The reliability of colorimetry is precise(ly) as expected.

Figure S6. Gene ontology (GO) analysis of the differentially expressed genes between C1 and C2. (JPG 403 kb

Table_1_The Essential Oils and Eucalyptol From Artemisia vulgaris L. Prevent Acetaminophen-Induced Liver Injury by Activating Nrf2–Keap1 and Enhancing APAP Clearance Through Non-Toxic Metabolic Pathway.xlsx

Artemisia has long been used in traditional medicine and as a food source for different functions in eastern Asia. Artemisia vulgaris L. (AV) is a species of the genus Artemisia. Essential oils (EOs) were extracted from AV by subcritical butane extraction. EO contents were detected by electronic nose and headspace solid-phase microextraction coupled with gas chromatography (HS-SPME-GC-MS). To investigate the hepatoprotective effects, mice subjected to liver injury were treated intragastrically with EOs or eucalyptol for 3 days. Acetaminophen (APAP) alone caused severe liver injury characterized by significantly increased serum AST and ALT levels, ROS and hepatic malondialdehyde (MDA), as well as liver superoxide dismutase (SOD) and catalase (CAT) depletions. EOs significantly attenuated APAP-induced liver damages. Further study confirmed that eucalyptol is an inhibitor of Keap1, the affinity KD of eucalyptol and Keap1 was 1.42 × 10−5, which increased the Nrf2 translocation from the cytoplasm into the mitochondria. The activated Nrf2 increased the mRNA expression of uridine diphosphate glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), also inhibiting CYP2E1 activities. Thus, the activated Nrf2 suppressed toxic intermediate formation, promoting APAP hepatic non-toxicity, whereby APAP was metabolized into APAP-gluc and APAP-sulf. Collectively, APAP non-toxic metabolism was accelerated by eucalyptol in protecting the liver against APAP-induced injury, indicating eucalyptol or EOs from AV potentials as a natural source of hepatoprotective agent.</p

Table_2_The Essential Oils and Eucalyptol From Artemisia vulgaris L. Prevent Acetaminophen-Induced Liver Injury by Activating Nrf2–Keap1 and Enhancing APAP Clearance Through Non-Toxic Metabolic Pathway.docx

Artemisia has long been used in traditional medicine and as a food source for different functions in eastern Asia. Artemisia vulgaris L. (AV) is a species of the genus Artemisia. Essential oils (EOs) were extracted from AV by subcritical butane extraction. EO contents were detected by electronic nose and headspace solid-phase microextraction coupled with gas chromatography (HS-SPME-GC-MS). To investigate the hepatoprotective effects, mice subjected to liver injury were treated intragastrically with EOs or eucalyptol for 3 days. Acetaminophen (APAP) alone caused severe liver injury characterized by significantly increased serum AST and ALT levels, ROS and hepatic malondialdehyde (MDA), as well as liver superoxide dismutase (SOD) and catalase (CAT) depletions. EOs significantly attenuated APAP-induced liver damages. Further study confirmed that eucalyptol is an inhibitor of Keap1, the affinity KD of eucalyptol and Keap1 was 1.42 × 10−5, which increased the Nrf2 translocation from the cytoplasm into the mitochondria. The activated Nrf2 increased the mRNA expression of uridine diphosphate glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), also inhibiting CYP2E1 activities. Thus, the activated Nrf2 suppressed toxic intermediate formation, promoting APAP hepatic non-toxicity, whereby APAP was metabolized into APAP-gluc and APAP-sulf. Collectively, APAP non-toxic metabolism was accelerated by eucalyptol in protecting the liver against APAP-induced injury, indicating eucalyptol or EOs from AV potentials as a natural source of hepatoprotective agent.</p

Simulated microgravity induces mouse oocytes cytoplasmic blebbing.

<p>Morphology of mouse oocytes with blebs in the perivitelline space cultured in simulated microgravity. A. a) Immunodetection of microtubules (green) and DNA (blue) in blebbing oocytes. b) Immunodetection of microfilaments (red) and DNA (blue) in blebbing oocytes. B. a) An oocyte cultured in simulated microgravity with many cytoplasmic blebs in the perivitelline space (arrow) under normal light microscopy. b) Morphology of oocyte with cytoplasmic blebs in the perivitelline space under transmission electron microscopy. Blebs protruded from the cytoplasm (asterisks) and a free bleb released from the cytoplasm (arrow). c) The ultrastructure of protruded bleb. The blebs do not appear to contain structures other than components of the cytoplasm, and microvilli around the bleb. d) The free bleb in the perivitelline space with microvilli around it. The components of the bleb are identical with the components of the cytoplasm.</p

Simulated microgravity disorders γ-tubulin in mouse oocytes.

<p>A. Location of γ-tubulin in mouse oocytes during meiosis cultured at different stages under 1 g gravity (a–c) and simulated microgravity (d–f).At 6 (a), 9 (b) and 16 (c) hours, γ-Tubulin accumulated at the spindle poles. d) At 6 hours, γ-Tubulin aggregated around the chromosomes. e) At 9 hours, γ-Tubulin dotted in cytoplasm markedly. f) At 16 hours, γ-Tubulin concentrated into two dots and located in the cytoplasm far away chromosomes. Blue = chromosomes, green = γ-tubulin. Each experiment was replicated 3 times with a minimum of 18 oocytes each group. B. Percent of disorder γ-tubulin configuration in mouse oocytes cultured at different time points under 1 g (control) and simulated microgravity (smg). At 6 h, n = 60(control), n = 91(smg). At 9 h, n = 79(control), n = 128(smg). At 16 h, n = 84(control), n = 117(smg). Every time point experiment was repeated 3 times. Different letters denote statistical differences between groups. a vs b, P<0.01; c vs d, P<0.05.</p

Simulated microgravity disrupts microtubules aggregation in mouse oocytes.

<p>A. Spindle morphology of mouse oocytes during meiosis cultured under 1 g gravity (a–c) and simulated microgravity (d–f). a) A mouse oocyte cultured at 6 hours. The barrel-shaped MI spindle is seen with microtubules and chromosomes, arranged on the equatorial plate. b) At 8 hours, chromosomes were separating. c) At 16 hours, the mouse oocyte extruded the first polar body and formed the second spindle. d) At 16 hours, the mouse oocyte showed chromosomes attached with few disordered microtubules. e) At 16 hours, chromosomes without microtubules attachment migrated. f) At 16 hours, the mouse oocyte extruded the first polar body and formed the second spindle. Blue = chromosomes, green = microtubules; Scale bar = 10 µm. B. Percent of microtubules subtypes. Oocytes were cultured in 1 g gravity (control, n = 98) and simulated microgravity (smg, n = 136). After 16 hours cultured, the percentage of different phenotypes were analyzed. Little unordered MTs means that there were little unordered microtubules attached to the chromosomes. Unseen MTs means that there were no obvious microtubules attached to chromosomes. This experiment was repeated 3 times. Different letters denote statistical differences between groups. a vs b, P<0.01; c vs d, P<0.05.</p

Development of GV oocytes under simulated microgravity.

<p>Oocytes were cultured in 1 g gravity (control) and simulated microgravity (smg). After 16 hours cultured, the percentage of different phenotypes were analyzed. This experiment was repeated more than 3 times. Different letters denote statistical differences between groups. a vs b, P<0.01; c vs d, P<0.05.</p

Simulated microgravity does not disorder microfilaments organization and function in mouse oocytes.

<p>Immunofluorescence localization of microfilaments (red) in mouse oocytes cultured at 16 hours under 1 g gravity (a, b) and simulated microgravity (c, d, e). a) Microfilaments formed the actins cap, the bright red stain area near the chromosomes (blue). Chromosomes arranged on the plate. b) Oocyte extruded first polar body, microfilaments aggregated in the cortex. c) Microfilaments formed actins cap near the chromosomes. Chromosomes condensed abnormally. d) Oocyte extruded the first polar body. Microfilaments aggregated in the cortex. e) Oocyte formed a protrusion with microfilaments aggregated in the oolemma near the abnormal chromosomes. Scale bar = 10 µm.</p

Additional file 11: of Previously claimed male germline stem cells from porcine testis are actually progenitor Leydig cells

Figure S9. Expression of adult Leydig cell (ALC)-associated markers in induced differentiated C2 clusters. They did not express the PLC markers PDGFRA1 (a) and LIFR (b), but expressed testosterone synthesis enzymes CYP11A1 (c), CYP17A1 (d), and StAR (e), demonstrated at both the protein and mRNA levels (f). (JPG 301 kb

Additional file 6: of Previously claimed male germline stem cells from porcine testis are actually progenitor Leydig cells

Figure S4. Expression of pluripotency-associated markers and germ cell-specific markers in the C1 and C2 clusters. Immunofluorescent staining showed that both the C1 and C2 clusters expressed the pluripotency-associated markers SSEA1, SSEA4, TRA-1-60, and TRA-1-81. However, the C1 clusters, but not the C2 clusters, expressed the germ cell-specific markers GFRA1 and PLZF. (JPG 249 kb