Axiocam 702

For the mitochondrial morphology assay, WT D1 mothers were treated with 5 μM rotenone (TargetMol, T-2970) and 2 μM CNB dye for 12 h. Offspring were collected for imaging at the embryonic, L2 and L4 stages on rotenone and CNB-free plates. The images of hypodermal mitochondria of L2s were taken with an Axiocam 702 mono camera on a Zeiss LSM800 (Airyscan) confocal microscope using a 63× oil objective. For body length measurement, WT D1 mothers were treated with 5 μM rotenone or 50 mM metformin (Sigma, PHR1084) for 12 h, and embryos were produced on rotenone and metformin-free NGM plates. The maximal adult body lengths of tested animals were measured and quantified.
For the mitochondrial recovery assay, WT OD5 animals were treated with 0.5 mM or 1 mM AICAR (Selleck, S1802) mixed with 2 μM CNB dye from embryos to the L2 stage. The images of hypodermal mitochondria of L2s were taken with an Axiocam 702 mono camera on a Zeiss LSM800 (Airyscan) confocal microscope using a 63× oil objective. For the body length assay, WT OD2 animals were treated with 1 mM AICAR from the embryonic to young adult stages (egg laying start) and then transferred to AICAR-free plates. The maximal adult body lengths of tested animals were measured and quantified.

Imaging experiments were performed at 37°C in 0.5 mM or 1 mM Ca²⁺ Ringer's buffer (as indicated in the figure legends) using either a Zeiss Cell Observer Z1 microscope equipped with a 40× oil “Fluar” (N.A.: 1.3) objective, multi‐filter system, fast acquisition EMCCD camera (Evolve® 512 Delta) and LED system (Colibri, Zeiss) or a Zeiss Observer D1 equipped with a 40X oil Neofluar (N.A.: 1.3) objective, Axiocam 702 mono and LED system (Colibri, Zeiss) or a Zeiss Axio Observer 7 equipped with 40× oil “Neofluar” (N.A.: 1.3), Axiocam 702 mono and LED system (Colibri 7, Zeiss). Subsequent obtained data were processed with the AxioVision, Zen 2.6, or Zen 3.2 softwares (Zeiss, Oberkochen, Germany). All plasmids and constructs used are listed in Table EV2.

PANC1 cells cultured on 25 mm coverslips in 35 mm culture dishes were transfected for 48 h with WT or L16R p16 in the pCMV-FLAG vector. Cells were then fixed in 3.2% formaldehyde for 40 min and permeabilized in 0.1% Triton-X-100 at 4 °C for 5 min. Subsequent steps were performed at room temperature. Samples were blocked for 1 h in blocking buffer (BB; 5% goat serum, 5% glycerol, 0.05% NaN₃ in PBS), incubated for 2 h in M2 anti-FLAG antibody diluted 1/200 in BB, washed three times in PBS, and then incubated for 1 h with ALEXAfluor-594 goat anti-mouse secondary antibody (Thermo) at 1:200 in BB. After staining, these were washed three times in PBC, and then samples were mounted on microscope slides in Prolong with DAPI (Thermo). Microscopy was performed using a Zeiss system including an Axiovert 200 microscope with a 20× Plan-APO Chromat lens and an Axiocam 702 mono camera. Images were captured using the Zen 2.3 application. Image fields were taken randomly in red (AF546; immunofluorescence) and blue (DAPI) channels, using only the blue signal for focusing. For each coverslip, 14 fields were acquired. DAPI and immunofluorescent images were overlaid and quantified using Adobe Photoshop CC 19.1.4. A minimum of 790 total cells were counted per coverslip. Results were expressed as percent transfection [100 × number of red (p16-positive) cells/total blue nuclei].