Journal: Research

Article Title: ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress

doi: 10.34133/2022/9834963

Figure Lengend Snippet: Upregulated HDAC2 phosphorylation is connected with downregulated ATP5O crotonylation in CS mice. (a, b) TMT-labeled phosphoproteomics showed that at the threshold of ≥1.2 or ≤ 0.833 , there are 117 downregulated and 60 upregulated phosphorylated proteins. (c) KEGG pathway showed that multiple pathways are lipid metabolism related. (d) We selected three deacetylases—HDAC2, KAT7, and CREBBP—to verify their relationship with ATP5O. Western blot showed that inhibition of HDAC2 phosphorylation at S424 with THP (CYGRKKRRQRRRSDSEDEGEGGRR) significantly decreased ATP5O, whereas inhibition of KAT7 phosphorylation at S102 with TKP (CYGRKKRRQRRRSSGSETEQVVDF) or CREBBP phosphorylation at S2361 and S2363 with TCP (CYGRKKRRQRRRQSQPPHSSPSPR) did not change ATP5O protein level at all. (e, f) We generated an ATP5O-S424 phosphorylation-specific antibody with a phosphorylated peptide SDS (phospho) EDEGEGGRR and verified the specificity through two ways. (e) Different amounts of non- and S424-phosphorylated HDAC2 peptide were loaded onto a dot blot PVDF membrane and detected by the HDAC2-S424 P antibody. Only phosphorylated HDAC2 peptide was detected in a dose-dependent manner. (f) When HDAC2-S424 P antibody was preblocked by the phosphorylated HDAC2 peptide, significantly less amount of endogenous ATP5O-K51 cr was detected. (g) Western blot showed that THP treatment significantly decreased HDAC2-S424 P (p-HDAC2) level and upregulated ATP5O-K51 cr (ATP5O cr ) level, whereas did not change KAT7-S102 P (p-KAT) level. Besides, THP treatment did not alter the level of HDAC1-S421 p at all, which shared the highest similarity with THP (Supp. Figure ). (h, i) By live imaging in Sf9 cells, when only ATP5O-WT-EGFP titer was added into sf9 culture media, as time progressed, green fluorescence rapidly increased; in contrast, when both ATP5O-EGFP and HDAC2-WT-TagRFP titers were added, as time went by, the red fluorescence (HDAC2) quickly increased, whereas the increment extent of green fluorescence (ATP5O) was significantly slowed down. (j) Coimmunoprecipitation and western blot showed that HDAC2 significantly interacted with ATP5O but rarely interacted with ATP5A1 or ATP5B. (k) GST-pulldown was carried out to verify the direct binding between ATP5O and HDAC2. GST-ATP5O and his-tagged HDAC2-TagRFP were cloned, expressed, and purified in E.coli (From left, the fourth and fifth lane, red asterisk-labeled) and sf9 cells (From left, the third lane, red asterisk-labeled), respectively. SDS-PAGE showed that GST-ATP5O could significantly pull down HDAC2-TagRFP (From left, the sixth lane, red asterisk-labeled). L-N. Western blot showed that when a fixed amount of ATP5O-WT and increasing amounts of HDAC2-WT were cotransfected into 293 T cells, ATP5O crotonylation level rapidly decreased (l, n); in contrast, when a fixed amount of ATP5O-WT and increasing amounts of HDAC2-S424A (a nonphosphorylable mutant) were cotransfected into 293 T cells, the decrement extent of ATP5O crotonylation level became much smaller (m, n). (o–s) When HDAC2-Flag and ATP5O-StrepII were cotransfected into 293 T cells at an increasing dosage, ATP5O crotonylation (o, p) and ATP levels both increased (q); accordingly, FAM126A and STAT5A levels also increased in a dose-dependent manner (r, s). (t–u) Western blot showed that compared with ATP5O-transfected 293 T cells, HDAC2 and ATP5O cotransfected cells had decreased ATP5O crotonylation, whereas inhibition of proteasome activity with MG132 significantly recovered ATP5O crotonylation level. (v, w) Western blot showed that in an in vitro ubiquitination assay with the ATP5O-E3 ubiquitin ligase complex immunoprecipitated from CS ovaries, the ATP5O ubiquitination level was significantly higher than the level in control ovaries. X. Model: ATP5O crotonylation is resistant to ubiquitination, whereas ATP5O-K51 cr decrotonylated by HDAC2 is susceptible to ubiquitination and protease degradation. Tubulin or actin was used as a loading control. * indicates p < 0.05 , *** indicates p < 0.001 , **** indicates p < 0.0001 .

Article Snippet: Other primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti- β -Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti- β -Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti-alpha Tubulin (Acetyl Lys40) (cat#: bsm-33235 M; Bioss, Beijing, China); Rabbit anti-Transferrin polyclonal antibody (Cat#: 17435-1-ap; Proteintech, Chicago, USA); Rabbit anti-ATP5O polyclonal antibody (Cat#: D126152; BBI Life science, Shanghai, China); Rabbit anti-ATP5A1 polyclonal antibody (Cat#: D154243; BBI Life science, Shanghai, China); Rabbit anti-ATPB polyclonal antibody (Cat#: A5286; Selleckchem, Shanghai, China); Mouse anti-COX4L1 monoclonal antibody (Cat#: D190618; BBI Life science, Shanghai, China); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Rabbit anti-Phosphpho RPS6 (Ser235/236, Cat#: D155178; BBI Life science, Shanghai, China); Rabbit anti-STAT5A polyclonal antibody (Cat#: D220085; BBI Life science, Shanghai, China); Rabbit anti-FAM126A polyclonal antibody (Cat#: bs-11554R; Bioss, Beijing, China); Rabbit anti-PTDSS1 polyclonal antibody (Cat#: BS-19583R; Bioss, Beijing, China); Rabbit anti-HDAC2 polyclonal antibody (Cat#: 12922-3-ap, Proteintech, Chicago, USA); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); Mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science, Shanghai, China); Rabbit anti-HDAC1-S421 p polyclonal antibody (Cat#: AP59578, Abcepta, San Diego, CA, USA); Rabbit anti-Ubinuclein polyclonal antibody (Cat#: DF8873, Affinity Biosciences, Melbourne, Australia); Fibrinogen alpha chain (FGA) Rabbit pAb (Cat#: A1453, ABclonal, Wuhan, Hubei, China); Albumin Polyclonal antibody (Cat#: 16475-1-AP; Proteintech, Rosemont, IL, USA); AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson, Cat#: 115-005-003, West Grove, PA, USA); Rabbit anti-ATP5O-K51 cr rabbit polyclonal antibody was made against ASK (crotonyl) EKKLDQVEKELLC by YuBo BioTech and purified by affinity purification; Rabbit anti-HDAC2-S424 p ,and anti-KAT7-S102 p polyclonal antibody was made against SDS(phospho)EDEGEGGRRC and CPLRQTRSSGS (phospho) ET by Zoonbio BioTech and purified by affinity purification.

Techniques: Labeling, Western Blot, Inhibition, Generated, Dot Blot, Membrane, Imaging, Fluorescence, Binding Assay, Clone Assay, Purification, SDS Page, Mutagenesis, Transfection, Activity Assay, In Vitro, Ubiquitin Assay, Immunoprecipitation

Journal: Research

Article Title: ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress

doi: 10.34133/2022/9834963

Figure Lengend Snippet: ATP5O crotonylation level was the most downregulated in the ovaries and plasma of CS mice. (a) We created a chronic stress (CS) female mouse model using a restraining cylinder. The treatment lasted for 28 days, 6 hours per day. No food and water were provided during the daily treatment. (b–d) Multiple assays indicated that the CS mouse modeling was successful. (b) The weight of the CS female mice was significantly lower than control group; (c) the serum corticosterone level of the CS female mice was significantly higher than control group; and (d) the mRNA levels of multiple HPA (hypothalamic–pituitary–adrenal) axis-related genes including RFRP (RF amide-related peptide), GnRH (gonadotropin-releasing hormone 1), and GPR147 (neuropeptide FF receptor 1), had significant differences between CS and control groups. (e–h) Crotonylation was significantly downregulated in multiple tissues of CS female mice, such as hypothalamus (Hth), livers, and ovaries. (e, h) Pan-crotonylation in hypothalamus; (f, h) pan-crotonylation in liver; (g, h) pan-crotonylation in ovary. (i, k) Pan-Kcr immunofluorescence in multiple tissues, including hypothalamus (i), livers (j), and ovaries (k), showed that pan-Kcr signal in CS group was significantly lower than in control group, whereas the morphology and condition of these tissues appeared normal in either group. Pan-Kcr in green, DNA in blue. (l, m) Quantitative crotonylomics showed that at a threshold of ≥ 1.3 or ≤ 0.77 , there were 44 upregulated and 90 downregulated crotonylation sites, with the latter covering 67.2% of all differentially crotonylation sites (DCSs). (n) Among the proteins with DCSs, metabolism-related proteins represented the largest percentage (15.97%), and there were 15 metabolism-related pathways in the top 30 KEGG pathway (50%). (o, p). Among the top 10 downregulated crotonylation sites, the crotonylation level of ATP5O at K51 decreased by about 10 folds in CS ovaries than in control ovaries. (q–h) we generated an ATP5O-K51 crotonylation-specific antibody with a crotonylated peptide ASK (crotonyl)EKKLDQVEKELL and verified the specificity through two ways. (q) Different amounts of non- and K51-crotonylated ATP5O peptide were loaded onto a dot blot PVDF membrane and detected by the ATP5O-K51 cr antibody. Only crotonylated ATP5O peptide was detected in a dose-dependent manner (Figure (q)). (r, s) When ATP5O-K51 cr antibody was preblocked by the crotonylated ATP5O peptide, significantly less amount of endogenous ATP5O-K51 cr was detected (Figures (r) and (s)). (t, u) Western blot by ATP5O-K51 cr antibody verified the decrement of ATP5O crotonylation in CS ovaries. (v) Immunofluorescence showed that ATP5O-K51 cr level was significantly lower in CS ovaries than in control. ATP5O-K51 cr in green, DNA in blue. (w) ATP5O mRNA level did not change in CS ovaries. (x, y) Western blot by gross ATP5O antibody showed that ATP5O level was also significantly lower in CS ovaries than in control. (z) Immunofluorescence showed that gross ATP5O level was significantly lower in CS ovaries than in control. ATP5O in green, DNA in blue. Tubulin was used as a loading control. * Indicates p < 0.05 , ** indicates p < 0.01 , *** indicates p < 0.001 .

Article Snippet: Other primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti- β -Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti- β -Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti-alpha Tubulin (Acetyl Lys40) (cat#: bsm-33235 M; Bioss, Beijing, China); Rabbit anti-Transferrin polyclonal antibody (Cat#: 17435-1-ap; Proteintech, Chicago, USA); Rabbit anti-ATP5O polyclonal antibody (Cat#: D126152; BBI Life science, Shanghai, China); Rabbit anti-ATP5A1 polyclonal antibody (Cat#: D154243; BBI Life science, Shanghai, China); Rabbit anti-ATPB polyclonal antibody (Cat#: A5286; Selleckchem, Shanghai, China); Mouse anti-COX4L1 monoclonal antibody (Cat#: D190618; BBI Life science, Shanghai, China); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Rabbit anti-Phosphpho RPS6 (Ser235/236, Cat#: D155178; BBI Life science, Shanghai, China); Rabbit anti-STAT5A polyclonal antibody (Cat#: D220085; BBI Life science, Shanghai, China); Rabbit anti-FAM126A polyclonal antibody (Cat#: bs-11554R; Bioss, Beijing, China); Rabbit anti-PTDSS1 polyclonal antibody (Cat#: BS-19583R; Bioss, Beijing, China); Rabbit anti-HDAC2 polyclonal antibody (Cat#: 12922-3-ap, Proteintech, Chicago, USA); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); Mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science, Shanghai, China); Rabbit anti-HDAC1-S421 p polyclonal antibody (Cat#: AP59578, Abcepta, San Diego, CA, USA); Rabbit anti-Ubinuclein polyclonal antibody (Cat#: DF8873, Affinity Biosciences, Melbourne, Australia); Fibrinogen alpha chain (FGA) Rabbit pAb (Cat#: A1453, ABclonal, Wuhan, Hubei, China); Albumin Polyclonal antibody (Cat#: 16475-1-AP; Proteintech, Rosemont, IL, USA); AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson, Cat#: 115-005-003, West Grove, PA, USA); Rabbit anti-ATP5O-K51 cr rabbit polyclonal antibody was made against ASK (crotonyl) EKKLDQVEKELLC by YuBo BioTech and purified by affinity purification; Rabbit anti-HDAC2-S424 p ,and anti-KAT7-S102 p polyclonal antibody was made against SDS(phospho)EDEGEGGRRC and CPLRQTRSSGS (phospho) ET by Zoonbio BioTech and purified by affinity purification.

Techniques: Immunofluorescence, Generated, Dot Blot, Membrane, Western Blot

Journal: Research

Article Title: ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress

doi: 10.34133/2022/9834963

Figure Lengend Snippet: Lipid metabolism was downregulated in the plasma of CS mice. (a). Liver ATP level was significantly lowers in CS group than in control. (b). Full-spectrum metabolome was performed on the female mouse serum of CS and control groups, six repeats per group. PCA (principal component analysis) plot showed that the metabolites in CS group were fairly distinct from control group. (c, d) Volcano Plot and Heat map showed that at a threshold of CS/control ≥ 1.5 or ≤ 0.67 , there were 435 downregulated and 189 upregulated metabolites. (e) Pie charts showed differential metabolite species, 68.91% (430/624) of the differential metabolites were lipids. Among the differential lipid metabolites, the first three categories are TG (triglycerides); PC & PE & PI (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol); and LPC & LPE (lysophosphatidylcholine, lysophosphatidylethanolamine). (f) Representative differentially downregulated PC and PE, which were beneficial for physical health, and representative differentially upregulated LPC and LPE, which were detrimental for physical health, are shown. (g–l). Western blot showed that among three lipid-metabolism-related enzymes, including STAT5A, FAM126A, and PTDSS1, STAT5A significantly decreased in the CS hypothalamus (Hth) (g, h), livers (i, j), and ovaries (k, l), whereas PTDSS1 and FAM126A significantly decreased only in CS ovaries. (m–o) Switching the 293 T cell culture media from high- to low-glucose DMEM significantly decreased cytoplasmic ATP level (m) as well as STAT5A level (n, o). (p–x) Blood biochemical index showed that multiple indexes, including TG (triglyceride), HDL-C (high-density lipoprotein cholesterol), LDL-C (low-density lipoprotein cholesterol), ALT (alanine aminotransferase), AST (aspartate aminotransferase), CREA (creatinine), TP (total protein), ALP (alkaline phosphatase), and UA (Uric Acid), were significantly different between CS and control groups. (y) Blood biochemical index assay showed that a month past CS treatment, LDL-C level in CS group was still significantly higher than control. Tubulin was used as a loading control. * indicates p < 0.05 , ** indicates p < 0.01 , *** indicates p < 0.001 , **** indicates p < 0.0001 .

Article Snippet: Other primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti- β -Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti- β -Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti-alpha Tubulin (Acetyl Lys40) (cat#: bsm-33235 M; Bioss, Beijing, China); Rabbit anti-Transferrin polyclonal antibody (Cat#: 17435-1-ap; Proteintech, Chicago, USA); Rabbit anti-ATP5O polyclonal antibody (Cat#: D126152; BBI Life science, Shanghai, China); Rabbit anti-ATP5A1 polyclonal antibody (Cat#: D154243; BBI Life science, Shanghai, China); Rabbit anti-ATPB polyclonal antibody (Cat#: A5286; Selleckchem, Shanghai, China); Mouse anti-COX4L1 monoclonal antibody (Cat#: D190618; BBI Life science, Shanghai, China); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Rabbit anti-Phosphpho RPS6 (Ser235/236, Cat#: D155178; BBI Life science, Shanghai, China); Rabbit anti-STAT5A polyclonal antibody (Cat#: D220085; BBI Life science, Shanghai, China); Rabbit anti-FAM126A polyclonal antibody (Cat#: bs-11554R; Bioss, Beijing, China); Rabbit anti-PTDSS1 polyclonal antibody (Cat#: BS-19583R; Bioss, Beijing, China); Rabbit anti-HDAC2 polyclonal antibody (Cat#: 12922-3-ap, Proteintech, Chicago, USA); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); Mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science, Shanghai, China); Rabbit anti-HDAC1-S421 p polyclonal antibody (Cat#: AP59578, Abcepta, San Diego, CA, USA); Rabbit anti-Ubinuclein polyclonal antibody (Cat#: DF8873, Affinity Biosciences, Melbourne, Australia); Fibrinogen alpha chain (FGA) Rabbit pAb (Cat#: A1453, ABclonal, Wuhan, Hubei, China); Albumin Polyclonal antibody (Cat#: 16475-1-AP; Proteintech, Rosemont, IL, USA); AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson, Cat#: 115-005-003, West Grove, PA, USA); Rabbit anti-ATP5O-K51 cr rabbit polyclonal antibody was made against ASK (crotonyl) EKKLDQVEKELLC by YuBo BioTech and purified by affinity purification; Rabbit anti-HDAC2-S424 p ,and anti-KAT7-S102 p polyclonal antibody was made against SDS(phospho)EDEGEGGRRC and CPLRQTRSSGS (phospho) ET by Zoonbio BioTech and purified by affinity purification.

Techniques: Western Blot, Cell Culture

Journal: Research

Article Title: ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress

doi: 10.34133/2022/9834963

Figure Lengend Snippet: Decreased ATP5O-K51 crotonylation conduced to reduced ATP5O stability and decreased lipid metabolism. (a–c) Transfection of ATP5O-WT or K51A (inactive mutant) plasmid (in pcDNA3.1) into 293 T cells and western blot by ATP5O-K51 cr antibody showed that ATP5O-K51A had significantly decreased crotonylation level (a, b), accordingly, ATP5O K51A-transfected 293 T cells had significantly lower cytoplasmic ATP level than ATP5O-WT-transfected 293 T cells. (d–g) Western blot showed that inhibition of ATP5O crotonylation with a competitive peptide TAC (CYGRKKRRQRRRATALYSAASKEKKL) significantly reduced ATP5O-K51 crotonylation (d, e) and gross ATP5O (f, g) levels, similar to CS treatment. (h, i). A region of Ubinuclein (UBN) shared the highest similarity with TAC peptide (Supp. Figure ); however, TAC treatment did not change UBN level at all. (j, k) Western blot showed that inhibition of ATP5O crotonylation with TAC significantly decreased STAT5A protein level. (l) TAC treatment significantly reduced liver ATP level. (m–o) Quantitative lipidome showed that at the threshold of ATP 5 O − TAC / Ctr ≥ 1.5 or ≤ 0.67 , there were 174 differentially upregulated or downregulated lipid metabolites (DULMs or DDLMs), among this, 22.41% (39/174) was phospholipid (PE & PC & PI), and 82.05% (32/39) of phospholipid was downregulated. (p) Representative downregulated phospholipid in TAC-treated group. (q–s) Blood biochemical index assay showed that multiple indexes in TAC group, including CREA, SOD, and HDL-C, were similar to CS group and significantly different from control group. Tubulin or actin was used as a loading control. * indicates p < 0.05 , ** indicates p < 0.01 , *** indicates p < 0.001 , **** indicates p < 0.0001 .

Article Snippet: Other primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti- β -Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti- β -Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti-alpha Tubulin (Acetyl Lys40) (cat#: bsm-33235 M; Bioss, Beijing, China); Rabbit anti-Transferrin polyclonal antibody (Cat#: 17435-1-ap; Proteintech, Chicago, USA); Rabbit anti-ATP5O polyclonal antibody (Cat#: D126152; BBI Life science, Shanghai, China); Rabbit anti-ATP5A1 polyclonal antibody (Cat#: D154243; BBI Life science, Shanghai, China); Rabbit anti-ATPB polyclonal antibody (Cat#: A5286; Selleckchem, Shanghai, China); Mouse anti-COX4L1 monoclonal antibody (Cat#: D190618; BBI Life science, Shanghai, China); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Rabbit anti-Phosphpho RPS6 (Ser235/236, Cat#: D155178; BBI Life science, Shanghai, China); Rabbit anti-STAT5A polyclonal antibody (Cat#: D220085; BBI Life science, Shanghai, China); Rabbit anti-FAM126A polyclonal antibody (Cat#: bs-11554R; Bioss, Beijing, China); Rabbit anti-PTDSS1 polyclonal antibody (Cat#: BS-19583R; Bioss, Beijing, China); Rabbit anti-HDAC2 polyclonal antibody (Cat#: 12922-3-ap, Proteintech, Chicago, USA); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); Mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science, Shanghai, China); Rabbit anti-HDAC1-S421 p polyclonal antibody (Cat#: AP59578, Abcepta, San Diego, CA, USA); Rabbit anti-Ubinuclein polyclonal antibody (Cat#: DF8873, Affinity Biosciences, Melbourne, Australia); Fibrinogen alpha chain (FGA) Rabbit pAb (Cat#: A1453, ABclonal, Wuhan, Hubei, China); Albumin Polyclonal antibody (Cat#: 16475-1-AP; Proteintech, Rosemont, IL, USA); AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson, Cat#: 115-005-003, West Grove, PA, USA); Rabbit anti-ATP5O-K51 cr rabbit polyclonal antibody was made against ASK (crotonyl) EKKLDQVEKELLC by YuBo BioTech and purified by affinity purification; Rabbit anti-HDAC2-S424 p ,and anti-KAT7-S102 p polyclonal antibody was made against SDS(phospho)EDEGEGGRRC and CPLRQTRSSGS (phospho) ET by Zoonbio BioTech and purified by affinity purification.

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Western Blot, Inhibition

Journal: Research

Article Title: ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress

doi: 10.34133/2022/9834963

Figure Lengend Snippet: Correcting HDAC2 phosphorylation rescued abnormal lipid metabolism. (a, b) Western blot showed that inhibition of HDAC2 activity (phosphorylation) by a peptide THP (CYGRKKRRQRRRSDSEDEGEGGRR) significantly reduced the level of p-HDAC2, whereas ATP5O crotonylation and gross ATP5O levels significantly increased. (c, d) ATP levels in CS ovaries (c) and livers (d) were significantly lower than control, whereas THP treatment significantly recovers the decreased ATP levels close to control. (e) Mating test showed that the first litter size was significantly lower than control, while THP injection recovered the decreased litter size close to control. (f–h) Lipid metabolomes were analyzed for the female mouse serum of control, CS, and CS + THP group; six repeats per group. PCA plot showed that the metabolites in the CS group are completely separate from control group, with the CS + THP group partially overlapping with the control group (f). Heat map showed that at a threshold of CS + THP vs. CS ≥ 1.5 or ≤ 0.67 , there were 91 differentially upregulated or downregulated lipid metabolites (DULMs or DDLMs) that were recovered close to the control (g). And the beneficial lipid form—PE, PA, and PC—covered the second largest percentage (30 of 91, 32.97%) (g, h). (i) Representative differentially downregulated PEs, which were beneficial for physical health, are shown. (j, k) Western blot showed that STAT5A level significantly decreased in CS group, whereas THP treatment significantly recovered the STAT5A level of CS group close to control group. Tubulin was used as a loading control. * indicates p < 0.05 , ** indicates p < 0.01 , *** indicates p < 0.001 , **** indicates p < 0.0001 .

Article Snippet: Other primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti- β -Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti- β -Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti-alpha Tubulin (Acetyl Lys40) (cat#: bsm-33235 M; Bioss, Beijing, China); Rabbit anti-Transferrin polyclonal antibody (Cat#: 17435-1-ap; Proteintech, Chicago, USA); Rabbit anti-ATP5O polyclonal antibody (Cat#: D126152; BBI Life science, Shanghai, China); Rabbit anti-ATP5A1 polyclonal antibody (Cat#: D154243; BBI Life science, Shanghai, China); Rabbit anti-ATPB polyclonal antibody (Cat#: A5286; Selleckchem, Shanghai, China); Mouse anti-COX4L1 monoclonal antibody (Cat#: D190618; BBI Life science, Shanghai, China); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Rabbit anti-Phosphpho RPS6 (Ser235/236, Cat#: D155178; BBI Life science, Shanghai, China); Rabbit anti-STAT5A polyclonal antibody (Cat#: D220085; BBI Life science, Shanghai, China); Rabbit anti-FAM126A polyclonal antibody (Cat#: bs-11554R; Bioss, Beijing, China); Rabbit anti-PTDSS1 polyclonal antibody (Cat#: BS-19583R; Bioss, Beijing, China); Rabbit anti-HDAC2 polyclonal antibody (Cat#: 12922-3-ap, Proteintech, Chicago, USA); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); Mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science, Shanghai, China); Rabbit anti-HDAC1-S421 p polyclonal antibody (Cat#: AP59578, Abcepta, San Diego, CA, USA); Rabbit anti-Ubinuclein polyclonal antibody (Cat#: DF8873, Affinity Biosciences, Melbourne, Australia); Fibrinogen alpha chain (FGA) Rabbit pAb (Cat#: A1453, ABclonal, Wuhan, Hubei, China); Albumin Polyclonal antibody (Cat#: 16475-1-AP; Proteintech, Rosemont, IL, USA); AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson, Cat#: 115-005-003, West Grove, PA, USA); Rabbit anti-ATP5O-K51 cr rabbit polyclonal antibody was made against ASK (crotonyl) EKKLDQVEKELLC by YuBo BioTech and purified by affinity purification; Rabbit anti-HDAC2-S424 p ,and anti-KAT7-S102 p polyclonal antibody was made against SDS(phospho)EDEGEGGRRC and CPLRQTRSSGS (phospho) ET by Zoonbio BioTech and purified by affinity purification.

Techniques: Western Blot, Inhibition, Activity Assay, Injection

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Primary and secondary antisera used for Western blot, IFL, and IHC.

Article Snippet: α-Tubulin mouse monoclonal IgG (E-AB-20036, Elabscience) , 1:5000.

Techniques: Western Blot, Plasmid Preparation

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Modulation of Kiss1R, Kiss1, Cyp19, GnRH , and miR expression in mediobasal hypothalamus homogenates. Representative Western blots ( n = 4) for Kiss1R, Kiss1, and Cyp19 (A) and normalization of Kiss1R (B) , Kiss1 (C) , and Cyp19 signals (D) carried out against α-tubulin. Quantitative expression ( n = 6) of GnRH (E) and miR-132-3p , Mir-145-5p , let-7a-5p , and let-7b-5p (F–I) carried out by qPCR. Data are expressed as protein OD/tubulin OD ± standard deviation or normalized fold expression (n.f.e.) ± SEM against the housekeeping genes HPRT / β-actin / U6 and the control group used as reference sample. C, control group (placebo injected animals); KP, animals injected with Kp10; AEA, animals injected with AEA; SR + AEA (SA), animals first received SR141716A and 30 min later received AEA treatment. ** p < 0.01, * p < 0.05 vs. the control group.

Article Snippet: α-Tubulin mouse monoclonal IgG (E-AB-20036, Elabscience) , 1:5000.

Techniques: Expressing, Western Blot, Standard Deviation, Control, Injection

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Modulation of the intratesticular markers of steroidogenesis and GnRH . Representative Western blots for 3βHsd and Cyp19 (A) and normalization of 3βHsd (B) and Cyp19 (C) signals carried out against α-tubulin. Quantitative expression of 3βHsd , Cyp19 , and GnRH mRNA carried out by qPCR (D) . Data are representative of n = 6 different animals and are expressed as protein OD/tubulin OD ± standard deviation or normalized fold expression (n.f.e.) ± SEM against the housekeeping genes HPRT / β-actin and the control group used as a reference sample. C, control group (placebo-injected animals); KP, animals injected with Kp10; * p < 0.05.

Article Snippet: α-Tubulin mouse monoclonal IgG (E-AB-20036, Elabscience) , 1:5000.

Techniques: Western Blot, Expressing, Standard Deviation, Control, Injection

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Testicular markers of proliferation, damage, and puberty onset. Representative Western blots ( n = 6) for PCNA, Lin28a, and Sirt1 (A) and normalization of PCNA (B) Lin28A (C) , and Sirt1 signals (D) carried out against α-tubulin. qPCR ( n = 4) for hsa-miR145-5p , has-miR-132-3p , has-let7a-5p , and has-let7b-5p (E) . Representative images of testicular morphology by H&E staining, ×10 magnification ( n = 4) (F) . Data are expressed as protein OD/tubulin OD ± standard deviation in Western blot and as normalized fold expression (n.f.e.) ± standard deviation against the housekeeping gene U6 in qPCR (reference sample arbitrarily assigned 1 to the control group). C, control group (placebo-injected animals); KP, animals injected with Kp10; AEA, animals injected with AEA; SR + AEA, animals first received SR141716A and 30 min later received AEA treatment. * p < 0.05; ** p < 0.01.

Article Snippet: α-Tubulin mouse monoclonal IgG (E-AB-20036, Elabscience) , 1:5000.

Techniques: Western Blot, Staining, Standard Deviation, Expressing, Control, Injection

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Modulation of the KS by KP (A–C) and AEA (D–F) in rat testis. Representative Western blots for Kiss1R and Kiss1 (A, D) and normalization of Kiss1R (B, E) and Kiss1 signals (C, F) carried out against α-tubulin. N = 6 different animals in KP treatments and n = 4 in AEA treatments; data are expressed as protein OD/tubulin OD ± standard deviation. C, control group (placebo-injected animals); KP, animals injected with Kp10; AEA, animals injected with AEA; SR + AEA, animals first received SR141716A and 30 min later received AEA treatment. * p < 0.05.

Article Snippet: α-Tubulin mouse monoclonal IgG (E-AB-20036, Elabscience) , 1:5000.

Techniques: Western Blot, Standard Deviation, Control, Injection

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Modulation of the ECS by KP (A–D) and AEA (E–G) in rat testis. Representative Western blots and normalization of the signals carried out against α-tubulin. n = 6 different animals in KP treatments and n = 4 in AEA treatments. Data are expressed as protein OD/tubulin OD ± standard deviation. C, control group (placebo-injected animals); KP, animals injected with Kp10; AEA, animals injected with AEA; SR + AEA, animals first received SR141716A and 30 min later received AEA treatment. * p < 0.05.

Article Snippet: α-Tubulin mouse monoclonal IgG (E-AB-20036, Elabscience) , 1:5000.

Techniques: Western Blot, Standard Deviation, Control, Injection

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: CPn0572 associates with the interphase and mitotic MT cytoskeleton. (A) Schematic representation of the Ctr TarP protein and full-length Cpn CPn0572 protein. Previously identified domains are shown: phosphorylation domain (phos), dark gray boxes; proline-rich domain (PRD), orange box; G-actin binding domain (ABD), light blue box; F-actin-binding domain (FAB), dark blue box; and vinculin-binding site (VBS), white box ( ; ). (B) Representative confocal images of HEp-2 cells expressing GFP-CPn0572 (green) for 24 h prior to fixation. Actin was visualized with Rhodamine–phalloidin staining (red) and MTs with anti-α-tubulin antibody. As secondary antibody anti-mouse-IgG conjugated to Alexa Fluor 647 was used (magenta). (C) Merged images of indicated single channels shown in (B). White boxes show enlargements. (D) Representative confocal images of HEp-2 cells transfected with plasmid expressing GFP–CPn0572 (green) for 24 h, prior to fixation. Shown is an exclusive predominant association of GFP–CPn0572 with actin. Actin was visualized with Rhodamine-phalloidin staining (red). (E) Shown is a predominant association of GFP–CPn0572 with interphase MTs visualized with anti-α-tubulin antibody (red). DAPI was used to visualize DNA (blue). (F) Quantification of HEp-2 cells transfected with a GFP–CPn0572 plasmid 24 h prior to fixation. Exclusive colocalization of GFP–CPn0572 with actin was scored in 81% (black) cells, whereas in 11% cells analyzed GFP–CPn0572 localized to both MTs and actin structures (dotted). In 7.7% cells, an exclusive GFP–CPn0572 localization to MT structures was observed (striped). Data represent a mean of three experiments, n =100 cells/experiment. (G) Diagrammatic representation of CPn0572 variants. Quantification of cells expressing GFP–CPn0572 variants and association with the MT cytoskeleton as a percentage (%) of cells counted. Association with the actin cytoskeleton is depicted as+(actin association) or−(no actin association). When no actin association was observed (four bottom CPn0572 variants), CPn0572 was either associated with MT structures (percentage given) or showed a non-specific cytoplasmic staining. * marks deletion variant with MT and actin phenotypes differing from the other protein variants (visualized in Fig. S1 , dot-like and curved-fiber phenotypes concentrated in proximity to the nucleus). (H) Schematic representation of full-length CPn0572 including the newly defined MT-binding region from aa 595–755 (purple box). (I) Confocal images of U2OS cells transfected with GFP–CPn0572 595-755 plasmid for 18 h. Mitotic metaphase is shown diagrammatically on the left. MTs were visualized by anti-α-tubulin antibody staining (red) and DNA with DAPI (blue). (J) Representative confocal image of mitotic U2OS cell expressing full length GFP-CPn0572 (green) full length for 18 h. MTs were visualized by using anti-α-tubulin antibody (magenta), actin with rhodamine-phalloidin staining (red) and DNA with DAPI. In A and G, the numbers indicate amino acid positions. For B–E, I and J images shown are representative of three or more repeats. Scale bars: 10 µm.

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Phospho-proteomics, Binding Assay, Expressing, Staining, Transfection, Plasmid Preparation, Variant Assay

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: Cpn TarP proteins from human and animal isolates can associate with the MT cytoskeleton. (A) Phylogenetic tree of members of the TarP protein family from different chlamydial species with the corresponding isolate indicated. Asterisks mark the TarP members selected for transfection experiment show in C. Phylogenetics were undertaken with Clustal Omega. (B) Schematic illustration of TarP family members and the C-terminus (blue) used for generation of GFP-fusion variants. (C) Representative confocal fluorescence images of HEp-2 cells expressing GFP-tagged versions of C-terminal fragments of the indicated TarP family members. Determination of colocalization with MTs is shown on the right. Cells were transfected with indicated plasmids for 18 h. MTs were visualized using anti-α-tubulin antibody (red) and DNA with DAPI (blue). White boxes show enlargements. Images shown are representative of three repeats. Scale bars: 10 µm.

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Transfection, Fluorescence, Expressing

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: Severity of alteration of MT structure correlates with time of CPn0572 595-755 expression. (A) Representative confocal fluorescence images of HEp-2 cells transfected with a plasmid encoding GFP for 18 h (top panels) or GFP–CPn0572 595-755 for the indicated times prior to fixation. MTs were visualized with anti-α-tubulin antibody (red) and DNA with DAPI (blue). Scale bars: 10 µm. (B) Western blot analysis of HEp-2 cells expressing GFP-CPn0572 595-755 for the indicated time points. Protein extracts were separated on a 10% SDS-PAGE followed by Western blot analysis. Western blot was probed with anti-GFP or anti-GAPDH antibodies. Blot shown is representative of two repeats. (C) Explanatory cartoon to show co-dependency of GFP–CPn0572 595-755 expression levels and the increase of aberrant MTs (only the 0 h and 18 h time points represent actual measurements). (D) Quantification (mean±s.e.m.) of MT phenotypes shown in A. Typical examples of MTs phenotypes are shown for (i) GFP-expressing control cells (18 h), (ii) GFP–CPn0572 595-755 -expressing cells (6 h) and (iii) GFP–CPn0572 595-755 -expressing cells (18 h). MT thickness was determined by calculating the average diameter measured at three different positions of a single MT bundle using ImageJ. Thin MTs were defined as MTs with <0.302 µm in diameter; thick MTs were defined as MTs with ≥0.302 µm in diameter. n =3 cells (30 MTs measured per condition). *** P <0.001; ** P <0.005; ns, not significant (two-tailed unpaired Student's t -test). (E) Quantification (mean±s.e.m.) of the percentage of GFP- or GFP–CPn0572 595-755 -expressing mitotic U2OS cells with spindle defects. n =3 independent experiments each representing 50 cells. *** P <0.001 (two-tailed unpaired Student's t -test). (F) Confocal images of U2OS cells transfected with a plasmid encoding GFP or GFP-CPn0572 595-755 for 18 h. Top images show a GFP-expressing mitotic cell in anaphase. Bottom images show a representative example of the main spindle defect observed in GFP–CPn0572 595-755 - expressing cells. MTs were visualized by using an α-tubulin antibody (red) and DNA was stained with DAPI (blue). Images shown are representative of three repeats. Scale bars: 10 µm.

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Expressing, Fluorescence, Transfection, Plasmid Preparation, Western Blot, SDS Page, Control, Two Tailed Test, Staining

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: CPn0572 associated with MTs during chlamydial infection impacts infection efficiency. (A) PCR and (B) western blot verification of Ctr expressing CPn0572–FLAG. For B cells infected with Ctr or Ctr transformed with plasmid encoding CPn0572–FLAG (pMH16) for 48 h were lysed and proteins were separated on 10% SDS-PAGE. For following western blot analysis anti-CPn0572 (generated in our lab), anti-FLAG and anti-DnaK antibodies were used. Images shown are representative of one (A) or two (B) repeats. (C) Confocal images of HEp-2 cells infected with Ctr or Ctr transformed with plasmid encoding CPn0572–FLAG for 15 min. CPn0572–FLAG was visualized with anti-FLAG antibody (green) and DNA with DAPI (blue). (D) Quantification (mean±s.e.m.) of inclusion size [µm 2 ] 24 hpi of Ctr or Ctr transformed with plasmid encoding CPn0572–FLAG. n =2, each representing 30 cells. * P <0.05 (two-tailed unpaired Student's t -test). (E) Representative images from three independent experiments showing single focal planes of HEp-2 cells infected with Ctr or Ctr transformed with plasmid encoding CPn0572–FLAG (MOI=0.5, 48 hpi). Cells were treated with 0.5% Triton X-100 for 30 s and fixed with 0.5% glutaraldehyde. Images show a focal plane between the inclusion and the plasma membrane. (F) Quantification (mean±s.e.m.) of the diameter of MTs measured at the inclusion or distal of the inclusion in cells infected with Ctr - or CPn0572-expressing Ctr . MT diameter was measured by measuring the diameter at three different positions of one MT fiber using ImageJ. Data represent measurements of MT diameter for 10 MTs ( n =4 cells). *** P <0.001; ns, not significant (two-tailed unpaired Student's t -test). (G) Representative images from three repeats of Caco-2 cells infected with Ctr transformed with plasmid encoding CPn0572–FLAG for 48 h. For E and G, CPn0572–FLAG was visualized with anti-FLAG (green), MTs with anti-α-tubulin antibody (red) and eukaryotic and chlamydial DNA with DAPI (blue). For C, E and G, white boxes show enlargements (zoom). Scale bars: 10 µm.

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Infection, Western Blot, Expressing, Transformation Assay, Plasmid Preparation, SDS Page, Generated, Two Tailed Test, Clinical Proteomics, Membrane

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: CPn0572 595-755 protects MTs against cold-induced MT depolymerization. (A,B) Representative confocal images of HEp-2 cells transfected with plasmids encoding GFP (A) or GFP–CPn0572 595-755 (B) for 18 h. MTs were wild-type (after incubation at 37°C) or depolymerized through incubation on ice for 15 or 60 min and then visualized by anti-α-tubulin antibody (red) and DAPI to stain DNA (blue). Boxed regions correspond to the enlarged images shown on the right. Scale bars: 10 µm. (C) Confocal images of HEp-2 cells transfected with a plasmid encoding GFP or GFP–CPn0572 595-755 for 18 h. MTs were visualized with an anti-acetylated-α-tubulin antibody (red) and DNA with DAPI (blue). Images shown in A–C are representative of three or more repeats. Scale bars: 10 µm. (D) A typical example of a Western blot analysis of HEp-2 cells expressing GFP or GFP-CPn0572 595-755 for 18 h. Protein extracts were separated on a 10% SDS-PAGE followed by western blot analysis. Western blot was probed with anti-GFP, anti-acetylated-α-tubulin, anti-α-tubulin or anti-GAPDH antibodies. (E) Quantification of relative acetylated-α-tubulin levels shown in D. Band intensities from Western blots of acetylated-α-tubulin in cells expressing GFP–CPn0572 595-755 were determined and compared relative to control cells (GFP alone=1). ImageJ 1.47v was used for band intensity quantification. Error bars denote ±s.e.m., n =3 independent experiments. * P <0.05 (two-tailed unpaired Student's t -test).

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Transfection, Incubation, Staining, Plasmid Preparation, Western Blot, Expressing, SDS Page, Control, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins

doi: 10.1083/jcb.201907183

Figure Lengend Snippet: FIT2 is required in the maintenance of ER homeostasis. (A) Sequence alignment of FIT2. The mutation site in fitm-2(erm002) is highlighted in red. The black box outlines the conserved SGH sequence containing the predicted active residue His. (B) Colocalization of FITM-2::RFP and ER marker GFP::TRAM-1 in the hypodermis. 3D-SIM images. Scale bar, 10 µm. (C) Depiction of fitm-2(av41) . Red dots show two stop codons on the N-terminus of FITM-2. (D) Representative images of fitm-2(av41 ) animals at the young adult stage carrying the qxIs439 (Phyp7::gfp::tram-1) transgene. The ER of WT animals shown in B was used for comparison. 3D-SIM images. Scale bar, 10 µm. (E) ER morphology of early embryos and oocytes in fitm-2(av41) animals carrying the ojIs23 (Ppie-1::gfp::sp12) transgene. Single confocal planes. Scale bar, 10 µm. (F) Relative mRNA level of FIT2 in control and FIT2-depleted COS-7 cells, indicating the knockdown efficiency. (G) ER morphology in control and FIT2-depleted COS-7 cells. Cells were transfected with control or FIT2 siRNA for 48 h, fixed, and stained with anti-calreticulin and anti–Climp-63 antibodies. Scale bar, 10 µm. (H) Representative images of wild-type and fitm-2 mutants at the young adult stage carrying the zcls4 (Phsp-4::gfp) transgene. Images were acquired under the same excitation light intensity. Scale bar, 100 µm. (I) Expression level of PDI and BIP in control and FIT2-knockdown COS-7 cells. (J) ER morphology in control and FIT2-HA–overexpressing COS-7 cells. Scale bar, 10 µm. (K) Bubble-like structures in COS-7, U2OS, and HEK293T cells overexpressing FIT2-HA. Single confocal planes. Scale bars, 10 µm. (L) COS-7 cells were overexpressed with FIT2-HA and stained with anti-HA antibody, as well as Oil Red. Arrows indicate bubble-like structures stained by Oil Red. Scale bar, 10 µm. (M) COS-7 cells were overexpressed with FIT2-HA and stained with anti-HA and anti-calreticulin antibodies. The insets show high-magnification details in the white squares. Scale bars: 10 µm (main panel) or 2 µm (inset). (N) Immunofluorescence assay using phalloidin (Dylight 405; Thermo Fisher Scientific) and antibodies targeting HA, septin 2, and α-tubulin in COS-7 cells transfected with FIT2-HA. Scale bar, 10 µm; 2 µm (inset). BIP, binding immunoglobulin protein; H. sapiens , Homo sapiens ; PDI, protein disulfide isomerase; siControl, small interfering control; siFIT2, small interfering FIT2.

Article Snippet: The primary antibodies used are mouse–anti-HA (ORIGENE; TA180128), rabbit–anti-HA (Abcam), rabbit–anti-Myc (Sigma; C3956), mouse–anti-Flag (Sigma), rabbit–anti-calreticulin (Abcam; ab2907), mouse–anti–Climp-63 (Enzo; ENZ-ABS669-0100), rabbit–anti–septin 2 (Abcam; ab187654), and mouse–anti–α-tubulin (Abcam; ab7291).

Techniques: Sequencing, Mutagenesis, Residue, Marker, Comparison, Control, Knockdown, Transfection, Staining, Expressing, Immunofluorescence, Binding Assay

Journal: Cells

Article Title: New Pieces in the Puzzle of uPAR Role in Cell Migration Mechanisms

doi: 10.3390/cells9122531

Figure Lengend Snippet: uPAR interactions with FPR1 and β1 integrins are involved in migration of prostate carcinoma PC3 cells. PC3 cells were lysed and 50 μg of cell extract was analyzed by Western blot with indicated antibodies; membranes were reprobed with an anti-tubulin mouse antibody for loading control ( A ). PC3 cells were pre-incubated with diluent (−) or 5 nM W Peptide (W Pep) or 50 µM P25 ( B ) or with 5 µg/mL nonimmune Ig (NI) or an antibody directed to 84–95 uPAR residues ( C ) or with diluent (−) or 10 µM Rho kinase inhibitor or 20 µM Rac1 inhibitor ( D ). Cells were then plated in Boyden chambers and allowed to migrate towards 10% fetal bovine serum (FBS). Migrated cells were fixed, stained with hematoxylin, and counted (( B – D ) left panels). Results of migration assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (( B – D ) right panels). The values are the mean + SD of three experiments performed in triplicate. (*) p < 0.05, as determined by the Student’s t -test.

Article Snippet: The mAb anti-tubulin antibody (bsm-50179M) was from Bioss antibodies (Woburn, MA, USA).

Techniques: Migration, Western Blot, Incubation, Staining

Journal: Cells

Article Title: New Pieces in the Puzzle of uPAR Role in Cell Migration Mechanisms

doi: 10.3390/cells9122531

Figure Lengend Snippet: Removal of uPAR GPI-anchor impairs uPAR-controlled mechanism of cell migration. PC3 ( A ) and uPAR-293 ( B ) cells were treated with 1 U/mL of phosphatidylinositol-specific phospholipase C (PI-PLC) or with diluent. Cells were then plated in Boyden chambers and allowed to migrate towards 10% fetal bovine serum (FBS). Migrated cells were fixed, stained with hematoxylin, and counted (left graphs). Results of migration assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (right graphs). The values are the mean ± SD of three experiments performed in triplicate. (*) p ≤ 0.05, as determined by the Student’s t -test. PC3 and uPAR-293 cells treated with or without PI-PLC were also lysed and analyzed by Western blot with an anti-uPAR monoclonal antibody to assess PI-PLC effect; membranes were then reprobed with anti-tubulin or anti-GAPDH rabbit antibodies for loading control (( A , B ), right panels).

Article Snippet: The mAb anti-tubulin antibody (bsm-50179M) was from Bioss antibodies (Woburn, MA, USA).

Techniques: Migration, Staining, Western Blot

Journal: Cells

Article Title: New Pieces in the Puzzle of uPAR Role in Cell Migration Mechanisms

doi: 10.3390/cells9122531

Figure Lengend Snippet: Disruption of lipid rafts impairs uPAR-controlled cell migration. PC3 ( A ) or uPAR-293 ( B ) cells were treated with or without 10 mM methyl-beta-cyclodextrin (MCD). Cells were then plated in Boyden chambers and allowed to migrate towards 10% fetal bovine serum (FBS). MCD treated or untreated uPAR-293 cells were also incubated with 5 μg/mL polyclonal anti-uPAR antibody or nonimmune Ig (NI) before migration ( C ). Migrated cells were fixed, stained with hematoxylin, and counted (left panels). Results of migration assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (right panels). The values are the mean + SD of three experiments performed in triplicate. (*) p < 0.05, as determined by the Student’s t -test. PC3 and uPAR-293 cells treated with or without MCD were also lysed in buffer containing 1% Triton X-100. Cell lysates (1 mg) were loaded onto a sucrose density gradient and subjected to ultracentrifugation. Then, 4.5 mL were harvested as 0.45 mL fractions. Equal volumes of each fraction were analyzed by Western blot with an anti-uPAR monoclonal antibody, or with anti-tubulin or anti-caveolin antibodies, as controls of non-raft associated proteins or raft associated proteins, respectively ( D ).

Article Snippet: The mAb anti-tubulin antibody (bsm-50179M) was from Bioss antibodies (Woburn, MA, USA).

Techniques: Migration, Incubation, Staining, Western Blot

Journal: Cells

Article Title: New Pieces in the Puzzle of uPAR Role in Cell Migration Mechanisms

doi: 10.3390/cells9122531

Figure Lengend Snippet: uPAR drives its signaling partners to lipid rafts. Cultured PC3 cells ( A ) or PC3 cells treated with 50 μM P25 or with its scrambled version (−) ( B ) were lysed in buffer containing 1% Triton X-100. Cell lysates were loaded onto a sucrose density gradient and subjected to ultracentrifugation. Then, 4.5 mL were harvested as 0.45 mL fractions; 45 μL of each fraction was analyzed by Western blot with the indicated antibodies; tubulin and caveolin were used as controls of non-raft associated proteins or raft associated proteins, respectively. ( C ) PC3 cells were pre-incubated with or without 50 μM C6 compound, then plated in Boyden chambers and allowed to migrate toward 100 ng/mL EGF Epidermal Growth Factor (EGF). Migrated cells were fixed, stained with hematoxylin, and counted (left panel). Results of migration assays are also expressed as percentage of cells migrated towards serum over the cells migrated without serum; 100% values represent cell migration in the absence of chemoattractants (right panels). The values are the mean + SD of three experiments performed in triplicate. (*) p < 0.05, as determined by the Student’s t -test. C6 treated or untreated cells were also lysed in buffer containing 1% Triton X-100, loaded onto a sucrose density gradient and subjected to ultracentrifugation. Then, 45 μL of each gradient fraction was analyzed by Western blot with the indicated antibodies; tubulin and caveolin were used as control of non-raft associated proteins and raft associated proteins, respectively ( D ).

Article Snippet: The mAb anti-tubulin antibody (bsm-50179M) was from Bioss antibodies (Woburn, MA, USA).

Techniques: Cell Culture, Western Blot, Incubation, Staining, Migration

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting SKAP2 restores sperm motility and morphology through modulating mitochondrial organization and cytoskeletal remodeling

doi: 10.1038/s41392-025-02513-3

Figure Lengend Snippet: Disruption of acrosome, nuclear, and manchette assembly in KI mouse sperm. a PAS (left) and PNA (right) staining of seminiferous tubules from WT and KI mice at different stages of spermatogenesis. Green arrowheads (PAS) and red arrowheads (PNA) indicate aberrant acrosomal morphology in KI spermatids. M: Metaphase. Scale bars, 10 μm. b Transmission electron microscopy (TEM) of sperm head development in WT and KI spermatids. Nu, nucleus; g, Golgi apparatus; av, proacrosomal vesicles; apx, acroplaxome; ac, acrosome. Red arrows indicate malformed acrosomes; blue asterisks denote abnormal nuclei. Scale bars, 1 μm. c Representative TEM images of manchette morphology during spermatid elongation in WT and KI mice. White double-headed arrows highlight the asymmetric manchette in KI spermatids. Abbreviations: Nu, nucleus; PR, perinuclear ring; M, manchette. Scale bars, 1 μm. d Immunofluorescence staining of tubulin in spermatozoa from adult WT and KI mice. Tubulin marks the manchette structure; DNA was counterstained with DAPI. Scale bars, 5 μm. e Quantification of manchette microtubule lengths from ( d ). For each group, 40 spermatozoa were randomly selected per mouse, and average values were calculated for every 10 sperms. Data are presented as mean ± SD. P -values were determined using a two-sided Student’s t -test. n = 36 per group

Article Snippet: We extend our gratitude to Bioss for the antibody of alpha Tubulin (bsm-33039M) and alpha Tubulin Antibody from MedChemExpress (Monmouth Junction, NJ, USA; Cat# HY- P86200 ).

Techniques: Disruption, Staining, Transmission Assay, Electron Microscopy, Immunofluorescence