Journal: Cell Genomics

Article Title: Multi-omics integration identifies cell-state-specific repression by PBRM1-PIAS1 cooperation

doi: 10.1016/j.xgen.2023.100471

Figure Lengend Snippet:

Article Snippet: Antibodies used for ChIP-seq include anti-PBRM1 (Bethyl, A700-019) and anti-PIAS1 (F-1) (Santa Cruz, sc-365127).

Techniques: Recombinant, SYBR Green Assay, Protease Inhibitor, In Situ, Blocking Assay, Isolation, Sequencing, shRNA, Software

Journal: Molecular Cell

Article Title: Stress-induced nuclear condensation of NELF drives transcriptional downregulation

doi: 10.1016/j.molcel.2021.01.016

Figure Lengend Snippet: NELF SUMOylation drives nuclear condensate formation (A) Fluorescence microscopy images of HeLa NELFA-GFP cells treated with vehicle or CDK9 inhibitor 5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) under no heat shock conditions (NHS). Scale bar indicates 10 μm. (B) Fluorescence microscopy images of HeLa NELFA-GFP cells exposed to heat shock (HS) and treated with vehicle or ML-792. Scale bar indicates 10 μm. (C) Percentage of cells with NELFA condensates after heat shock in vehicle or ML-792 treated samples. Data quantification of (B). Error bars represent SD ( n = 2 independent cell cultures). Asterisks denote p value <0.001 as calculated by two-tailed unpaired t test. (D) Fluorescence microscopy images of heat-shocked HeLa cells expressing NELFA-GFP. Cells were treated with siRNAs against SUMO E2 conjugating enzyme (UBC9), SUMO E3 ligases (PIAS1, PIAS4, and ZNF451), non-targeting siRNA (siNON), or left untreated. Scale bar indicates 10 μm. (E) Percentage of heat-shocked HeLa cells with NELFA condensates from experiment described in (D). Error bars represent SD ( n = 2 independent replicates). Conditions compared to siNON. Asterisks denote p value of ( ∗∗∗ , 0.001; ∗∗ , 0.01) as calculated by one-way ANOVA with Dunnett’s multiple comparison tests. (F) Western blot analyses of NELF after performing in vitro SUMOylation reactions using NELFA (top) and NELFC/D antibodies (bottom). Either immunoprecipitated full-length E3 ligase ZNF451 or a recombinant N-terminal fragment were used at two different concentrations . Reactions lacking ATP or ZNF451 serve as negative controls. (G) Western blot analysis of ZNF451 and NELFE is shown for indicated fractions from HeLa cells exposed to heat shock (HS) or no heat shock (NHS). SNRNP70 and RPB3 are used as loading controls. (H) Effect of SUMO E2 (UBC9) depletion on HS-induced changes in RNA Pol II ChIP-seq occupancy in gene body regions of top 250 expressed genes. Regions from transcription start site (TSS) +1.5 kb to transcription end site (TES) –0.5 kb are shown. p value from Wilcoxon test is indicated (n = 2 independent cell cultures). (I) Effect of SUMO E3 ligase ZNF451 depletion on HS-induced changes in nascent transcript counts as quantified by SLAM-seq in HeLa cells. Significantly downregulated genes in siNON cells as calculated by differential gene expression analysis based on the negative binomial distribution (DESeq2) are used. The red line indicates no change in expression. The violin plot depicts median, interquartile range, and 95% confidence interval with white dot, black bar, and thin black line, respectively. p value from Wilcoxon test is indicated (n = 3 independent cell cultures). See also .

Article Snippet: PIAS1 siGenome siRNA , Origene , SR305620.

Techniques: Fluorescence, Microscopy, Two Tailed Test, Expressing, Western Blot, In Vitro, Immunoprecipitation, Recombinant, ChIP-sequencing

Journal: Molecular Cell

Article Title: Stress-induced nuclear condensation of NELF drives transcriptional downregulation

doi: 10.1016/j.molcel.2021.01.016

Figure Lengend Snippet:

Article Snippet: PIAS1 siGenome siRNA , Origene , SR305620.

Techniques: Recombinant, Cell Viability Assay, Sequencing, Expressing, Software

Journal: BMC complementary medicine and therapies

Article Title: Effect and underlying mechanism of Huangjing Qianshi decoction in pre-diabetes mouse model.

doi: 10.1186/s12906-025-04893-z

Figure Lengend Snippet: Fig. 5 NR3C2 protein level in the tissues of pre-diabetic mice was reduced, whereas protein inhibitor of activated STAT1 (PIAS1) protein level was in creased after Huangjing Qianshi decoction (HJQST) and MET treatment. PIAS1 (A) and NR3C2 (B) proteins were measured using immunohistochemistry. Magnification ×100

Article Snippet: Antibodies against GLUT-4 (1:100 dilution, 66846-1-Ig), INS (1:1000 dilution, 66198-1-Ig), NR3C2 (1:2000 dilution for western blot and 1:100 dilution for IHC, 21854-1-AP), PIAS1 (1:2000 dilution for western blot and 1:100 dilution for IHC, 23395-1-AP), STAT1 (1:2000 dilution, 66545-1- Ig), p-STAT1 (1:2000 dilution, 28977-1-AP), PGC-1a (1:5000 dilution, 66369-1-Ig), and GAPDH (1:20000 dilution, 60004-1-Ig) were purchased from the Proteintech (Wuhan Sanying, Wuhan, China).

Techniques: Immunohistochemistry

Journal: BMC complementary medicine and therapies

Article Title: Effect and underlying mechanism of Huangjing Qianshi decoction in pre-diabetes mouse model.

doi: 10.1186/s12906-025-04893-z

Figure Lengend Snippet: Fig. 6 NR3C2, protein inhibitor of activated STAT1 (PIAS1), p-STAT1, and PGC-1α protein level was affected in the tissues of pre-diabetic mice after Huangjing Qianshi decoction (HJQST) and MET treatment. Proteins in the liver tissue and skeletal muscle tissues of pre-diabetic mice after Huangjing Qianshi decoction (HJQST) and MET treatment were measured using western blot. Full-length blots are presented in Supplementary Fig. 1. *p < 0.05, **p < 0.01, and ***p < 0.001

Article Snippet: Antibodies against GLUT-4 (1:100 dilution, 66846-1-Ig), INS (1:1000 dilution, 66198-1-Ig), NR3C2 (1:2000 dilution for western blot and 1:100 dilution for IHC, 21854-1-AP), PIAS1 (1:2000 dilution for western blot and 1:100 dilution for IHC, 23395-1-AP), STAT1 (1:2000 dilution, 66545-1- Ig), p-STAT1 (1:2000 dilution, 28977-1-AP), PGC-1a (1:5000 dilution, 66369-1-Ig), and GAPDH (1:20000 dilution, 60004-1-Ig) were purchased from the Proteintech (Wuhan Sanying, Wuhan, China).

Techniques: Western Blot

Journal:

Article Title: The zinc finger protein Gfi-1 can enhance STAT3 signaling by interacting with the STAT3 inhibitor PIAS3

doi: 10.1093/emboj/19.21.5845

Figure Lengend Snippet: Fig. 4. Endogenous PIAS3 and Gfi-1 proteins can be coprecipitated from human cells. (A and B) Nuclear and cytoplasmic extracts (see lanes marked N and C) were prepared from human KIT 225 T cells and immunoprecipitations (IP) were performed with anti-PIAS3c (Chung et al., 1997) or anti-PIAS1/3 (α-Pias3N) antibodies (Santa Cruz). To detect Gfi-1 (arrowheads) the precipitates were analyzed by SDS–PAGE and western blotting (We) using either the anti-Gfi-1 N20 antibody (Santa Cruz) or affinity-purified anti-Gfi-1 rabbit antibodies (Schmidt et al., 1998). (C) The expression level of endogenous PIAS3 was determined in nuclear and cytoplasmic extracts (lanes N and C) from human KIT 225 T cells, HepG2 cells and HeLa cells by western blotting using the anti-PIAS3c antibody. (D) Immunoprecipitations were performed with the nuclear extracts from (C) and the anti-PIAS3c antibody. The extracts and the precipitates were analyzed by SDS–PAGE and western blotting (We) using the anti-Gfi-1 N20 antibody. Gfi-1 could be precipitated only from KIT 225 extracts (arrowhead) but not from HepG2 or HeLa cell extracts. All data shown are representative of several independent experiments.

Article Snippet: With these extracts, we performed immunoprecipitations with the anti-PIAS3c antiserum ( Chung et al ., 1997 ) or the anti-PIAS1/3 (Santa Cruz) antibody, which recognize the C- or N-terminus, respectively, of PIAS3 (Figure A, B and D).

Techniques: SDS Page, Western Blot, Affinity Purification, Expressing

Journal: JCI insight

Article Title: The E3 ubiquitin ligase Siah1 regulates adrenal gland organization and aldosterone secretion.

doi: 10.1172/jci.insight.97128

Figure Lengend Snippet: Figure 5. Downregulation of LXR/RXR and FXR/RXR expression and cholesterol biosynthesis correlates with induction of PIAS1 SUMO E3 ligase pro- tein in the adrenal glands of Siah1a–/– mice. (A) Heatmap of RNA-seq data from the adrenal glands of female and male WT and Siah1a KO mice, showing the top canonical pathways upregulated and downregulated in Siah1a KO mice. Selection criteria were P ≤ 0.05 and ≥2-fold change in expression. (B) PIAS1 staining (red) in sections of adrenal glands from 21-day-old WT and Siah1a KO mice. Scale bar: 100 μm. Original magnification, ×3 (insets). Images shown are representative of images from 4 different mice per group. (C) Western blot analysis of PIAS1 and tubulin expression in protein lysates of pooled adrenal

Article Snippet: Antibodies to α-tubulin (Santa Cruz, catalog sc-8035), β-actin (Santa Cruz, catalog sc-47778), PIAS1 (Cell Signaling for Western blot analysis, catalog 3550; Boster Biological Technology for mouse immunofluorescence, catalog PA2252), Flag (Millipore, catalog F1804), myc (Cell Signaling, catalog 227G), DAB2 (Cell Signaling, catalog 12906), β-catenin (Abcam, catalog ab6302), MECA 32 (BD biosciences, catalog BDB550563), eNACα (Bioss antibodies, catalog bs-2957R), SGK1 (Boster biological Technology, catalog PA2184), and V5 (Invitrogen, catalog R96025) were used according to the manufacturers’ recommendations.

Techniques: Expressing, RNA Sequencing, Selection, Staining, Western Blot

Journal: JCI insight

Article Title: The E3 ubiquitin ligase Siah1 regulates adrenal gland organization and aldosterone secretion.

doi: 10.1172/jci.insight.97128

Figure Lengend Snippet: Figure 6. Reduced activity of Siah1 I251L and increased PIAS1 expression in a patient with primary aldosteronism. (A) Cartoon representation of Siah1 surrounding the I151L mutation site (yellow sticks, blue label) that lies on a “helix-turn-helix” element that connects the dimer interface to the linker between the 2 Zn fingers (indicated by dashed black double-headed arrow). Ile151 is partly buried but also forms part of a prominent pocket (“D”; brown dashed circle). Loss-of-function mutation sites are colored red, and a red circle emphasizes how they cluster across the SBD dimer interface (part of second SBD is in gray). Three crystal forms are overlaid (see Methods and Results) to demonstrate conformational hetero- geneity in the second Zn finger (ZnF-2). For clarity, ZnF-2 is represented by a single helix (each structure is labeled by its PDB code, see Methods), but the motion is simple rigid-body rotation about the axis labeled “R,” circled in orange. The RING domain is N-terminal to ZnF-2, and its structure has not been determined. (B) Western blot analysis of PIAS1 expression in 293T cells transfected with PIAS1-V5 alone or in combination with either Siah1-myc or Siah1-I251L-myc. A cycloheximide (CHX) chase was performed for the indicated times. Blots were probed with antibodies against V5 or myc. Tubulin served as a loading control. Dashed lines denote the different degradation rate of PIAS1 coexpressed with WT Siah1 versus PIAS1 coexpressed with Siah1-I251L. The graph shows quantification of PIAS1 degradation from 5 independent experiments. *P < 0.05, compared with

Article Snippet: Antibodies to α-tubulin (Santa Cruz, catalog sc-8035), β-actin (Santa Cruz, catalog sc-47778), PIAS1 (Cell Signaling for Western blot analysis, catalog 3550; Boster Biological Technology for mouse immunofluorescence, catalog PA2252), Flag (Millipore, catalog F1804), myc (Cell Signaling, catalog 227G), DAB2 (Cell Signaling, catalog 12906), β-catenin (Abcam, catalog ab6302), MECA 32 (BD biosciences, catalog BDB550563), eNACα (Bioss antibodies, catalog bs-2957R), SGK1 (Boster biological Technology, catalog PA2184), and V5 (Invitrogen, catalog R96025) were used according to the manufacturers’ recommendations.

Techniques: Activity Assay, Expressing, Mutagenesis, Labeling, Western Blot, Transfection, Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Protein S -glutathionylation stimulate adipogenesis by stabilizing C/EBPβ in 3T3L1 cells

doi: 10.1096/fj.201902575R

Figure Lengend Snippet: Regulation of C/EBPβ protein stability by S-glutathionylation. (A) Detection of S-glutathionylation on C/EBPβ. Cell lysate of control and Glrx KO 3T3L1 cells after adipocyte differentiation was immunoprecipitated with an anti-GSH antibody. Then Western blot analysis was performed by using an anti-C/EBPβ antibody. (B) Scheme of C/EBPβ regulation by SUMO E3 ligase PIAS1, SUMOlation, ubiquitination, and proteasome degradation. (C) Induction of S-glutathionylation in HEK293T cell lysate. HEK293T cell were treated with GSH/GSSG mixture to promote S-glutathionylation. Western blot was performed by using anti-GSH antibody. (D) Inhibition of PIAS1 attachment by S-glutathionylation on C/EBPβ. Western blotting analysis following Co-IP of Myc-FLAG-tagged mouse PIAS1 and GSH/GSSG mixture or vehicle treated HA-tagged mouse C/EBPβ by anti-Myc antibody-conjugated magnetic beads. Detection of HA-tagged mouse C/EBPβ and Myc-FLAG-tagged mouse PIAS1 was performed by anti-HA antibody and anti-Flag antibody, respectively. (E) Identification of a region of PIAS1 attachment in C/EBPβ. HA-tagged LIP isoform or LIP deleted full-length mutant (ΔLIP) overexpressed HEK293T cell lysate was mixed with Myc-FLAG-tagged mouse PIAS1 overexpressed HEK293T cell lysate followed by Co-IP using anti-Myc antibody-conjugated magnetic beads. Western blot analysis was performed using anti-HA antibody for LIP and ΔLIP, anti-FLAG antibody for PIAS1. (F) Detection of S-glutathionylation on Cys201 (Upper) and Cys296 (lower) by MS. S-glutathionylation of Cys201 and Cys296 were detected from trypsin fragments 197AAPACFAGPPAAPAK211 and 289QLPEPLLASAGCTR298. The actual mass of these fragments was 1,714.78 (m/z=858.3992+) and 1,896.89 (m/z=949.4532+), respectively; the MW increased by 305 Da for S-glutathionylation. (G) C201S and C296S double mutation of C/EBPβ diminished effect of S-glutathionylation on inhibition of PIAS1 attachment. HA-tagged mouse C/EBPβ, HA-tagged C201S, C296S single mutant C/EBPβ, and HA-tagged C201S and C209S double mutant C/EBPβ were transfected to HEK293T cells. These cell lysate incubated with vehicle or GSH/GSSG mixture for induction S-glutathionylation. Then, Co-IP experiment was performed using these cells lysate, and Myc and FLAG-tagged mouse PIAS1 overexpressed cell lysate. Experiments were repeated three times with similar results.

Article Snippet: Myc-FLAG-tagged mouse C/EBPβ plasmid (MR227563), Myc -FLAG-tagged mouse PIAS1 plasmid (MR209770), and C-terminal HA-tag vector (PS100004) were from Origene (Rockville, MD, USA).

Techniques: Immunoprecipitation, Western Blot, Inhibition, Co-Immunoprecipitation Assay, Magnetic Beads, Mutagenesis, Transfection, Incubation