar region in adult (PN60) LyzM-Cre;Rosa26-mTmG;Fgfr2fl/fl mouse stained for apoptosis marker cleaved Caspase-3 (cCaspase-3). Note GFPpos AT2 deleted for Fgfr2 undergoing apoptosis (asterisk) but not unlabeled control AT2 nearby (arrowhead). Bar, 10 µm. c–f MASTR system for the conditional, complete deletion of Fgfr2 in AT2. c AAV-Sftpc-Flp virus (top) expresses Flp from Sftpc promoter. MASTR allele (bottom), following Flp-mediated deletion (at frt, triangles) of Pgk-neo-pA cassette, constitutively expresses GFP-Cre fusion. d Scheme. Two weeks after AAV-Sftpc-Flp instillation into lungs of adult Fgfr2fl/fl;Rosa26mTmG/MASTR mice to induce constitutive GFP-Cre and complete deletion of Fgfr2fl/fl in AT2, lungs were immunostained (GFP, Muc1, Fgfr2, cCaspase-3). e Alveolar regions from Fgfr2fl/+ control (top) or Fgfr2fl/fl (bottom) mice treated as in (d). Note the reduction in GFPpos AT2 (Muc1pos) (bottom). Bar, 25 µm. f Quantification of AT2 loss in (e). n = 890 Muc1pos cells scored/condition (three experiments, mean ± SD). g, h Quantification (h) of Fgfr2 immunostaining (n = 100 GFPpos cells scored/condition, three experiments, mean ± SD). Nearly all (96%) GFPpos AT2 remaining after MASTR-mediated Fgfr2fl/fl deletion (e, f) have not fully lost Fgfr2. Micrograph (g) shows an example of rare GFPposFgfr2neg AT2, which were all rounded and extruded into the lumen. Bar, 10 µm. i, j Quantification (j) of cCaspase-3 immunostaining (n = 470 GFPpos cells scored/condition, mean ± SD). Note that 36% of GFPpos AT2 cells remaining after MASTR-mediated Fgfr2fl/fl deletion were cCaspase-3pos, indicating apoptosis initiated (micrograph, i). Bar, 10 µm. k–n Acute inhibition of Fgfr2 signaling by FIIN-1. k Scheme for co-instillation of FIIN-1 and fluorescent marker WGA-405 into lungs of adult SftpcCreER/+;Rosa26mTmG/+ mice tamoxifen(Tam)-treated to label AT2 then analyzed over 2 weeks. Bar, 200 µm. l Lung regions exposed to FIIN-1 (WGA-labeled, left) and shown merged (right) with mTmG fluorescence (AT2 lineage GFP-labeled, other cells tdTomato (mT)-labeled). Bar, 250 µm. m WGA-labeled alveolar regions exposed 2 h to the vehicle (top) or FIIN-1 (bottom). FIIN-1-exposed AT2 cells (GFPpos) undergo apoptosis (cCaspase-3pos, arrowheads). Bar, 20 µm. n Kinetics of AT2 apoptosis in (m). In vehicle control, <2.5% of AT2 were cCaspase-3pos (n = 650 cells scored/condition, three experiments, mean ± SD). No conversion of labeled AT2 to AT1 was detected during this period (n = 390 cells scored/condition, three experiments) (see also Suppl. Fig. 7). o–q Effect of acute Fgfr2 inhibition in AT2 cells by Fgfr2iiib-Fc protein. o Scheme. After Tam-induction of adult SftpcCreER/+;Rosa26mTmG/+ mice to GFP-label AT2, Fgfr2iiib-Fc (or vehicle) and WGA-405 were co-instilled and lungs subsequently stained for cCaspase-3. p WGA-labeled alveolar regions from vehicle control (top) and Fgfr2iiib-Fc (bottom) instilled lungs. Bar, 25 µm. q Quantification of p. n, cells scored, six experiments (mean ± SD). ***p = 4.3 × 10−4 for (f), **p = 3.4 × 10−3 for (j), and ***p = 2.5 × 10−4 for (q) (Student’s two-sided t-test) (panels f, j, q).

Fig 3: Lipofection of top broccoletti-miR candidates does not influence basal and induced apoptosis.(A) BxPc-3 and Bx-Gem cells were transfected as described in Figure 3. Seventy-two hours later, the cells were stained with Annexin V-PE and 7-AAD, followed by FACS analysis. The percentage of Annexin V- and 7-AAD-positive cells is shown. (B) Lipofected BxPc-3 and Bx-Gem cells were stained with an antibody specific for the proliferation marker Ki-67 (green) or the apoptosis marker cleaved fragment of caspase-3 (red), which indicates apoptosis. Representative images at ×100 magnification are shown. The percentage of Ki-67- or caspase-3-positive cells was counted in 18 visual fields, and the means ± SD are shown in the diagram below. (C) BxPc-3 and Bx-Gem were lipofected as described above, and at 24 h later, the cells were treated with gemcitabine (10 nM) or were left untreated. Ninety-six hours after gemcitabine treatment, viability was determined by MTT assay. The data are presented as the means ± SD (** P < 0.01).

Fig 4: Cell-death patterns of C6 cells treated with GNR-ANGI-2 under laser or sham exposure. (A) Cell cytotoxicity pattern of C6 cells preincubated with caspase (Z-VAD) inhibitor or RIPK1 (Nec-1) inhibitor for 4 h and irradiated with laser (12 min); cell viability was evaluated using the MTT assay; cells treated with media were used as control. The bar graph represents the percentages of cell viability respective to the treatment conditions. Error bars represent the standard deviations of 3 experiments (n = 3, * p < 0.05). (B) C6 cells incubated with medium (control), laser (control+ NIR), GNR-ANGI-2 (GA), and GR-ANGI-2 (GA+NIR) were treated with 5 µg/mL AO for 30 min; then, images were recorded under fluorescence microscopy. Red fluorescence (white arrows) indicates acid vesicular organelles stained with acridine orange within the cells. (C) LC3-II processing in C6 cells treated with GNR-ANGI-2 under laser or sham exposure, and control and control treated with laser incubated for 24 h using Western blotting. (D) Changes in protein expression in C6 cells treated with GNR-ANGI-2 under sham or laser exposure. Representative Western blot results show the expression of p53, caspase-3, caspase-7, and Bcl-2 proteins. ImageJ was used to analyze the fold change in protein expression.

Fig 5: Photomicrograph of hepatic tissues immunostained with caspase-3 antibody. Liver of control basal group showing mild cytoplasmic expression of caspase-3 within hepatocytes (A) (arrowhead). Liver of SPC0.25 group showing mild expression of caspase-3 within hepatocytes (B) (arrowhead). The liver of the SPC0.5 group showed a decrease in the cytoplasmic immunostaining of caspase-3 within hepatocytes (C) (arrowhead). Liver of SPC0.75 group showing mild cytoplasmic expression of caspase-3 within hepatocytes (D) (arrowhead). Liver of SPC1 group showing mild cytoplasmic expression of caspase-3 within hepatocytes (E) (arrowhead). Bar = 50 µm. (F) shows morphometric measures of caspase-3 immunostaining expression (%). a,b Means carrying different superscripts were significantly different (p ≤ 0.05).