Journal: Heliyon

Article Title: GDF11 inhibits adipogenesis of human adipose-derived stromal cells through ALK5/KLF15/β-catenin/PPARγ cascade

doi: 10.1016/j.heliyon.2023.e13088

Figure Lengend Snippet: GDF11 activated SMAD2/3 signaling pathway. (A) The effect of GDF11 GDF11 (25 ng/ml) exposure on the SMAD2/3 pathway showed by the Western blot. Uncropped images were provided in the supplementary file. (B) Statistical analysis of pSMAD2 levels. (C) Statistical analysis of pSMAD3 levels. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The following antibodies were used: anti-β-actin antibody (Cell Signaling Technology, 4970), anti-PERILIPIN antibody (Cell Signaling Technology, 9349), anti-PPARγ antibody (Cell Signaling Technology, 2435), anti-FABP4 antibody (Cell Signaling Technology, 2120), anti-CEBPα antibody (Cell Signaling Technology, 2843), anti-pAKT antibody (Cell Signaling Technology, 4060), anti-AKT antibody (Cell Signaling Technology, 9272), anti-pERK1/2 antibody (Cell Signaling Technology, 4370), anti-ERK1/2 antibody (Cell Signaling Technology, 4695), anti-pP38 antibody (Cell Signaling Technology, 4511), anti-P38 antibody (Cell Signaling Technology, 9212), anti-pJNK antibody (Cell Signaling Technology, 9255), anti-JNK antibody (Cell Signaling Technology, 9252), anti-pSMAD1/5 antibody (Cell Signaling Technology, 9516), anti-pSMAD2 antibody (Cell Signaling Technology, 18338), anti-pSMAD3 antibody (Cell Signaling Technology, 9520), anti-SMAD2/3 antibody (Cell Signaling Technology, 8685), anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, 7074) and anti-mouse IgG, HRP-linked Antibody (Cell Signaling Technology, 7076).

Techniques: Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial–mesenchymal transition of breast cancer cells

doi: 10.1007/s00018-022-04672-w

Figure Lengend Snippet: The USP19 cytosolic isoform (USP19-CY) promotes TGF-β signaling; conversely, the endoplasmic reticulum (ER)-localized USP19 isoform (USP19-ER) inhibits this TGF-β pathway. A Schematic diagram showing the depicting USP19-ER and USP19-CY isoforms with common structural domains, including a catalytic domain bearing the essential cysteine (C), aspartic acid (D) and histidine triad of amino acid residues required for catalysis, and unique C-terminal regions. The C-terminal transmembrane domain (TMD) causes the ER localization of the USP19-ER isoform. The catalytic domain also bears a putative ubiquitin-like (UBL) domain as well as a MYND Zn finger domain that is involved in protein‒protein interactions. B Immunofluorescence analysis of the localization of USP19 (red) and calnexin (green) in U2OS cells transfected with FLAG-tagged wild-type USP19-CY and USP19-ER expression plasmids. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, blue). Images were captured with confocal microscopy. Scale bar = 5 μm. C Effect of USP19-CY-wt or USP19-CY-CA on the SMAD3-dependent CAGA 12 -luciferase transcriptional response induced by TGF-β (2.5 ng/mL; overnight treatment) in HEK293T cells. The data are expressed as the mean ± SD, n = 3 (biological replicates). *** P < 0.001, based on unpaired Student’s t test. D Immunoblotting analysis of the p-SMAD2, total (t)-SMAD2, USP19-CY and total USP19 levels in MDA-MB-231 cells that were infected with empty vector (pLV-EV), wild-type USP19-CY (CY-wt) or USP19-CY enzyme inactive mutant (CY-CA) lentivirus after stimulation with vehicle control or TGF-β (2.5 ng/mL) for 1 h. GAPDH, loading control. E qRT‒PCR analysis of TGF-β target genes, i.e., CCN2 , SERPINE1 and SMAD7 , in MDA-MB-231 cells stably infected with pLV-EV, CY-wt, or CY-CA in the presence of vehicle control or TGF-β (2.5 ng/mL) for 6 h. The data are expressed as the mean ± SD, n = 3 (biological replicates). * P ≤ 0.05, ** P < 0.01, based on unpaired Student’s t test. F Western blotting analysis of the p-SMAD2, t-SMAD2 and USP19 levels in MDA-MB-231 cells with or without shRNA-mediated specific knockdown of USP19-CY (sh-CY) treated with vehicle control or TGF-β (2.5 ng/mL) for 1 h. GAPDH, loading control. G Expression levels of the TGF-β target genes, CCN2 , SERPINE1 and SMAD7 in pLKO-EV control or USP19-CY-deficient MDA-MB-231 cells treated with vehicle control or TGF-β (2.5 ng/mL) for 6 h. The data are expressed as the mean ± SD, n = 3 (biological replicates). * P ≤ 0.05, *** P < 0.001, based on unpaired Student’s t test. H Immunoblotting analysis of the p-SMAD2, t-SMAD2 and USP19 levels in MDA-MB-231 cells infected with pLV-EV, wild-type USP19-ER (ER-wt) and USP19-ER enzyme inactive mutant (ER-CS) and treated with vehicle control or TGF-β (2.5 ng/mL) for 1 h. GAPDH, loading control. I Measurement of the SMAD3-dependent CAGA 12 -luciferase transcriptional activity induced by overnight treatment with TGF-β (2.5 ng/mL) in HEK293T cells that were transfected with ER-wt or ER-CS or pLV-EV expression plasmids. The data are expressed as the mean ± SD, n = 3 (biological replicates). *** P < 0.001, based on unpaired Student’s t test. J qRT‒PCR analysis of the expression of TGF-β target genes, i.e., CCN2 , SERPINE1 and SMAD7 , in MDA-MB-231 cells stably infected with pLV-EV, ER-wt or ER-CS in the presence of vehicle control or TGF-β (2.5 ng/mL) for 6 h. The data are expressed as the mean ± SD, n = 3 (biological replicates). * P ≤ 0.05, based on unpaired Student’s t test. K Immunoblotting of the p-SMAD2, t-SMAD2 and USP19 levels in MDA-MB-231 cells with or without shRNA-mediated knockdown of USP19-ER (sh-ER) treated with vehicle control or TGF-β (2.5 ng/mL) for 1 h. GAPDH, loading control. L Expression levels of TGF-β target genes, i.e., CCN2 , SERPINE1 and SMAD7 , in PLKV-EV control or USP19-ER-deficient MDA-MB-231 cells treated with vehicle control or TGF-β (2.5 ng/mL) for 6 h. The data are expressed as the mean ± SD, n = 3 (biological replicates). * P ≤ 0.05, *** P < 0.001, based on unpaired Student’s t test

Article Snippet: The antibodies used for immunoprecipitation (IP), immunoblotting (IB), and immunofluorescence (IF) were as follows: phosphor-SMAD2 (1:1000; IB; 3108, Cell Signaling), total-SMAD2 (1:1000; IB; 3103S, Cell Signaling), USP19 (1:1000; IB; IF: ab189518, Abcam), GAPDH (1:1000; IB; MAB374, Millipore), Tubulin (1:1000; IB; 2148, Cell Signaling), E-cadherin (1:1000; IB; 610181, BD Biosciences), N-cadherin (1:1000; IB; 610920, BD Biosciences), vimentin (1:1000; IB; 5741, Cell Signaling), SNAIL (1:1000; IB; 3879, Cell Signaling), vinculin (1:1000; IB; V9131, Sigma), c-MYC (1:200; IP; sc-40, Santa Cruz), FLAG (1:1000; IB; F3165), HA (1:1000; IB; 1583816, Roche), TβRI (1:1000; IB; sc-398, Santa Cruz), calnexin (1:1000; IF; ab22595, Abcam), Alexa Fluor 555 secondary antibody (1:250 or 1:1000; IF; A-31572, Thermo), Alexa Fluor 488 secondary antibody (1:1000; IF; A-11001, Thermo).

Techniques: Immunofluorescence, Transfection, Expressing, Confocal Microscopy, Luciferase, Western Blot, Infection, Plasmid Preparation, Mutagenesis, Stable Transfection, shRNA, Activity Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial–mesenchymal transition of breast cancer cells

doi: 10.1007/s00018-022-04672-w

Figure Lengend Snippet: USP19-CY is highly expressed in breast cancer tissues, and USP19 mRNA splicing is regulated by herboxidiene. Representative images of USP19-CY (red) immunofluorescence staining in a human breast cancer tissue microarray containing 34 pairs of cancer adjacent tissues and cancer tissues ( A ) or cancer tissues of different stages (stage IIA, IIB, IIIA, IIIB) ( B ). Nuclei were counterstained with DAPI (blue). Large field and magnified pictures (outlined with a dotted square) are shown. Scale bar = 250 μm, 50 μm or 250 μm. C Quantification of the percent USP19-CY expression in pairs of breast tissues (adjacent and cancer tissues). Red lines indicate significant upregulation, and blue lines indicate downregulation of USP19-CY in cancer tissues compared to adjacent tissues; black lines indicate no significant change in USP19-CY in tissue pairs. The data are represented as the mean ± SD, tissue pairs, n = 34, ** P < 0.01, based on a paired Student’s t test. D Quantification of percent USP19-CY expression in breast cancer adjacent tissues and cancer tissues of different stages. The data are expressed as the mean ± SD, adjacent tissues, n = 10; adenocarcinoma (stage IIA), n = 49; adenocarcinoma (stage IIB), n = 22; adenocarcinoma (stage IIIA), n = 16; adenocarcinoma (stage IIIB), n = 6; * P ≤ 0.05, **** P < 0.0001, based on unpaired Student’s t test. E qRT‒PCR analysis of the expression of the USP19 , USP19-CY and USP19-ER in A549-VIM-RFP cells treated with 0.2 or 1 μM herboxidiene. The data are expressed as the mean ± SD, n = 3 (technical replicates). F MDA-MB-231 cells stably infected with pLV-EV or USP19-CY-wt were pretreated with 1 μM herboxidiene (Herbo) for 24 h and then, combined with vehicle control or TGF-β (2.5 ng/mL) for 1 h, followed by immunoblot analysis of the p-SMAD2 and t-SMAD2 expression levels. GAPDH: loading control. G HEK293T cells transfected with pRK5 or USP19-CY-wt were pretreated with 1 μM herboxidiene (Herbo) for 24 h and then, combined with vehicle control or TGF-β (2.5 ng/mL) overnight, followed by the analysis of CAGA 12 -luciferase transcriptional responses. The data were expressed as the mean ± SD, n = 3 (biological replicates). * P ≤ 0.05, ** P < 0.01, *** P < 0.001, based on unpaired Student’s t test. H A549-VIM-RFP cells stably infected with pLV-EV or USP19-CY-wt were pretreated with 1 μM herboxidiene (Herbo) for 24 h and then, treated with vehicle control or TGF-β (2.5 ng/mL) for 48 h. Then, immunoblotting analysis of the expression of the epithelial marker E-cadherin and mesenchymal markers N-cadherin, vimentin and SNAIL was performed. GAPDH: loading control. I A549-VIM-RFP cells with pLV-EV and USP19-CY-wt plasmids were pretreated with 1 μM herboxidiene for 24 h and then, incubated with vehicle control or TGF-β (2.5 ng/mL) for the indicated times. The results of the scratch assay time course were analyzed by IncuCyte. The relative wound density (closure) is presented as the mean ± SD, n = 3 (biological replicates). * P ≤ 0.05, ** P < 0.01, based on unpaired Student’ s t test

Article Snippet: The antibodies used for immunoprecipitation (IP), immunoblotting (IB), and immunofluorescence (IF) were as follows: phosphor-SMAD2 (1:1000; IB; 3108, Cell Signaling), total-SMAD2 (1:1000; IB; 3103S, Cell Signaling), USP19 (1:1000; IB; IF: ab189518, Abcam), GAPDH (1:1000; IB; MAB374, Millipore), Tubulin (1:1000; IB; 2148, Cell Signaling), E-cadherin (1:1000; IB; 610181, BD Biosciences), N-cadherin (1:1000; IB; 610920, BD Biosciences), vimentin (1:1000; IB; 5741, Cell Signaling), SNAIL (1:1000; IB; 3879, Cell Signaling), vinculin (1:1000; IB; V9131, Sigma), c-MYC (1:200; IP; sc-40, Santa Cruz), FLAG (1:1000; IB; F3165), HA (1:1000; IB; 1583816, Roche), TβRI (1:1000; IB; sc-398, Santa Cruz), calnexin (1:1000; IF; ab22595, Abcam), Alexa Fluor 555 secondary antibody (1:250 or 1:1000; IF; A-31572, Thermo), Alexa Fluor 488 secondary antibody (1:1000; IF; A-11001, Thermo).

Techniques: Immunofluorescence, Staining, Microarray, Expressing, Stable Transfection, Infection, Western Blot, Transfection, Luciferase, Marker, Incubation, Wound Healing Assay

Journal: Nature Communications

Article Title: In vivo induction of activin A-producing alveolar macrophages supports the progression of lung cell carcinoma

doi: 10.1038/s41467-022-35701-8

Figure Lengend Snippet: a Flow cytometry plots of AMs isolated from C57BL/6 wild-type (WT) mice and MyD88 knockout (KO) mice after inoculation with lung cancer cells. b Inhba RT-PCR analysis of AM isolated from tumor-bearing wild-type mice and MyD88 knockout mice ( n = 6 mice for wild-type, n = 5 mice for MyD88 KO). c Inhba RT-PCR analysis in the AM cell line AMJ2-C11 after incubation with or without LLC cell line supernatant (Sup.) and JNK inhibitor SP600125 ( n = 4 per group). d – f Immunoblotting of JNK phosphorylation in AM cell line with or without stimulation of LLC cell line supernatant after incubation with each inhibitor ( d ; TAK-242, TLR4 inhibitor, e ; ST2825, MyD88 inhibitor, f ; takinib, TAK1 inhibitor). These images were representative of three independent experiments with similar results. g , Schematic representation of Inhba signaling in AM. h Viability of LLC cells treated with the ALK4 inhibitor SB505124 and recombinant activin A (rActA) was assessed using WST-1 assay ( n = 4 per group except for the one with only rActA administration ( n = 3)). i , j Immunoblotting of ERK ( i ) and Smad2 phosphorylation ( j ) in LLC cells stimulated with rActA after incubation with the ALK4 inhibitor SB505124. These images were representative of three independent experiments with similar results. Means ± s.e.m. for each group are shown. Symbols represent individual mice ( b ) and wells ( c , h ). Statistical significance was determined using unpaired two-tailed Mann–Whitney U- test ( b ) or one-way ANOVA with Bonferroni’s post hoc test ( c , h ).

Article Snippet: Antibodies were obtained from the following sources: anti-CD45-PECy7 (1:50, clone 30-F11, #103114), anti-F4/80-BV421 (1:50, clone BM8, #123132), anti-CD11c-APC (1:50, clone N418, #117310), anti-CD11b-BV421 (1:50, clone M1/70, #101235), BV421-rat IgG2a, κ isotype control (1:50, clone RTK2758, #400535), and APC-human IgG1, κ isotype control (1:50, clone QA16A12, #403505) were from BioLegend (San Diego, CA); anti-Siglec-F-APC (1:50, clone REA798, #130-112-333), and anti-CD11b-APC (1:50, clone REA592, #130-113-802) were from Miltenyi Biotec (Bergisch Gladbach, NRW, Germany); anti-p-JNK (1:1000, #9251), anti-JNK (1:1000, #9252), anti-p-Erk1/2 (1:1000, #4370), anti-Erk1/2 (1:1000, #4695), anti-p-Smad2 (1:1000, #3108), anti-Smad2 (1:1000, #5339), anti-β-actin (1:1000, #5125), and anti-rabbit IgG, HRP-linked antibody (1:1000, #7074) were from Cell Signaling Technology (Danvers, MA); anti-Inhibin beta A (1:200, ab56057), and anti-TTF-1 (1:100, clone SP141, ab227652) were from Abcam (Cambridge, UK); anti-CD163 antibody (1:800, clone 10D6) was from Leica biosystems (Wetzlar, Germany); anti-MARCO antibody (1:500, clone 2359 A, MAB29561) was from R&D systems (Minneapolis, MN); AlexaFlour647 AffiniPure donkey anti-rabbit IgG (1:200, #711-605-152) was from Jackson Immuno Research (West Grove, PA); and anti-CD16/32 antibody (1:100, clone 2.4G2, #553141) was from BD Biosciences (San Jose, CA).

Techniques: Flow Cytometry, Isolation, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Incubation, Western Blot, Recombinant, WST-1 Assay, Two Tailed Test, MANN-WHITNEY