Comparison of acrosome integrity of frozen-thawed spermatozoa among groups.

a<p>indicates a statistical difference when compared with Group A (P<0.05).</p

Photographs showing DNA integrity of frozen-thawed spermatozoa in each group at a magnification of 400×.

<p>The symbol “−” and “+” represent cathode and anode respectively during electrophoresis of negatively charged DNA. <b>a.</b> pre-frozen spermatozoa; <b>b.</b> post-thawed spermatozoa of Group A; <b>c.</b> comet tail of post-thawed spermatozoa in group B; <b>d.</b> obvious long comet tail in post-thawed spermatozoa of Group C; <b>e.</b> post-thawed spermatozoa of Group D.</p

Comparison of viability and motility of frozen-thawed spermatozoa among the groups.

a<p>indicates a statistical difference when compared with post-thawing spermatozoa from Group A to Group D (P<0.05);</p>b<p>indicates a statistical difference when compared with Group A (P<0.05).</p

Photographs of four categories of acrosome status evaluated by FITC-PNA staining at a magnification of 1000×.

<p><b>a.</b> I-intact acrosome; <b>b.</b> II-intermediate form of minimal acrosome reactivity; <b>c.</b> III-intermediate form of severe acrosome reactivity; <b>d.</b> IV-reacted acrosome.</p

Workflow of PDMS chip.

<p><b>a.</b> a PDMS chip compared with a coin; <b>b.</b> sample loading with a micro-injector; <b>c.</b> thawing of frozen spermatozoa; <b>d.</b> push the syringe and thawed spermatozoa in micro-channel is observed to be transferred into the tissue culture dish under the microscope.</p

Schema of spermatozoa store in PDMS chip and freezing tube.

<p><b>a.</b> spermatozoa cryopreserved in micro-channel of which height is 10 µm (group A) one by one; <b>b.</b> spermatozoa cryopreserved in micro-channel of which height is 50 µm (group B); <b>c.</b> spermatozoa cryopreserved in micro-channel of which height is 100 µm (group C); <b>d.</b> spermatozoa cryopreserved disorderly in a 1.8 ml freezing tube (group D).</p

Schematic illustration of the volume distribution of sperm suspension in each step when cryopreserved in 10 µm height channel.

<p>The density of spermatozoa sample was adjusted to 10⁵/µL before loading. 5×10⁻³ µL medium containing 1000 spermatozoa can be stored in 10 µm height channel and 800–900 spermatozoa can be finally transferred out in 1 mL fertilization medium.</p

Comparison of DNA integrity of frozen-thawed spermatozoa among groups.

a<p>indicates a statistical difference when compared with Group A (P<0.05).</p

Silicosis is associated with GSDME, GSDMD and caspases cleavage.

a GSDMD, GSDME, CASP1, CASP3, CASP6, CASP8, IL1B and IL18 cleavage in controls (#1~#6) and silicosis patients (#7~#14) lung tissues. b Grayscale analysis of (a). c Relative protein cleavage in the BALF of controls (207, 209, 223, 233 and 257) and silicosis patients (356, 387, 392, 398, 194897 and cf). d Grayscale analysis of (c). e Gsdmd, Gsdme, Caspase-1, Caspase-3, Caspase-6, Caspase-8, IL-1β and IL-18 cleavage in mouse BALF, n = 4. f Grayscale analysis of (e). Human data were presented as the median ± 95% CI, and analyzed with Mann-Whitney U test. e was expressed as mean ± SD. NS, not significant; *PPP<0.001.</p

Apoptosis caspases are required for silica-induced pyroptosis.

a, Cell survival of primed Nlrp3-/- BMDMs pretreated with inhibitor mixture as indicated and stimulated with silica. b, Cell survival of silica-stimulated Nlrp3-/- macrophages pretreated with indicated caspase inhibitors. c, Cell survival of primed Caspase-1-/- BMDMs pretreated with inhibitor mixture as indicated and stimulated with silica. d, Cell survival of silica-stimulated Caspase-1-/- macrophages pretreated with indicated caspase inhibitors. a-d, The cell viability was measured through extracellular LDH release assay. e, BMDMs derived from WT, Caspase-1-/- and Gsdmd-/- mice on chambered coverslips were stimulated with silica and monitored for morphological changes over time by differential interference contrast (DIC) and fluorescence microscopy. Loss of membrane integrity was indicated by PI (red) staining of nuclear DNA. Scale bar represents 100 μm. z-VAD-FMK, pan-caspase inhibitor; C1i, VX765 (Caspase-1 inhibitor); C2i, z-VDVAD-FMK (Caspase-2 inhibitor); C3i, z-DEVD-FMK (Caspase-3 inhibitor); C6i, z-VEID-FMK (Caspase-6 inhibitor); C8i, z-IETD-FMK (Caspase-8 inhibitor); C9i, z-LEHD-FMK (Caspase-9 inhibitor); C10i, z-AEVD-FMK (Caspase-10 inhibitor); C12i, z-ATAD-FMK (Caspase-12 inhibitor). CIMix represents the mixture of all the indicated caspase inhibitors, while CIMix-C1i means lack of Caspase-1 specific inhibitor and the rest can be deduced by analogy. Results are expressed as mean ± SD from three independent experiments. *PPP (TIF)</p