Journal: Nature Communications

Article Title: Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24

doi: 10.1038/s41467-022-29581-1

Figure Lengend Snippet: a AcrIF24 but not AcrIF24ΔMD inhibits the in vitro cleavage activity of the type I-F CRISPR-Cas system. Reactions were performed with 0.32 μM Csy complex, 0.16 μM Cas2/3, and 0.04 μM 54-bp dsDNA (5′-FAM in the non-target DNA strand, NTS). AcrIF24 or AcrIF24ΔMD was added with concentrations of 0.16, 0.32, and 0.64 μM following the order indicated by the black triangles. Fraction DNA cleavage was calculated as mentioned in the Methods section. Data are presented as mean values ± SD; n = 3. Two-sided t test was performed. The “*“ indicates that there is a significant difference between the quantitative data ( P < 0.05). * P = 0.0199, 0.0018, 0.0008 (left to right). b The crystal structure of AcrIF24 is shown in the cartoon model, together with its schematic illustration. The NTD and CTD are colored in yellow and cyan, respectively. The HTH motif is colored in orange. The unmodelled MD is indicated by the dashed line and colored hot pink in the schematic illustration. c Static light scattering (SLS) studies of AcrIF24 and AcrIF24ΔMD. The calculated molecular weights of the main peaks of the two profiles are shown above the peaks. d The AcrIF24ΔMD dimer is shown in the cartoon and surface model. Two perpendicular views are shown. e Structural superimposition between AcrIF24 CTD and the clp gene regulator ClgR from Corynebacterium glutamicum (PDB: 3F51). AcrIF24 CTD and ClgR are colored in cyan and marine, respectively. Two perpendicular views are shown.

Article Snippet: For cryo-EM sample preparation, 4 μL aliquots of Csy-AcrIF24, Csy-AcrIF24-dsDNA SP (3 mg/mL), and Csy-AcrIF24-dsDNA NS sample (2.5 mg/mL) were applied to discharged 200-mesh Au R1.2/1.3 grids (Quantifoil, Micro Tools GmbH, Germany).

Techniques: In Vitro, Activity Assay, CRISPR

Journal: Nature Communications

Article Title: Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24

doi: 10.1038/s41467-022-29581-1

Figure Lengend Snippet: a Inverted repeat sequence are conserved in the upstream promoters of AcrIF24 homologs. Genomic context of the acrIF23–acrIF24 locus of Pseudomonas aeruginosa prophage with inverted repeat sequence (shades of orange and red for the two half-sites of the respective repeat). Predicted regulatory sequences are shaded in blue and the predicted transcription start site (+1) is indicated by an arrow. The lower panel shows the alignment of acrIF23–acrIF24 operon promoter from P. aeruginosa prophage with those of other species in which inverted repeats are displayed as above. Invariant residues are indicated by an asterisk. b EMSA shows that AcrIF24 and AcrIF24ΔMD both exhibit high affinity to the IR23-24 DNA. The K D values are 20.18 and 49.06 nM for AcrIF24 and AcrIF24ΔMD, respectively. AcrIF24 displays a weak binding to non-sequence-specific dsDNA (dsDNA NS ), with a K D value of 3.461 μM. Data are presented as mean values ± SD; n = 3. Raw data for these curves are shown in Supplementary Fig. . c EMSA used to test the binding affinities of AcrIF24 for IR23-24 and mutated IR23-24. The sequences of WT and mutated IR23-24 are shown above the gel. Reactions were performed with 1 μM WT or mutated 23-bp IR23-24 and AcrIF24 concentrations of 0.125, 0.5, 2, 8, and 32 μM following the order indicated by the black triangle. d Structural superimposition between AcrIF24 and a restriction-modification controller protein C.Csp231I complexed with DNA (PDB: 4JQD). The two protomers of C.Csp231I are colored in pink and gray, respectively. The NTD and CTD of AcrIF24 are colored in yellow and cyan, respectively. N194, R196, and K197 of AcrIF24 are shown in sticks. e EMSA was used to test the IR23-24 binding activity of AcrIF24 mutants. Reactions are performed as in c with IR23-24 WT. Please see the left panel of c for comparison with WT control. f The promoter region containing IR23-24 sequence from P. aeruginosa prophage was cloned upstream of a promoterless lacZ gene. β-galactosidase activity was measured in P. aeruginosa PA14 strain. The β-galactosidase activity relative to IR23-24 is shown as mean ± SD; n = 3. Two-sided t test was performed. The “*“ indicates that there is a significant difference between the quantitative data ( P < 0.05). * P = 6.937e-7, 2.599e-7, 3.447e-7, 4.101e-7 (left to right). g EMSA was used to test the IR23-24 binding activity of AcrIF24ΔMD and its mutants. Reactions were performed as in e . FHY/AAA and FHY/EEE represent F212A/H216A/Y217A and F212E/H216E/Y217E, respectively.

Article Snippet: For cryo-EM sample preparation, 4 μL aliquots of Csy-AcrIF24, Csy-AcrIF24-dsDNA SP (3 mg/mL), and Csy-AcrIF24-dsDNA NS sample (2.5 mg/mL) were applied to discharged 200-mesh Au R1.2/1.3 grids (Quantifoil, Micro Tools GmbH, Germany).

Techniques: Sequencing, Binding Assay, Modification, Activity Assay, Clone Assay

Journal: Nature Communications

Article Title: Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24

doi: 10.1038/s41467-022-29581-1

Figure Lengend Snippet: a Binding kinetics of AcrIF24 with the Csy complex, measured by surface plasmon resonance (SPR) method. SPR curves (colored curves) were fit kinetically using a 1:1 Langmuir binding model (black lines). The data shown are representative of three independent experiments. b Static light scattering (SLS) studies of the mix of AcrIF24 and the Csy complex under different molar ratios. The predicted composition of each peak based on the calculated molecular weight is shown above the peaks. The calculated molecular weights of the peaks are shown in Supplementary Table . c Cryo-EM map of the Csy-AcrIF24 complex with each subunit color-coded. Two views are shown. d Atomic structure of Csy-AcrIF24 in cartoon representation with each subunit colored as in c . Two views are shown. e The AcrIF24 dimer in the Csy-AcrIF24 structure. Cryo-EM density is shown in a slate mesh. The MD of AcrIF24 is marked in a box. f Close-up view of the MD of AcrIF24. Cryo-EM density is shown in slate mesh.

Article Snippet: For cryo-EM sample preparation, 4 μL aliquots of Csy-AcrIF24, Csy-AcrIF24-dsDNA SP (3 mg/mL), and Csy-AcrIF24-dsDNA NS sample (2.5 mg/mL) were applied to discharged 200-mesh Au R1.2/1.3 grids (Quantifoil, Micro Tools GmbH, Germany).

Techniques: Binding Assay, SPR Assay, Molecular Weight, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24

doi: 10.1038/s41467-022-29581-1

Figure Lengend Snippet: a Overall structure of the Csy-AcrIF24 complex with one AcrIF24 bound to one Csy complex. The Csy complex is shown in the surface model with each subunit colored as in Fig. . AcrIF24 is shown in cartoon model colored in yellow, hot pink, and cyan for its NTD, MD, and CTD, respectively. b – e Close-up view of the interfaces between AcrIF24 and the Csy complex. The interfaces of AcrIF24 NTD -Cas7.6f ( b ), AcrIF24 NTD -Cas7.5f ( c ), AcrIF24 MD -Cas7.4f ( d ), and AcrIF24 MD -Cas7.2f ( e ) are shown. AcrIF24 is colored as in a, and interacting residues are shown as sticks with cryo-EM density map shown in mesh. f Native gel was used to test the binding between the Csy complex and AcrIF24 or its mutants. Reactions were performed with 0.32 μM Csy complex and AcrIF24 concentrations of 0.16, 0.32, and 0.64 μM following the order indicated by the black triangle. DDRF/AAAA represents the D104A/D105A/R106A/F107A mutant. The gel was stained with Coomassie blue staining. g Mutations of the interface residues of AcrIF24 decreased its inhibition capacity of the in vitro cleavage activity of the type I-F CRISPR system. Reactions are performed as in Fig. . DDRF/AAAA represents the D104A/D105A/R106A/F107A mutant.

Article Snippet: For cryo-EM sample preparation, 4 μL aliquots of Csy-AcrIF24, Csy-AcrIF24-dsDNA SP (3 mg/mL), and Csy-AcrIF24-dsDNA NS sample (2.5 mg/mL) were applied to discharged 200-mesh Au R1.2/1.3 grids (Quantifoil, Micro Tools GmbH, Germany).

Techniques: Cryo-EM Sample Prep, Binding Assay, Mutagenesis, Staining, Inhibition, In Vitro, Activity Assay, CRISPR

Journal: Nature Communications

Article Title: Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24

doi: 10.1038/s41467-022-29581-1

Figure Lengend Snippet: a EMSA was used to test the induction of non-specific DNA binding by AcrIF24 using dsDNA SP and dsDNA NS . Reactions were performed with 1 μM dsDNA SP or dsDNA NS , 8 μM Csy, and concentrations of AcrIF24 were set as 1, 2, 4, and 8 μM following the order indicated by the black triangle. The “+” represents 8 μM of AcrIF24. b EMSA was used to test the induction of non-specific DNA binding by AcrIF24 mutants using dsDNA SP and dsDNA NS . Reactions were performed as in a . DDRF/AAAA represents the D104A/D105A/R106A/F107A mutant. Please see the left and right panels of a for WT controls of the experiments using dsDNA SP and dsDNA NS , respectively. c – d EMSA was used to test the DNA binding of Csy complex and Csy-AcrIF24 using dsDNA SP ( c ) and dsDNA NS ( d ). dsDNA SP and dsDNA NS were added as 1 μM, and the concentrations of Csy complex and Csy-AcrIF24 were set as 0.5, 1, 2, 4, 8, and 16 μM following the order indicated by the black triangle. e EMSA was used to test the binding competition between dsDNA SP and dsDNA NS on Csy or Csy-AcrIF24 complex. Reactions were started with incubation of 1 μM dsDNA SP and 10 μM Csy or Csy-AcrIF24 complex, and dsDNA NS without FAM label was then added with concentrations of 5, 10, and 20 μM following the order indicated by the black triangle. f EMSA was used to test the binding competition between ssDNA SP and ssDNA NS on Csy or Csy-AcrIF24 complex. Reactions were exactly the same as in e except that all DNA molecules involved were single-stranded.

Article Snippet: For cryo-EM sample preparation, 4 μL aliquots of Csy-AcrIF24, Csy-AcrIF24-dsDNA SP (3 mg/mL), and Csy-AcrIF24-dsDNA NS sample (2.5 mg/mL) were applied to discharged 200-mesh Au R1.2/1.3 grids (Quantifoil, Micro Tools GmbH, Germany).

Techniques: Binding Assay, Mutagenesis, Incubation

Journal: Nature Communications

Article Title: Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24

doi: 10.1038/s41467-022-29581-1

Figure Lengend Snippet: a Atomic structure of Csy-AcrIF24-dsDNA SP in cartoon representation with each subunit colored as in Fig. . The bound dsDNA SP molecules are shown in surface model colored in yellow. b Structural superimposition between Csy-AcrIF24-dsDNA SP and Csy-AcrIF24. The structures of Csy-AcrIF24-dsDNA SP and Csy-AcrIF24 are colored in orange and gray, respectively. The dsDNA SP is shown in the surface model colored in yellow. c The densities corresponding to dsDNA molecules in the Csy-AcrIF24-dsDNA SP structure are shown in yellow mesh. The Csy and AcrIF24 are colored as in Fig. . d Close-up view of the binding site of dsDNA SP near the PAM recognition loop in Cas8f in the Csy-AcrIF24-dsDNA SP complex. The cryo-EM density maps for dsDNA SP , K247 of Cas8f and U1 of the crRNA are shown in mesh. e Close-up view of the interfaces between AcrIF24 and the dsDNA SP density within the Csy-AcrIF24-dsDNA SP complex. The density corresponding to dsDNA is shown as yellow mesh and N194/R196/K197 is shown in sticks. f The densities corresponding to dsDNA molecules in the Csy-AcrIF24-dsDNA NS structure are shown in the slate mesh. The Csy and AcrIF24 are colored as in Fig. . g Close-up view of the binding site of dsDNA NS near the PAM recognition loop in Cas8f in the Csy-AcrIF24-dsDNA NS complex. The cryo-EM density maps for dsDNA NS , K247, and N250 of Cas8f and U1 of the crRNA are shown in the mesh. The base groups of dsDNA NS are shown in cartoon, although the sequence of dsDNA NS cannot be determined. h Structural superimposition between Csy-AcrIF24-dsDNA SP and Csy-AcrIF24-dsDNA NS . Only dsDNA NS (colored slate) molecules are shown in Csy-AcrIF24-dsDNA NS structure. The dsDNA SP is colored in yellow.

Article Snippet: For cryo-EM sample preparation, 4 μL aliquots of Csy-AcrIF24, Csy-AcrIF24-dsDNA SP (3 mg/mL), and Csy-AcrIF24-dsDNA NS sample (2.5 mg/mL) were applied to discharged 200-mesh Au R1.2/1.3 grids (Quantifoil, Micro Tools GmbH, Germany).

Techniques: Binding Assay, Cryo-EM Sample Prep, Sequencing

Journal: Nature Communications

Article Title: Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24

doi: 10.1038/s41467-022-29581-1

Figure Lengend Snippet: a EMSA used to test the non-specific DNA binding ability of Csy-AcrIF24 with mutations in the PAM recognition loop of Cas8f using dsDNA SP and dsDNA NS . Reactions were performed as in Fig. . Please see the right panels of Figs. c, for WT controls of the experiments using dsDNA SP and dsDNA NS , respectively. b EMSA used to test the induction of non-specific DNA binding by AcrIF24 or its mutants using dsDNA SP and dsDNA NS . Reactions were performed as in Fig. . c EMSA used to test the non-specific DNA binding ability of Csy-AcrIF24 with mutations both in AcrIF24 and Csy using dsDNA SP and dsDNA NS . Reactions were performed as in Fig. . Please see the right panels of Figs. c, for WT controls of the experiments using dsDNA SP and dsDNA NS , respectively. Mutations impairing the induction of non-specific DNA binding of AcrIF24 decreased its capacity of inhibition in the in vitro cleavage activity assay. Reactions were performed as in Fig. except that the concentrations of AcrIF24 or its mutants were set as 0.16 μM. Data are presented as mean values ± SD; n = 3. Two-sided t test was performed. The “*“ indicates that there is a significant difference between the quantitative data ( P < 0.05). * P = 0.0098, 0.0125 (left to right). n.s, not significant , P = 0.7685. e Phage plaque assay verified that mutations impairing the induction of non-specific DNA binding of AcrIF24 decreased its capacity of inhibition of the type I-F system in vivo. Data are presented as mean values ± SD; n = 3. A two-sided t test was performed. The “*“ indicates that there is a significant difference between the quantitative data ( P < 0.05). * P = 0.0003, 0.0015, 0.0006, 0.0005 (left to right). f Schematic illustration of working mechanisms of AcrIF24 on the inhibition of type I-F CRISPR-Cas system. Direct interaction with Csy and induction of its non-specific DNA binding both contribute to the inhibition by AcrIF24.

Article Snippet: For cryo-EM sample preparation, 4 μL aliquots of Csy-AcrIF24, Csy-AcrIF24-dsDNA SP (3 mg/mL), and Csy-AcrIF24-dsDNA NS sample (2.5 mg/mL) were applied to discharged 200-mesh Au R1.2/1.3 grids (Quantifoil, Micro Tools GmbH, Germany).

Techniques: Binding Assay, Inhibition, In Vitro, Activity Assay, Plaque Assay, In Vivo, CRISPR

Journal: Nature Communications

Article Title: Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24

doi: 10.1038/s41467-022-29581-1

Figure Lengend Snippet: a Schematic illustration of the dsDNA SP with different lengths is used in the present study. Red and green lines represent PAM and protospacer sequences, respectively. b – c EMSA was used to test the non-specific DNA binding of Csy-AcrIF24 or Csy-AcrIF9 using dsDNA NS with 5′ FAM labels ( b ) and dsDNA SP with 3′ FAM labels ( c ) of different lengths. Reactions were performed as in Fig. . d – e EMSA was used to test the non-specific DNA binding of Csy-AcrIF24 mutants using dsDNA NS with 5′ FAM labels ( d ) and dsDNA SP with 3′ FAM labels ( e ) of different lengths. Reactions were performed as in Fig. . Please see the right two panels of b for WT controls of the experiments using dsDNA NS 35 bp and 45 bp in d , respectively.

Article Snippet: For cryo-EM sample preparation, 4 μL aliquots of Csy-AcrIF24, Csy-AcrIF24-dsDNA SP (3 mg/mL), and Csy-AcrIF24-dsDNA NS sample (2.5 mg/mL) were applied to discharged 200-mesh Au R1.2/1.3 grids (Quantifoil, Micro Tools GmbH, Germany).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24

doi: 10.1038/s41467-022-29581-1

Figure Lengend Snippet: a – b EMSA used to test the non-specific DNA binding affinity and mode of Csy-AcrIF24 and Csy-AcrIF9 using dsDNA SP /dsDNA NS ( a ), or ssDNA SP /ssDNA NS ( b ). Reactions were performed as in Fig. . c EMSA used to test the binding ability of AcrIF24 and AcrIF9 using dsDNA SP or dsDNA NS . Reactions were performed as in Fig. . d Structural superimposition of the Csy-AcrIF24-dsDNA SP and Csy-AcrIF9-dsDNA NS complexes. Only the Cas8f, Acr, and dsDNA regions are shown. K247 of Cas8f and the residues involved in non-specific DNA binding of AcrIF9 and AcrIF24 are shown in the sphere model. e EMSA was used to test the non-specific DNA binding of Csy-AcrIF9 with mutations in AcrIF9 or Csy using 3′ FAM-labeled dsDNA SP or 5′ FAM-labeled dsDNA NS . Reactions were performed as in Fig. . Please see the left two panels of Fig. for the WT control of the experiments using 3′ FAM dsDNA SP 15 bp and 25 bp, respectively. Please see the left two panels of Fig. for the WT control of the experiments using 5′ FAM dsDNA SP 15 bp and 25 bp, respectively.

Article Snippet: For cryo-EM sample preparation, 4 μL aliquots of Csy-AcrIF24, Csy-AcrIF24-dsDNA SP (3 mg/mL), and Csy-AcrIF24-dsDNA NS sample (2.5 mg/mL) were applied to discharged 200-mesh Au R1.2/1.3 grids (Quantifoil, Micro Tools GmbH, Germany).

Techniques: Binding Assay, Labeling

Journal: International Journal of Biological Sciences

Article Title: Human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling in pancreatic cancer cells

doi: 10.7150/ijbs.70007

Figure Lengend Snippet: Transcriptional regulation of CPTP expression by Sp1/Sp3. (A) 5' deletion analysis of the CPTP promoter in the PANC-1 cell line. A series of CPTP promoter deletion mutants were generated utilizing firefly luciferase plasmid. Each construct was co-transfected with pRL-TK plasmid as the internal control. Numbering is relative to the major transcriptional start site. (B) EMSA for the -282/-273 binding site. The assays were performed with nuclear extracts from the PANC-1 or Miacapa-2 cell lines. Competitive assays were executed with a 200-fold molar excess of unlabeled double-strand probes. Supershift assay was carried out with adding Sp1 or Sp3 antibody. The arrows show specific transcription factor binding. # indicates supershifted bands. #? indicates the position of expected supershifted Sp1 complex. (C) EMSA for the -258/-249 binding site. (D) Sp1/Sp3 interacts with the CPTP promoter in vivo was identified by ChIP assay, mouse IgG and the CPTP region (+67/+251) which does not contain binding sites of Sp1/Sp3 acted as negative control. Input, sheared DNA preceding immunoprecipitation posed as positive control. (E) RT-qPCR analysis for CPTP mRNA expression levels in the PANC-1 cells treated with mithramycin A for 24 h. (F) Knockdown of Sp1 or Sp3 decreased CPTP promoter activity. Cells transfected with pGL3(-454/-1), then transfected with Sp1 or Sp3 siRNA, and cultured for 24 h before dual-luciferase activity assay and Western blot analysis. *P < 0.05, **P < 0.01.

Article Snippet: Small interfering RNA targeted to Sp1 or Sp3 (Santa Cruz Biotechnology, Inc.) was used to knockdown the expression levels of Sp1 and Sp3, respectively.

Techniques: Expressing, Generated, Luciferase, Plasmid Preparation, Construct, Transfection, Control, Binding Assay, In Vivo, Negative Control, Immunoprecipitation, Positive Control, Quantitative RT-PCR, Knockdown, Activity Assay, Cell Culture, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling in pancreatic cancer cells

doi: 10.7150/ijbs.70007

Figure Lengend Snippet: Sp3 attenuates the decrease in cell proliferation, migration and invasion induced by CPTP knockdown. (A) Decreased cell viability in CPTP knockdown PC cells was restored by Sp3 overexpression. The cell viability was measured using a CCK-8 assay. (B-C) Sp3 overexpression rescued the decrease in cell motility in CPTP knockdown PC cells. (D) Sp3 overexpression rescued upregulation of epithelial-related markers and downregulation of mesenchymal-related markers expression induced by CPTP knockdown as detected by Western blot analysis. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Article Snippet: Small interfering RNA targeted to Sp1 or Sp3 (Santa Cruz Biotechnology, Inc.) was used to knockdown the expression levels of Sp1 and Sp3, respectively.

Techniques: Migration, Knockdown, Over Expression, CCK-8 Assay, Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling in pancreatic cancer cells

doi: 10.7150/ijbs.70007

Figure Lengend Snippet: Sp1/Sp3 expression is concurrently increased with CPTP expression in PC tissues. Representative images and IHC scores of Sp1 (A-B) or Sp3 (C-D) expression in PC (90 cases) and adjacent normal tissues (60 cases) were detected using IHC. Scale bar, 500 μm. Correlation analysis of protein expression in PC tissue samples between CPTP and Sp1 (E) or Sp3 (F). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Small interfering RNA targeted to Sp1 or Sp3 (Santa Cruz Biotechnology, Inc.) was used to knockdown the expression levels of Sp1 and Sp3, respectively.

Techniques: Expressing

Journal: International Journal of Biological Sciences

Article Title: Human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling in pancreatic cancer cells

doi: 10.7150/ijbs.70007

Figure Lengend Snippet: Schematic diagram illustrating the proposed mechanism of CPTP in PC cells. CPTP expression affects the levels of sphingolipid metabolite ceramide as well as the PI4KA/AKT signaling pathways. At the point when CPTP expression is upregulated, activation of the PI4KA/AKT signaling pathway and decreased levels of the sphingolipid metabolite ceramide promote PC cell growth and metastasis, respectively. Furthermore, in PC cells, the transcription factors Sp1 and Sp3 operate as upstream positive regulators of CPTP expression. Cer, ceramide.

Article Snippet: Small interfering RNA targeted to Sp1 or Sp3 (Santa Cruz Biotechnology, Inc.) was used to knockdown the expression levels of Sp1 and Sp3, respectively.

Techniques: Expressing, Protein-Protein interactions, Activation Assay

Journal: PLOS Pathogens

Article Title: The elevated expression of ORF75, a KSHV lytic gene, in Kaposi sarcoma lesions is driven by a GC-rich DNA cis element in its promoter region

doi: 10.1371/journal.ppat.1012984

Figure Lengend Snippet: A). (Left) schematic of truncation mutants, deletion mutants, and binding site mutants of the ORF75 promoter used to make luciferase constructs. (Right) promoter luciferase activity of these constructs in 293T cells. Numbers on the top of each bar indicates average fold change relative to empty vector pGL3 control. B) Schematic showing the location and sequence of the proximal Sp1 (pSp1) element and ARE elements in the ORF75 promoter and the mutants. C) Sequences of the pSp1 consensus element of ORF75 promoter probe and the mutant probes used in EMSA. The promoter probe is a 50 nt long dsDNA labelled with 5’ IR dye CW700. The mutated pSp1 element is in purple and underlined. The terminal ATG is the start codon of ORF75 protein. D) EMSA showing binding of Sp1, Sp3, and Sp4 proteins with the double-stranded DNA (dsDNA) IR-probe of ORF75 promoter in a 8% native PAGE gel. Equal concentration of HEK293T lysates over-expressing the various Sp proteins were used in the assay. E) Same as D), but with competitive specific and Sp1 element mutated probes. Shown are the means ± standard deviations of at least 3 separate experiments. P -values (* p ≤ 0.05, ** p ≤ 0.01, ns not significant) are calculated using two-sided paired t -test. Representative gel shift assay blots shown. All gel shift assay observations were reproduced in at least 3 separate experiments. Beta-galactosidase was used for transfection normalization. See for full blots.

Article Snippet: 10 μg of Sp1 antibody (Proteintech, 21962-1-Ap), Sp3 antibody (Proteintech, 26584-1-Ap), IgG antibody (CST), and H3 antibody (Abcam, ChIP grade) were incubated with the sheared chromatin overnight at 4°C in a rotary shaker.

Techniques: Binding Assay, Luciferase, Construct, Activity Assay, Plasmid Preparation, Control, Sequencing, Mutagenesis, Clear Native PAGE, Concentration Assay, Expressing, Gel Shift, Transfection

Journal: PLOS Pathogens

Article Title: The elevated expression of ORF75, a KSHV lytic gene, in Kaposi sarcoma lesions is driven by a GC-rich DNA cis element in its promoter region

doi: 10.1371/journal.ppat.1012984

Figure Lengend Snippet: A) Promoter luciferase assay of ORF75 promoter constructs along with over-expression of various Sp proteins in HepG2 cells. Sp proteins were expressed from a CMV-driven pN3 vector. ORF75 full-length (p75) and truncated (p75-T2) promoters are shown. Numbers on the top of each bar represents average fold change normalized to untreated pGL3 for each group of expression plasmid set as 1. The panel below the histogram represents the representative western blot for the over-expression proteins. B) ORF75 promoter (p75-T2) luciferase activity in the presence of Sp1-specific inhibitor Mithramycin A in 293T cells. 48h post transfection. C) Supershift assay. Equal amounts of HEK293T lysates (30ug) were preincubated with 1, 1.5 and 2µg of anti-Sp1 (21962-1-AP, Proteintech) or anti-Sp3 (26584-1-AP, Proteintech) before incubation with ORF75 promoter probe (p75 pSp1 probe) followed by gel shift assay. 6% native TBE gel. D) Same as in C) but with HEK293T nuclear lysates and 3µg each of different Sp1 antibodies. Sp1 antibody (AP) (21962-1-AP, Proteintech), Sp1 antibody (1C6) (sc-420, Santa Cruz), Sp1 antibody (E3) (sc-17824, Santa Cruz). Shown are the means ± standard deviations of 3 separate experiments. P -values (** p ≤ 0.01, **** p ≤ 0.0001, ns not significant) are calculated using two-sided paired t -test. Beta-galactosidase was used for transfection normalization. See for full blots.

Article Snippet: 10 μg of Sp1 antibody (Proteintech, 21962-1-Ap), Sp3 antibody (Proteintech, 26584-1-Ap), IgG antibody (CST), and H3 antibody (Abcam, ChIP grade) were incubated with the sheared chromatin overnight at 4°C in a rotary shaker.

Techniques: Luciferase, Construct, Over Expression, Plasmid Preparation, Expressing, Western Blot, Activity Assay, Transfection, Incubation, Gel Shift

Journal: PLOS Pathogens

Article Title: The elevated expression of ORF75, a KSHV lytic gene, in Kaposi sarcoma lesions is driven by a GC-rich DNA cis element in its promoter region

doi: 10.1371/journal.ppat.1012984

Figure Lengend Snippet: A) Promoter luciferase assay of ORF75 full length promoter in TIVE (endothelial cell line) and BJAB (B-cell line) cells. Results shown are the fold change over the respective pGL3 empty vector for each cell type. Data shown are ± standard deviations of 3 separate experiments. P -values (** p ≤ 0.01) are calculated using two-sided paired t -test B) Western blot analysis of Sp1, Sp3 and Sp4 protein levels from different cell lines using whole cell lysates. Blue and black arrows indicates full-length Sp1 and alternate SP3 forms, respectively. C) EMSA and WB analysis. Nuclear lysates of various uninfected cell lines were analysed for their Sp1 binding activity to the proximal Sp1 element of ORF75 promoter through EMSA. D) Same as in C) but with nuclear lysates of KSHV infected and uninfected cell lines. The lower three panels represent parallel WB of the nuclear lysates. *In the LANA WB, the asterisk indicates a non-specific band. E) Same as A), except four different ORF75 promoters were used for the promoter luciferase assay in BJAB and TIVE cells. Assayed at 72h post transfection. Numbers on the top of each bar indicates average fold upregulation relative to empty vector pGL3 for each cell type set as 1. Shown are the means ± standard deviations of 3 separate experiments. P -values (** p ≤ 0.01, **** p ≤ 0.0001, ns not significant) are calculated using two-sided unpaired t -test. All blots and gel shift assays were at least replicated in three separate experiments. Blots stripped and reprobed, see for more information.

Article Snippet: 10 μg of Sp1 antibody (Proteintech, 21962-1-Ap), Sp3 antibody (Proteintech, 26584-1-Ap), IgG antibody (CST), and H3 antibody (Abcam, ChIP grade) were incubated with the sheared chromatin overnight at 4°C in a rotary shaker.

Techniques: Luciferase, Plasmid Preparation, Western Blot, Binding Assay, Activity Assay, Infection, Transfection, Gel Shift

Journal: PLOS Pathogens

Article Title: The elevated expression of ORF75, a KSHV lytic gene, in Kaposi sarcoma lesions is driven by a GC-rich DNA cis element in its promoter region

doi: 10.1371/journal.ppat.1012984

Figure Lengend Snippet: A) WB of whole cell lysate (RIPA buffer) of early passage cell lines showing alternate forms of Sp1 protein in KSHV infected B-cell lines (4-12% BIS-TRIS gel, MES buffer). Sp1, Sp4 and ACTIN are same blot stripped and reprobed. Sp3 blot was run parallel. B) WB of BCBL-1 cells at different time points of lytic reactivation blotted with anti-Sp1 antibody. ORF45 and vIL6 were used here as control for lytic reactivation (10% BIS-TRIS gel, MES buffer). Sp1, ORF45 and ACTIN are same blot stripped and reprobed. vIL6 blot was run separately with the same samples. C) EMSA and western blot analysis. 10 µg of whole cell lysates (M-PER, Invitrogen) of BCBL-1 cells at different time points of lytic reactivation were analysed for their Sp1 protein binding activity to the proximal Sp1 element of ORF75 promoter through EMSA in a 8% TBE gel. The bottom three panel represent parallel WBs showing accumulation pattern of full-length Sp1 and 45 kDa Sp1 protein isoform during NaB induced lytic induction (10% BIS-TRIS gel, MES buffer). Black and red triangle indicates full-length Sp1 and 45 kDa Sp1 form binding to the proximal Sp1 DNA element of the ORF75 promoter, respectively. Sp1 and ACTIN are same blot stripped and reprobed. D) WB of uninfected TIME and infected TIME.219 cell line at different time points of lytic reactivation using TPA. LANA was used as control for infected TIME.219 cells and vIL6 was used as a control for lytic reactivation (8% Bolt BIS-TRIS gel, MES buffer). Black and red arrow indicates full-length and alternate Sp1 forms in WBs, respectively. SP1, vIL6, and ACTIN were on one blot, which was stripped and reprobed, while SP3 and LANA were on a parallel blot, which was also stripped and reprobed. Shown are representative blots, all observations were reproduced in at least 3 separate experiments. See and for full blots.

Article Snippet: 10 μg of Sp1 antibody (Proteintech, 21962-1-Ap), Sp3 antibody (Proteintech, 26584-1-Ap), IgG antibody (CST), and H3 antibody (Abcam, ChIP grade) were incubated with the sheared chromatin overnight at 4°C in a rotary shaker.

Techniques: Infection, Control, Western Blot, Protein Binding, Activity Assay, Binding Assay

Journal: Journal of Advanced Research

Article Title: Sp3 ameliorated experimental autoimmune encephalomyelitis by triggering Socs3 in Th17 cells

doi: 10.1016/j.jare.2025.01.051

Figure Lengend Snippet: Sp3 is high expressed in normal tissues and was blocked in the spleen, overexpression of Sp3 attenuates the clinical symptoms and inflammation in the CNS of EAE mice. a The expression of SP3 in brain from 42 health and 37 MS patients in GEO ( GSE131282 ). b The expression of SP3 from 92 normal tissues or compartments of human in genecards ( www.genecards.org ). c The expression of Sp3 was checked by real-time PCR in the spleen from normal wild type C57BL/6 and EAE mice. Changes in mRNA levels were quantified using the 2-ΔΔCT method with mGapdh mRNA as a control. Data were from three independent experiments (mean ± SD). * p < 0.05; ** p < 0.01, as determined using the Student’s t test. d The protein levels of Sp3 was tested by immunoblotting. β-actin was used as the loading control. EAE was induced by MOG 35-55 in female C57BL/6 mice (n = 6).The clinical scores of all the EAE mice were assessed daily according to the same criteria for 24 continuous days. Incidence ( e ), disease onset ( f ), daily clinical scores ( g ), cumulative disease scores ( h ), and peak disease scores ( i ) were monitored. e The incidence of EAE (mice with a clinical score ≥ 1 for 2 continuous days). The disease incidence is represented by the percentage of mice suffering from EAE. f The line graph depicts the survival rate of EAE between WT and LV-Sp3 mice. g The clinical scores of all the mice for 24 continuous days. h The mean disease cumulative score. i The mean maximum clinical score. j Hematoxylin and eosin (H&E) staining of representative spinal cord sections from LV-Sp3 mice and WT mice showing the infiltration of inflammatory cells in the white matter. k , m Showing the infiltration of inflammatory cells or demyelination in the white matter. l Luxol fast blue staining of intact myelin (blue) and demyelination (pink). The data are representative of at least two experiments with similar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Sp3 fl/fl , Sp3 +/- gene knockout mouse, Sp3 +/+CD4Cre knock in mouse and Sp3 -/-CD4Cre knockout mouse were purchased from the Cyagen Biosciences (Guangzhou, China).

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Staining

Journal: Journal of Advanced Research

Article Title: Sp3 ameliorated experimental autoimmune encephalomyelitis by triggering Socs3 in Th17 cells

doi: 10.1016/j.jare.2025.01.051

Figure Lengend Snippet: Overexpression of Sp3 suppressed Th1 and Th17cells in vivo and in vitro . Flow cytometry analysis of activated signature markers of Th1 and Th17 (IFN-γ and IL-17A) cells on the range of CD4 + cells isolated from the spleen, lymph nodes and CNS of LV-Ctrl and LV-Sp3-treated EAE mice (n = 6 mice per group) detected on the day 18 after the induction of EAE. Data are shown in a representative plot of the Th1 and Th17 cell subpopulations in three types of tissues ( a - c ). The percentage of respective subpopulations in CD4 + cells summarized by histograms. Treatment of cells isolated from the spleen, lymph nodes and CNS of LV-Ctrl and LV-Sp3-treated EAE mice was performed according to the description in the “Intracellular cytokine staining” section. Cells from the spleen ( d ) and lymph nodes ( e ) of LV-Ctrl and LV-Sp3-treated EAE mice were collected, and total RNA was extracted based on the manufacturer’s instructions using TRIzol reagent. The quantity of mRNA was recorded using real-time PCR. Gene expression was normalized based on the mGapdh content. f Naïve CD4 + T cells were separated from the spleen of female C57BL/6 mice, and then Th1 and Th17 cells were induced under various conditions according to the “Naïve CD4 + T cell preparation and differentiation” section. The cells were evaluated per the description in the “Intracellular cytokine staining” section. g , h ELISA was used to assess the expression of IFN-γ and IL-17A in the supernatants of Th1 and Th17 cells. Data were from three independent experiments (mean ± SEM). * p < 0.05; ** p < 0.01, as determined using the Student’s t test.

Article Snippet: Sp3 fl/fl , Sp3 +/- gene knockout mouse, Sp3 +/+CD4Cre knock in mouse and Sp3 -/-CD4Cre knockout mouse were purchased from the Cyagen Biosciences (Guangzhou, China).

Techniques: Over Expression, In Vivo, In Vitro, Flow Cytometry, Isolation, Staining, Real-time Polymerase Chain Reaction, Gene Expression, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of Advanced Research

Article Title: Sp3 ameliorated experimental autoimmune encephalomyelitis by triggering Socs3 in Th17 cells

doi: 10.1016/j.jare.2025.01.051

Figure Lengend Snippet: Sp3 as a direct target of miR-223. a Potential target genes of miR-223 as predicted by miRanda, TargetRank and TargetScan. b The miR-223 target sequence in the 3′UTR of Sp3 and Hsp72 in mouse. c EL4 cells were transfected with LV-Ctrl or LV-anti-miR-223. Quantitative PCR (real-time PCR) analysis of Hsp72, Sp3 and Sp1 mRNA levels. d The protein level of Sp3 and Hsp72 was tested by immunoblotting. H3 and β-actin were used as the nuclear and cytoplasm loading controls. e A schematic representing the luciferase constructs with the wild-type (CN) or mutant 3′UTR miR-223 MRE sequences of Sp3 and Hsp72. Mutations are marked in lowercase letters and underlined. f Normalized luciferase activity in lysates from 293 T cells co-transfected with wild-type or mutant Sp3-luciferase constructs and miR-223 or control mimics (mean ± SEM, n = 3, * p < 0.05, ** p < 0.01). g - i EL4 cells were transfected with LV-Ctrl or LV-anti-miR-223 and stimulated with PMA/I for 0 min, 10 min, 30 min, and 60 min. Quantitative PCR analysis of IFN-γ, IL-17A and IL-17F mRNA levels (g). h , i The protein levels of Smad2/3, phospho-Smad2/3, and Stat3 were tested by immunoblotting. β-actin was used as the loading control. The data are expressed as the mean ± SEM; experiments were performed in triplicate (* p < 0.05, ** p < 0.01).

Article Snippet: Sp3 fl/fl , Sp3 +/- gene knockout mouse, Sp3 +/+CD4Cre knock in mouse and Sp3 -/-CD4Cre knockout mouse were purchased from the Cyagen Biosciences (Guangzhou, China).

Techniques: Sequencing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Luciferase, Construct, Mutagenesis, Activity Assay, Control

Journal: Journal of Advanced Research

Article Title: Sp3 ameliorated experimental autoimmune encephalomyelitis by triggering Socs3 in Th17 cells

doi: 10.1016/j.jare.2025.01.051

Figure Lengend Snippet: Sp3 regulated the Smad2/3 and Stat3 signaling pathways during Th17 cell differentiation through Socs3. a , b The protein levels of Smad2/3, phospho-Smad2, Stat3 and phospho-Stat3 were tested by immunoblotting in the spleen and CNS from LV-Ctrl, LV-Sp3 or LV-anti-Sp3-treated EAE mice. c RORγt, a key transcription factor in Th17 cells, was tested in CNS by immunoblotting. d EL4 cells were transfected with LV-Ctrl or LV-Sp3 and stimulated with PMA/I for 0 min, 10 min, 30 min, and 60 min. The protein levels of Smad2/3, phospho-Smad2/3, and Stat3 were assessed by immunoblotting. e Analysis SP3 binding genes by TRRUST, FIMO_JASPAR, GSE131282 database. f Analysis SOCS3 gene expression in MS patients in GEO ( GSE131282 ). g JASPAR database predicts that transcription factor SP3 binds to the upstream 1740–1750 bp position of the SOCS3 gene. h The protein level of Socs3 was tested by immunoblotting in EL4 cells. i The protein level of Sp3 was tested by immunoblotting in EL4 cells and 293 T cells. β-actin was used as the loading control. Densitometric ratios were quantified using ImageJ software. The data are expressed as the mean ± SEM (* p < 0.05, ** p < 0.01).

Article Snippet: Sp3 fl/fl , Sp3 +/- gene knockout mouse, Sp3 +/+CD4Cre knock in mouse and Sp3 -/-CD4Cre knockout mouse were purchased from the Cyagen Biosciences (Guangzhou, China).

Techniques: Protein-Protein interactions, Cell Differentiation, Western Blot, Transfection, Binding Assay, Gene Expression, Control, Software

Journal: Journal of Advanced Research

Article Title: Sp3 ameliorated experimental autoimmune encephalomyelitis by triggering Socs3 in Th17 cells

doi: 10.1016/j.jare.2025.01.051

Figure Lengend Snippet: Sp3 in CD4 T cells attenuated or aggravated the clinical symptoms of Sp3 +/+CD4Cre or Sp3 -/-CD4Cre EAE mice. The clinical scores of all the EAE mice were assessed daily according to the same criteria for 24 continuous days. Incidence ( a ), Daily clinical scores ( b ), peak disease scores ( c ), and cumulative disease scores ( d ) were monitored Sp3 fl/fl and Sp3 +/+CD4Cre EAE mice. The data are representative of at least two experiments with similar results. Flow cytometry analysis of CD4 + cells isolated from the spleen and lymph node of Sp3 fl/fl and Sp3 +/+CD4Cre EAE mice (n = 6 mice per group) at day 18 after the induction of EAE. Data are shown in a representative plot of the CD4 + cell subpopulations in spleen and lymph node ( e, f, g ). Knockout of Sp3 in CD4 T cells of Sp3 -/-CD4Cre EAE mice. Incidence ( h ), Daily clinical scores ( i ), peak disease scores ( j ), and cumulative disease scores ( k ) were monitored. The data are representative of at least two experiments with similar results. Flow cytometry analysis of CD4 + cells isolated from the spleen of Sp3 fl/fl and Sp3 -/-CD4Cre EAE mice at day 15 after the induction of EAE. Data are shown in a representative plot of the CD4 + cell subpopulations in spleen ( l ). m Naïve CD4 + T cells were separated from the spleen of female Sp3 fl/fl and Sp3 -/-CD4Cre mice and then induced to differentiate into Th17 cells. Data were from three independent experiments (mean ± SEM). * p < 0.05; ** p < 0.01, as determined using the Student’s t test. n Schematic of the molecular mechanism by which Sp3 regulates Th17 cell differentiation. As the target of miR-223, Sp3 positively regulates Socs3, which regulates Sp3 via a feedback mechanism. Socs3 negatively regulates the IL-6/JAK2/Stat3 and TGF-β/Smad2/3 signal transduction pathways to control Th17 cell differentiation. Thus, RORγt contributes to Th17 cell differentiation and EAE induction.

Article Snippet: Sp3 fl/fl , Sp3 +/- gene knockout mouse, Sp3 +/+CD4Cre knock in mouse and Sp3 -/-CD4Cre knockout mouse were purchased from the Cyagen Biosciences (Guangzhou, China).

Techniques: Flow Cytometry, Isolation, Knock-Out, Cell Differentiation, Transduction, Control

Journal: Journal of Biological Chemistry

Article Title: Regulation of the Bone-restricted IFITM-like (Bril) Gene Transcription by Sp and Gli Family Members and CpG Methylation

doi: 10.1074/jbc.m113.457010

Figure Lengend Snippet: FIGURE 4. Transcriptional activation of the rat, mouse, and human Bril promoter by Sp family members. The promoter Luc constructs (100 ng) were co-transfected with 300 ng of each expression plasmids encoding Sp1, Sp3-L1 (long form 1), Sp3-L2 (long form 2), Sp3-S (short form), and OSX. Luc activity was measured 48 h after transient transfection into MC3T3 osteo- blasts (A) or HEK293 (B and C). C, the human 1434 bppromoterLucconstruct (100 ng) was co-transfected with 150 ng of each plasmid encoding either GFP or Sp1, in combination with the other Sp members. The first two bars represent transfection of Sp1 (150 ng) with GFP (150 ng) or with itself (300 ng). Results are presented as -fold increase relative to the negative control plasmid encoding GFP. Results shown are mean S.E. (error bars) (n 3–5).

Article Snippet: The following plasmids were purchased from Origene: human Sp3 variant 1 (Sp3-L1) 2 P. Moffatt, unpublished data.

Techniques: Activation Assay, Construct, Transfection, Expressing, Activity Assay, Plasmid Preparation, Negative Control