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prps2 (Sino Biological)

Name: prps2
Brand: Sino Biological
Rating: 90 (2 reviews)

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Sino Biological prps2

(A-C) Volcano plots indicating the proteins identified from PRPS1, <t>PRPS2</t> (A) , PRPSAP1, PRPSAP2 (B) , tNASP and sNASP (C) FLAG-affinity purification followed by mass spectrometry. PRPP-synthetase components are indicated in green whereas key histone chaperones and histones are indicated in blue color. All other enriched proteins are shown in yellow, while grey datapoints represent proteins not enriched in FLAG compared to parental. n=3 , p values were calculated using Student’s t-test with Welch correction. Also see method details . (D-E) Co-immunoprecipitations demonstrating the interaction of stably expressing FLAG-tagged PRPS1 (D) or PRPSAP1 (E) with histone chaperones HSP90, NASP, IPO4, HAT and RBBP7 in HEK293T cells. (F) Interaction of t and s isoforms of NASP with PRPS1 as detected through co-immunoprecipitation of FLAG-tagged t- or sNASP from HEK293T cells followed by immunoblotting with PRPS1 antibody. (G) Co-immunoprecipitation showing the interaction of FLAG-PRPS WT, ΔCD, ΔR5P with t- or sNASP in HEK293T cells. (H-K) GST pulldown demonstrating the in vitro binding between recombinant GST-sNASP and PRPS1 (H) , GST-PRPSAP1 and HAT1 (I) , GST-PRPSAP1 and RBBP7 (J) and GST-PRPS1 or GST-PRPSAP1 with H3-H4 tetramer (K) . (L) Depiction of PRPP-synthetase, histone and histone chaperone complex based on the direct interaction of PRPS1 with H3-H4 and NASP and PRPSAP1 with H3-H4, HAT1 and RBBP7. (M) In vitro HAT assay showing the stimulatory effect of PRPSAP1 on HAT1-RBBP7 activity towards histone H4 acetylation. Molecular weight (kDa) .

Prps2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/prps2/product/Sino Biological
Average 90 stars, based on 2 article reviews

prps2 - by Bioz Stars, 2026-02

90/100 stars

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1) Product Images from "Rate Limiting Enzymes in Nucleotide Metabolism Synchronize Nucleotide Biosynthesis and Chromatin Formation"

Article Title: Rate Limiting Enzymes in Nucleotide Metabolism Synchronize Nucleotide Biosynthesis and Chromatin Formation

Journal: Molecular cell

doi: 10.1016/j.molcel.2025.11.009

(A-C) Volcano plots indicating the proteins identified from PRPS1, PRPS2 (A) , PRPSAP1, PRPSAP2 (B) , tNASP and sNASP (C) FLAG-affinity purification followed by mass spectrometry. PRPP-synthetase components are indicated in green whereas key histone chaperones and histones are indicated in blue color. All other enriched proteins are shown in yellow, while grey datapoints represent proteins not enriched in FLAG compared to parental. n=3 , p values were calculated using Student’s t-test with Welch correction. Also see method details . (D-E) Co-immunoprecipitations demonstrating the interaction of stably expressing FLAG-tagged PRPS1 (D) or PRPSAP1 (E) with histone chaperones HSP90, NASP, IPO4, HAT and RBBP7 in HEK293T cells. (F) Interaction of t and s isoforms of NASP with PRPS1 as detected through co-immunoprecipitation of FLAG-tagged t- or sNASP from HEK293T cells followed by immunoblotting with PRPS1 antibody. (G) Co-immunoprecipitation showing the interaction of FLAG-PRPS WT, ΔCD, ΔR5P with t- or sNASP in HEK293T cells. (H-K) GST pulldown demonstrating the in vitro binding between recombinant GST-sNASP and PRPS1 (H) , GST-PRPSAP1 and HAT1 (I) , GST-PRPSAP1 and RBBP7 (J) and GST-PRPS1 or GST-PRPSAP1 with H3-H4 tetramer (K) . (L) Depiction of PRPP-synthetase, histone and histone chaperone complex based on the direct interaction of PRPS1 with H3-H4 and NASP and PRPSAP1 with H3-H4, HAT1 and RBBP7. (M) In vitro HAT assay showing the stimulatory effect of PRPSAP1 on HAT1-RBBP7 activity towards histone H4 acetylation. Molecular weight (kDa) .

Figure Legend Snippet: (A-C) Volcano plots indicating the proteins identified from PRPS1, PRPS2 (A) , PRPSAP1, PRPSAP2 (B) , tNASP and sNASP (C) FLAG-affinity purification followed by mass spectrometry. PRPP-synthetase components are indicated in green whereas key histone chaperones and histones are indicated in blue color. All other enriched proteins are shown in yellow, while grey datapoints represent proteins not enriched in FLAG compared to parental. n=3 , p values were calculated using Student’s t-test with Welch correction. Also see method details . (D-E) Co-immunoprecipitations demonstrating the interaction of stably expressing FLAG-tagged PRPS1 (D) or PRPSAP1 (E) with histone chaperones HSP90, NASP, IPO4, HAT and RBBP7 in HEK293T cells. (F) Interaction of t and s isoforms of NASP with PRPS1 as detected through co-immunoprecipitation of FLAG-tagged t- or sNASP from HEK293T cells followed by immunoblotting with PRPS1 antibody. (G) Co-immunoprecipitation showing the interaction of FLAG-PRPS WT, ΔCD, ΔR5P with t- or sNASP in HEK293T cells. (H-K) GST pulldown demonstrating the in vitro binding between recombinant GST-sNASP and PRPS1 (H) , GST-PRPSAP1 and HAT1 (I) , GST-PRPSAP1 and RBBP7 (J) and GST-PRPS1 or GST-PRPSAP1 with H3-H4 tetramer (K) . (L) Depiction of PRPP-synthetase, histone and histone chaperone complex based on the direct interaction of PRPS1 with H3-H4 and NASP and PRPSAP1 with H3-H4, HAT1 and RBBP7. (M) In vitro HAT assay showing the stimulatory effect of PRPSAP1 on HAT1-RBBP7 activity towards histone H4 acetylation. Molecular weight (kDa) .

Techniques Used: Affinity Purification, Mass Spectrometry, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, In Vitro, Binding Assay, Recombinant, HAT Assay, Activity Assay, Molecular Weight

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Journal: Molecular cell

Article Title: Rate Limiting Enzymes in Nucleotide Metabolism Synchronize Nucleotide Biosynthesis and Chromatin Formation

doi: 10.1016/j.molcel.2025.11.009

Figure Lengend Snippet: (A-C) Volcano plots indicating the proteins identified from PRPS1, PRPS2 (A) , PRPSAP1, PRPSAP2 (B) , tNASP and sNASP (C) FLAG-affinity purification followed by mass spectrometry. PRPP-synthetase components are indicated in green whereas key histone chaperones and histones are indicated in blue color. All other enriched proteins are shown in yellow, while grey datapoints represent proteins not enriched in FLAG compared to parental. n=3 , p values were calculated using Student’s t-test with Welch correction. Also see method details . (D-E) Co-immunoprecipitations demonstrating the interaction of stably expressing FLAG-tagged PRPS1 (D) or PRPSAP1 (E) with histone chaperones HSP90, NASP, IPO4, HAT and RBBP7 in HEK293T cells. (F) Interaction of t and s isoforms of NASP with PRPS1 as detected through co-immunoprecipitation of FLAG-tagged t- or sNASP from HEK293T cells followed by immunoblotting with PRPS1 antibody. (G) Co-immunoprecipitation showing the interaction of FLAG-PRPS WT, ΔCD, ΔR5P with t- or sNASP in HEK293T cells. (H-K) GST pulldown demonstrating the in vitro binding between recombinant GST-sNASP and PRPS1 (H) , GST-PRPSAP1 and HAT1 (I) , GST-PRPSAP1 and RBBP7 (J) and GST-PRPS1 or GST-PRPSAP1 with H3-H4 tetramer (K) . (L) Depiction of PRPP-synthetase, histone and histone chaperone complex based on the direct interaction of PRPS1 with H3-H4 and NASP and PRPSAP1 with H3-H4, HAT1 and RBBP7. (M) In vitro HAT assay showing the stimulatory effect of PRPSAP1 on HAT1-RBBP7 activity towards histone H4 acetylation. Molecular weight (kDa) .

Article Snippet: N-terminus FLAG-tagged PRPS1 or PRPS2 entry clones were generated by sub-cloning N-FLAG-PRPS1 (Sinobiologicals, HG17214-NF) and PRPS2 (Sinobiologicals, HG15128-NF) into pDONR221 vector (Invitrogen, 12536017).

Techniques: Affinity Purification, Mass Spectrometry, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, In Vitro, Binding Assay, Recombinant, HAT Assay, Activity Assay, Molecular Weight

a) Phylogenetic distribution profiles of PRPS homologs in eukaryotes and PRPS homologs containing NHR in opisthokonts (presence/absence) are noted across the tree. PRPSAP2 represent orthologs of mammalian PRPSAP2 while Prs1 and Prs5 represent orthologs of S. cerevisiae Prs1 and Prs5, respectively. b) Analysis of conserved splice site junction among PRPS homologs across different representative organisms of Opisthokonta. Exons from each of the PRPS, PRPSAP2 and Prs5 encoding genes are merged to highlight the highly splice site junction with coding amino acids above. In PRPS1, red dots mark the regulatory flexible loop where insertions occur in Prs5 orthologs (red hatch marks), and black dots mark the catalytic flexible loop where insertions occur in PRPSAP2 orthologs (black hatch marks). Notably, some Prs5 orthologs possess additional insertions in the catalytic flexible loop. c) Phylogenetic distribution profiles of PRPS homologs in chordates (presence/absence) are noted across the tree. d) The structure of dimeric human PRPS1 (PDB ID: 2HCR), and a zoom in of the catalytic site highlighting metal binding site (Cd 2+ ), AMP (representing the AMP moiety of the ATP), SO - (representing the 5’-phosphate of R5P), and several conserved active site residues (magenta). D171 coordinates metal binding, K194 interacts with the ATP, R196 and T225 interact with the R5P, and N200 stabilizes the catalytic loop . e) Predicted dimeric structure of human PRPSAP1 (Accession #AAH09012.1) from the AlphaFold2 model, and a zoom in highlights four non-conserved residues in PRPSAP1 (magenta) at the corresponding positions of residues for PRPS1 shown in (d). AMP was modeled into the dimer to denote the putative ATP binding site.

Journal: bioRxiv

Article Title: Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

doi: 10.1101/2024.10.01.616059

Figure Lengend Snippet: a) Phylogenetic distribution profiles of PRPS homologs in eukaryotes and PRPS homologs containing NHR in opisthokonts (presence/absence) are noted across the tree. PRPSAP2 represent orthologs of mammalian PRPSAP2 while Prs1 and Prs5 represent orthologs of S. cerevisiae Prs1 and Prs5, respectively. b) Analysis of conserved splice site junction among PRPS homologs across different representative organisms of Opisthokonta. Exons from each of the PRPS, PRPSAP2 and Prs5 encoding genes are merged to highlight the highly splice site junction with coding amino acids above. In PRPS1, red dots mark the regulatory flexible loop where insertions occur in Prs5 orthologs (red hatch marks), and black dots mark the catalytic flexible loop where insertions occur in PRPSAP2 orthologs (black hatch marks). Notably, some Prs5 orthologs possess additional insertions in the catalytic flexible loop. c) Phylogenetic distribution profiles of PRPS homologs in chordates (presence/absence) are noted across the tree. d) The structure of dimeric human PRPS1 (PDB ID: 2HCR), and a zoom in of the catalytic site highlighting metal binding site (Cd 2+ ), AMP (representing the AMP moiety of the ATP), SO - (representing the 5’-phosphate of R5P), and several conserved active site residues (magenta). D171 coordinates metal binding, K194 interacts with the ATP, R196 and T225 interact with the R5P, and N200 stabilizes the catalytic loop . e) Predicted dimeric structure of human PRPSAP1 (Accession #AAH09012.1) from the AlphaFold2 model, and a zoom in highlights four non-conserved residues in PRPSAP1 (magenta) at the corresponding positions of residues for PRPS1 shown in (d). AMP was modeled into the dimer to denote the putative ATP binding site.

Article Snippet: Primary antibodies used were: CAD (Cell Signaling #93925), TCP1-η (Santa Cruz #sc-271951), FASN (Cell Signaling #3180), FLC (Santa Cruz #sc-390558), HK2 (Cell Signaling #2867), AK2 (Santa Cruz #sc-374095), PRPS1/2 (Santa Cruz #sc-100822), PRPS1 (Proteintech #15549-1-AP), PRPS2 (Sigma #SAB2107995), PRPS1/2/3 (Santa Cruz #sc-376440), PRPSAP1 (Santa Cruz #sc-398422), PRPSAP2 (Proteintech #17814-1-AP), HSP90 (Cell Signaling #4877), β-Actin (Cell Signaling #4970; Cell Signaling #3700), ALFA-HRP (SynapticSystems # N1505-HRP), XO (Abcam #109235), Ras (G12V Mutant Specific) (Cell Signaling #14412), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling #4376), p44/42 MAPK (Erk1/2) (Cell Signaling #9102), β-Tubulin (Cell Signaling #2128), Phospho-AMPKα (Thr172) (Cell Signaling #2535), AMPKα (Cell Signaling #2532), cleaved PARP1 (Abcam #32064), GAPDH (Cell Signaling #5174), HPRT (Abcam #109021).

Techniques: Binding Assay

a) The catalytic flexible (CF) loop from the sequence alignments of PRPS homologs from representative organisms of Opisthokonta, Amoebozoa (Evosea), Apusozoa, and CRuMs. PrsA in Evosea is the PRPS enzyme while PrsB represents the PRPS homologs with insertions in the CF loop (NHRs), similar to Opisthokonta PRPSAP2. PRPS homologs in Apusozoa and CRuMs that share features with Opisthokonta PRPSAP2 are termed “PRPSAP-like”. Underlined residues denote the NHRs. For S. cerevisiae Prs1 and D. rotans PRPSAP-like sequence, only a part of the insertion is shown, and the rest of the sequence is represented by hatch marks. b) PRPS paralogs in Saccharomyces cerevisiae and Homo sapiens showing relative positions of non-homologous regions – NHR1 of Prs1, NHR5-1 and NHR5-2 of Prs5, NHRs of PRPSAP1 and PRPSAP2. The open bar represents the polypeptide for Prs2, Prs3, and Prs4 in S. cerevisiae , and for PRPS1 and PRPS2 in H. sapiens . The insertion points for Prs1, Prs5, PRPSAP1, and PRPSAP2 are marked, with NHRs indicated as triangles either above or below the open bar. The residue numbers on the NHRs indicate the amino acid positions on the respective homologs. c) and d) The N-terminal residues from a sequence alignment of PRPS homologs from representative organisms of Amorphea. Opisthokonta PRPSAP2 sequences are more similar to Opisthokonta PRPS enzymes (blue box) than to PRPS from Apusozoa, Breviatea and Amoebozoa (c) while Evosea PrsB sequences are more similar to Evosea PrsA (red box) than to PRPS from Apusozoa, Breviatea and Opisthokonta (d) indicating that PRPSAP2 and PrsB likely emerged independently from ancestral Opisthokonta PRPS1 and Evosea PrsA, respectively. e) Analysis of conserved splice site junction among PrsA and PrsB homologs across different representative organisms of Evosea. Exons from each of the Prs encoding genes are merged to highlight the splice site junctions. PrsA shares a conserved splice site junction – LH_(A/S)S – with PrsB, further supporting that PrsB likely originated from a gene duplication event in the ancestral Evosea PrsA.

Journal: bioRxiv

Article Title: Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

doi: 10.1101/2024.10.01.616059

Figure Lengend Snippet: a) The catalytic flexible (CF) loop from the sequence alignments of PRPS homologs from representative organisms of Opisthokonta, Amoebozoa (Evosea), Apusozoa, and CRuMs. PrsA in Evosea is the PRPS enzyme while PrsB represents the PRPS homologs with insertions in the CF loop (NHRs), similar to Opisthokonta PRPSAP2. PRPS homologs in Apusozoa and CRuMs that share features with Opisthokonta PRPSAP2 are termed “PRPSAP-like”. Underlined residues denote the NHRs. For S. cerevisiae Prs1 and D. rotans PRPSAP-like sequence, only a part of the insertion is shown, and the rest of the sequence is represented by hatch marks. b) PRPS paralogs in Saccharomyces cerevisiae and Homo sapiens showing relative positions of non-homologous regions – NHR1 of Prs1, NHR5-1 and NHR5-2 of Prs5, NHRs of PRPSAP1 and PRPSAP2. The open bar represents the polypeptide for Prs2, Prs3, and Prs4 in S. cerevisiae , and for PRPS1 and PRPS2 in H. sapiens . The insertion points for Prs1, Prs5, PRPSAP1, and PRPSAP2 are marked, with NHRs indicated as triangles either above or below the open bar. The residue numbers on the NHRs indicate the amino acid positions on the respective homologs. c) and d) The N-terminal residues from a sequence alignment of PRPS homologs from representative organisms of Amorphea. Opisthokonta PRPSAP2 sequences are more similar to Opisthokonta PRPS enzymes (blue box) than to PRPS from Apusozoa, Breviatea and Amoebozoa (c) while Evosea PrsB sequences are more similar to Evosea PrsA (red box) than to PRPS from Apusozoa, Breviatea and Opisthokonta (d) indicating that PRPSAP2 and PrsB likely emerged independently from ancestral Opisthokonta PRPS1 and Evosea PrsA, respectively. e) Analysis of conserved splice site junction among PrsA and PrsB homologs across different representative organisms of Evosea. Exons from each of the Prs encoding genes are merged to highlight the splice site junctions. PrsA shares a conserved splice site junction – LH_(A/S)S – with PrsB, further supporting that PrsB likely originated from a gene duplication event in the ancestral Evosea PrsA.

Techniques: Sequencing, Residue

a) Analysis of splice site junctions among PRPS1 and PRPS2 across different representative organisms of jawed vertebrates. Exons from each of the PRPS1 and PRPS2 encoding genes are merged to highlight the highly conserved splice site junctions. b) Analysis of splice site junction among PRPSAP2 and PRPSAP1 across different representative organisms of jawed vertebrates. Exons from each of the PRPSAP2 and PRPSAP1 encoding genes are merged to highlight the highly conserved splice site junctions. Hatch marks representing the NHRs are not intended to accurately represent the variable length of the NHRs.

Journal: bioRxiv

Article Title: Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

doi: 10.1101/2024.10.01.616059

Figure Lengend Snippet: a) Analysis of splice site junctions among PRPS1 and PRPS2 across different representative organisms of jawed vertebrates. Exons from each of the PRPS1 and PRPS2 encoding genes are merged to highlight the highly conserved splice site junctions. b) Analysis of splice site junction among PRPSAP2 and PRPSAP1 across different representative organisms of jawed vertebrates. Exons from each of the PRPSAP2 and PRPSAP1 encoding genes are merged to highlight the highly conserved splice site junctions. Hatch marks representing the NHRs are not intended to accurately represent the variable length of the NHRs.

Techniques:

Journal: bioRxiv

Article Title: Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

doi: 10.1101/2024.10.01.616059

Figure Lengend Snippet: a) WebLogo depicting the multiple sequence alignment of the active site residues of PRPS1 and PRPSAP2 from representative organisms in Opisthokonta (n = 44 for PRPS1 and n = 44 for PRPSAP2). Residues from the FLAG region, regulatory flexible loop, pyrophosphate (PP) loop, CF loop, and R5P binding loop are indicated. Many residues shown are conserved in PRPS1 but not in PRPSAP2. The numbers below the logo sequences indicate the corresponding residue positions of the human PRPS1 (NP_002755.1) and PRPSAP2 (NP_001340030.1). Asterisks denote some evolutionarily conserved active site residues that are exclusive to PRPS enzymes and highlighted in (d) but replaced with different residues at the corresponding positions in PRPSAPs, highlighted in (e). b) The trimeric structure of human PRPS1 (PDB: 2HCR). In the dashed box, red- and magenta-colored residues represent those involved in the formation of bent (B and C subunits) and parallel (A and B subunits) dimers, respectively. The amino acid sequence of B. subtilis PRPS was aligned with the Opisthokonta PRPS homologs, and the corresponding dimer interface residues were selected for generating the WebLogo. Sequences for PRPS1 (n = 44) and PRPSAP2 (n = 44) are derived from representative organisms within opisthokonts, while PRPS2 (n = 46) and PRPSAP1 (n = 92) sequences are derived from representative organisms within jawed vertebrates. The numbers below the logo sequences indicate the corresponding residues positions of the human PRPS1 (NP_002755.1), PRPS2 (NP_002756.1), PRPSAP1 (AAH09012.1), and PRPSAP2 (NP_001340030.1). The significant sequence conservation between PRPS and PRPSAP indicates the potential for heteromeric associations.

Techniques: Sequencing, Binding Assay, Residue, Derivative Assay

$a) SDS-PAGE followed by Coomassie stain of the eluates from GFP immunoprecipitation (IP) performed in NIH3T3 and HEK293T cells stably expressing PRPS1-GFP, PRPS2-GFP and NES-GFP. NES (Nuclear export signal)-GFP cells were used as controls. Asterisk indicates a non-specific band in the eluates. b) Scatter plot from mass spectrometry (MS) runs of eluates from GFP immunoprecipitation (IP) in NIH3T3 and HEK293T cells stably expressing PRPS1-GFP on the x-axis and y-axis, respectively. The axes represent the square root-transformed SEQUEST HT scores of PRPS1-GFP normalized to control. PRPS1L1 isoform was detected in HEK29T cells only. c) Western blot analysis of size exclusion chromatography (SEC) fractions collected from NIH3T3 native whole cell lysates. Cell lysates were fractionated using a Superose 6 Increase 3.2/300 column. Immunoblots probing indicated PRPS complex members and internal standards are shown. d) Protein expression profiles of PRPS complex components in various mouse tissues. HSP90 was used as a loading control. 12-week-old male C57BL/6 mice were used. e) and f) and g) represent western blot analysis of SEC fractions collected from native tissue lysates of liver, kidney, and lung, respectively. 12-week-old male C57BL/6 mice were used. Cell lysates were fractionated on a Superose 6 Increase 3.2/300 column. Immunoblots probing PRPS complex members and internal standards are shown. The circular pictograms on the bottom of the SEC immunoblots represent the schematized configurations of PRPS complex. A double circle means multiple copies of the protein are interacting within the heteromeric complex.$

Journal: bioRxiv

Article Title: Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

doi: 10.1101/2024.10.01.616059

Figure Lengend Snippet: a) SDS-PAGE followed by Coomassie stain of the eluates from GFP immunoprecipitation (IP) performed in NIH3T3 and HEK293T cells stably expressing PRPS1-GFP, PRPS2-GFP and NES-GFP. NES (Nuclear export signal)-GFP cells were used as controls. Asterisk indicates a non-specific band in the eluates. b) Scatter plot from mass spectrometry (MS) runs of eluates from GFP immunoprecipitation (IP) in NIH3T3 and HEK293T cells stably expressing PRPS1-GFP on the x-axis and y-axis, respectively. The axes represent the square root-transformed SEQUEST HT scores of PRPS1-GFP normalized to control. PRPS1L1 isoform was detected in HEK29T cells only. c) Western blot analysis of size exclusion chromatography (SEC) fractions collected from NIH3T3 native whole cell lysates. Cell lysates were fractionated using a Superose 6 Increase 3.2/300 column. Immunoblots probing indicated PRPS complex members and internal standards are shown. d) Protein expression profiles of PRPS complex components in various mouse tissues. HSP90 was used as a loading control. 12-week-old male C57BL/6 mice were used. e) and f) and g) represent western blot analysis of SEC fractions collected from native tissue lysates of liver, kidney, and lung, respectively. 12-week-old male C57BL/6 mice were used. Cell lysates were fractionated on a Superose 6 Increase 3.2/300 column. Immunoblots probing PRPS complex members and internal standards are shown. The circular pictograms on the bottom of the SEC immunoblots represent the schematized configurations of PRPS complex. A double circle means multiple copies of the protein are interacting within the heteromeric complex.

Techniques: SDS Page, Staining, Immunoprecipitation, Stable Transfection, Expressing, Mass Spectrometry, Transformation Assay, Control, Western Blot, Size-exclusion Chromatography

$a) and b) and c) represent the scatter plots from mass spectrometry (MS) runs of eluates from GFP IP in stably expressing PRPS2-GFP, PRPSAP1-GFP, and PRPSAP2-GFP, respectively in NIH3T3 (x-axis) and HEK293T (y-axis) cells. The axes represent the square root-transformed SEQUEST HT scores normalized to control. d) Schematic of Prps1 gene with an endogenous ALFA tag knocked in frame at the C-terminus. mNG-11 represents the short fragment of the split monomeric Neon Green protein and ALFA is an epitope tag containing residues – PSRLEEELRRRLTEP. Western blot validating full-length expression of the endogenously tagged PRPS1-ALFA protein is shown. e) ALFA pulldown from the whole cell extracts of knock-in NIH3T3 cells demonstrating the interaction of endogenous PRPS1 with PRPS2, PRPSAP1, and PRPSAP2. f) Western blot analysis of SEC fractions collected from HEK293T native whole cell lysates. Cell lysates were fractionated on a Superose 6 Increase 3.2/300 column. Immunoblots probing PRPS complex members and internal standards are shown. In the pictogram, a double circle means multiple copies of the protein are interacting within the heteromeric complex. g) Chromatogram showing SEC traces of HEK293T whole cell lysates fractionated on a Bio SEC-5 2000Å column, which offers better fractionation for proteins/protein complexes in high molecular weight (HMW) range. The proteins that eluted in the fractions shown as hatch marks were sent for mass-spectrometry analyses for identification of proteins in HMW range. h) Classification of HMW proteins based on their functions (manual curation) from the mass-spectrometry dataset obtained from fractions collected in (g). i) Subclassification of enzymes from (h) based on their cellular localization. j) Normalized transcript per million (nTPM) of PRPS complex components in various human tissues obtained from Human Protein Atlas.$

Journal: bioRxiv

Article Title: Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

doi: 10.1101/2024.10.01.616059

Figure Lengend Snippet: a) and b) and c) represent the scatter plots from mass spectrometry (MS) runs of eluates from GFP IP in stably expressing PRPS2-GFP, PRPSAP1-GFP, and PRPSAP2-GFP, respectively in NIH3T3 (x-axis) and HEK293T (y-axis) cells. The axes represent the square root-transformed SEQUEST HT scores normalized to control. d) Schematic of Prps1 gene with an endogenous ALFA tag knocked in frame at the C-terminus. mNG-11 represents the short fragment of the split monomeric Neon Green protein and ALFA is an epitope tag containing residues – PSRLEEELRRRLTEP. Western blot validating full-length expression of the endogenously tagged PRPS1-ALFA protein is shown. e) ALFA pulldown from the whole cell extracts of knock-in NIH3T3 cells demonstrating the interaction of endogenous PRPS1 with PRPS2, PRPSAP1, and PRPSAP2. f) Western blot analysis of SEC fractions collected from HEK293T native whole cell lysates. Cell lysates were fractionated on a Superose 6 Increase 3.2/300 column. Immunoblots probing PRPS complex members and internal standards are shown. In the pictogram, a double circle means multiple copies of the protein are interacting within the heteromeric complex. g) Chromatogram showing SEC traces of HEK293T whole cell lysates fractionated on a Bio SEC-5 2000Å column, which offers better fractionation for proteins/protein complexes in high molecular weight (HMW) range. The proteins that eluted in the fractions shown as hatch marks were sent for mass-spectrometry analyses for identification of proteins in HMW range. h) Classification of HMW proteins based on their functions (manual curation) from the mass-spectrometry dataset obtained from fractions collected in (g). i) Subclassification of enzymes from (h) based on their cellular localization. j) Normalized transcript per million (nTPM) of PRPS complex components in various human tissues obtained from Human Protein Atlas.

Techniques: Mass Spectrometry, Stable Transfection, Expressing, Transformation Assay, Control, Western Blot, Knock-In, Fractionation, High Molecular Weight

$a) Western blot validating CRISPR-Cas9-generated isogenic knockout cell lines. b) Proliferation for the panel of NIH3T3 knockout cell lines generated in a). c) Western blot validating HRAS G12V -overexpression in parental and P2/AP1/AP2 KO cell lines. Phospho-MAPK (Erk1/2) (T202/Y204) was used as a marker for activation of signaling pathways upon HRAS G12V overexpression. d) Representative colony images from soft agar colony formation assay performed in NIH3T3 parental and P2/AP1/AP2 KO cells expressing HRAS G12V . e) Colony numbers from d). Error bars represent SD; n = 3 (***P < 0.001 by unpaired two-tailed t-test). f) Oxygen consumption rate (OCR) measured by Seahorse ATP Rate Assay in parental and P2/AP1/AP2 KO cell lines. Error bars represent SD; n = 7. g) Oxygen consumption rate (OCR) measured by Seahorse mitochondrial stress tests for NDI1-expressing P2/AP1/AP2 KO cell lines. Error bars represent SD; n = 7. h) Proliferation for nucleoside supplemented and NDI1 expressing P2/AP1/AP2 cell lines. Error bars represent SD; n = 3. (**P < 0.01; ***P < 0.001 by one-way ANOVA with post-hoc test). i) 13 C6-glucose metabolic labeling performed in NIH3T3 parental and P2/AP1/AP2 KO cell lines for 30 minutes (30’) and 5 hours (5h’). Unlabeled, 13 C-labeled (30 mins), and 13 C-labeled (5 hours) sets are represented in brown, green and blue colors, respectively. 13 C-enrichments were quantified from 1 H-NMR spectra. Error bars represent SE; n = 3 (**P < 0.01; ***P < 0.001 by unpaired two-tailed t-test). j) Western blot analysis of SEC fractions collected from NIH3T3 P2/AP1/AP2 KO native whole-cell lysates. Cell lysates were fractionated on a Superose 6 Increase 3.2/300 column. In the pictogram, a double circle with a dotted inner circle means multiple copies of PRPS1 are forming homo-oligomers.$

Journal: bioRxiv

Article Title: Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

doi: 10.1101/2024.10.01.616059

Figure Lengend Snippet: a) Western blot validating CRISPR-Cas9-generated isogenic knockout cell lines. b) Proliferation for the panel of NIH3T3 knockout cell lines generated in a). c) Western blot validating HRAS G12V -overexpression in parental and P2/AP1/AP2 KO cell lines. Phospho-MAPK (Erk1/2) (T202/Y204) was used as a marker for activation of signaling pathways upon HRAS G12V overexpression. d) Representative colony images from soft agar colony formation assay performed in NIH3T3 parental and P2/AP1/AP2 KO cells expressing HRAS G12V . e) Colony numbers from d). Error bars represent SD; n = 3 (***P < 0.001 by unpaired two-tailed t-test). f) Oxygen consumption rate (OCR) measured by Seahorse ATP Rate Assay in parental and P2/AP1/AP2 KO cell lines. Error bars represent SD; n = 7. g) Oxygen consumption rate (OCR) measured by Seahorse mitochondrial stress tests for NDI1-expressing P2/AP1/AP2 KO cell lines. Error bars represent SD; n = 7. h) Proliferation for nucleoside supplemented and NDI1 expressing P2/AP1/AP2 cell lines. Error bars represent SD; n = 3. (**P < 0.01; ***P < 0.001 by one-way ANOVA with post-hoc test). i) 13 C6-glucose metabolic labeling performed in NIH3T3 parental and P2/AP1/AP2 KO cell lines for 30 minutes (30’) and 5 hours (5h’). Unlabeled, 13 C-labeled (30 mins), and 13 C-labeled (5 hours) sets are represented in brown, green and blue colors, respectively. 13 C-enrichments were quantified from 1 H-NMR spectra. Error bars represent SE; n = 3 (**P < 0.01; ***P < 0.001 by unpaired two-tailed t-test). j) Western blot analysis of SEC fractions collected from NIH3T3 P2/AP1/AP2 KO native whole-cell lysates. Cell lysates were fractionated on a Superose 6 Increase 3.2/300 column. In the pictogram, a double circle with a dotted inner circle means multiple copies of PRPS1 are forming homo-oligomers.

Techniques: Western Blot, CRISPR, Generated, Knock-Out, Over Expression, Marker, Activation Assay, Protein-Protein interactions, Soft Agar Assay, Expressing, Two Tailed Test, Labeling

$a-f) Western blot analysis of SEC fractions collected from native whole-cell lysates of NIH3T3 P2/AP2 KO cells (a), AP1/AP2 KO cells (b), AP1 KO cells (c) AP1/AP2 KO cells (d), AP1/AP2 KO cells stably expressing chimeric AP1 with AP2’s N-terminus (residues 1-95) (e) and chimeric AP2 with AP1’s N-terminus (residues 1-95) (f). g) Multiple isoforms for AP1 and AP2 were detected under optimal SDS-PAGE resolution. The arrows in immunoblots indicate the longer isoforms of AP1 and AP2, which are N-terminal leader sequences of 29 and 12 amino acids, respectively. h) and i) Western blot analysis of SEC fractions collected from NIH3T3 AP1/AP2 KO cells stably expressing the short isoform of AP1 (h) and long isoform of AP1 (i). Cell lysates for (a), (b), (e), (f), (h), and (i) were fractionated on a Superose 6 Increase 3.2/300 column whereas cell lysates for (c) and (d) were fractionated on a Yarra SEC-2000 column. The circular pictograms at the bottom of the SEC immunoblots illustrate the different configurations of the PRPS complex. A double circle means multiple copies of the protein are interacting within the heteromeric complex. A double circle with a dotted inner circle means multiple copies of the protein are forming homo-oligomers. A single circle means a single protein is interacting within the complex. A circle with lines inside indicates that the proteins may be forming a trimer or tetramer. j) Association of different PRPS complex configurations with cellular fitness. (I) PRPS1 alone undergoes self-assembly to form homo-oligomers. (II) PRPS1 and AP1 cannot interact directly and form separate homo-oligomers. (III) PRPS2 binds PRPS1 to form a dimer and disrupts PRPS1 homotypic assemblies. (IV) AP2 preferentially binds with PRPS1 to form a trimer/tetramer to nucleate the complex. (V) AP1 preferentially binds with AP2 and PRPS2 to elongate the PRPS complex via its N-terminus. (VI) The longer isoform of AP1 with flexible N-terminal extension likely caps the complex. The weight of the connectors between the circles representing proteins reflects the predicted strength of protein-protein interactions between them. The heavier the line, the stronger the predicted interaction between the members of the complex.$

Journal: bioRxiv

Article Title: Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

doi: 10.1101/2024.10.01.616059

Figure Lengend Snippet: a-f) Western blot analysis of SEC fractions collected from native whole-cell lysates of NIH3T3 P2/AP2 KO cells (a), AP1/AP2 KO cells (b), AP1 KO cells (c) AP1/AP2 KO cells (d), AP1/AP2 KO cells stably expressing chimeric AP1 with AP2’s N-terminus (residues 1-95) (e) and chimeric AP2 with AP1’s N-terminus (residues 1-95) (f). g) Multiple isoforms for AP1 and AP2 were detected under optimal SDS-PAGE resolution. The arrows in immunoblots indicate the longer isoforms of AP1 and AP2, which are N-terminal leader sequences of 29 and 12 amino acids, respectively. h) and i) Western blot analysis of SEC fractions collected from NIH3T3 AP1/AP2 KO cells stably expressing the short isoform of AP1 (h) and long isoform of AP1 (i). Cell lysates for (a), (b), (e), (f), (h), and (i) were fractionated on a Superose 6 Increase 3.2/300 column whereas cell lysates for (c) and (d) were fractionated on a Yarra SEC-2000 column. The circular pictograms at the bottom of the SEC immunoblots illustrate the different configurations of the PRPS complex. A double circle means multiple copies of the protein are interacting within the heteromeric complex. A double circle with a dotted inner circle means multiple copies of the protein are forming homo-oligomers. A single circle means a single protein is interacting within the complex. A circle with lines inside indicates that the proteins may be forming a trimer or tetramer. j) Association of different PRPS complex configurations with cellular fitness. (I) PRPS1 alone undergoes self-assembly to form homo-oligomers. (II) PRPS1 and AP1 cannot interact directly and form separate homo-oligomers. (III) PRPS2 binds PRPS1 to form a dimer and disrupts PRPS1 homotypic assemblies. (IV) AP2 preferentially binds with PRPS1 to form a trimer/tetramer to nucleate the complex. (V) AP1 preferentially binds with AP2 and PRPS2 to elongate the PRPS complex via its N-terminus. (VI) The longer isoform of AP1 with flexible N-terminal extension likely caps the complex. The weight of the connectors between the circles representing proteins reflects the predicted strength of protein-protein interactions between them. The heavier the line, the stronger the predicted interaction between the members of the complex.

Techniques: Western Blot, Stable Transfection, Expressing, SDS Page, Protein-Protein interactions

$a) Western blot analysis of SEC fractions collected from native whole cell lysates of NIH3T3 P2/AP1/AP2 KO cells stably expressing AP1 b) ALFA pulldown from the whole cell extracts of NIH3T3 parental and P2/AP2 KO cells transiently transfected with PRPS1-ALFA c-i) Western blot analysis of SEC fractions collected from native whole cell lysates of NIH3T3 P2/AP1 KO cells (c), P1/AP2 KO cells (d), P1 KO cells (e), P2 KO cells (f), AP2 KO cells (g), AP1 KO cells (h), and P1/AP1 KO cells (i). All the fractionation experiments were performed on Superose 6 Increase 3.2/300 column. The circular pictograms at the bottom of the SEC immunoblots illustrate the different configurations of the PRPS complex. A double circle means multiple copies of the protein are interacting within the heteromeric complex. A double circle with a dotted inner circle means multiple copies of the protein are forming homo-oligomers. A single circle means a single protein is interacting within the complex. A circle with lines inside indicates that the proteins may be forming a trimer or tetramer.$

Journal: bioRxiv

Article Title: Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

doi: 10.1101/2024.10.01.616059

Figure Lengend Snippet: a) Western blot analysis of SEC fractions collected from native whole cell lysates of NIH3T3 P2/AP1/AP2 KO cells stably expressing AP1 b) ALFA pulldown from the whole cell extracts of NIH3T3 parental and P2/AP2 KO cells transiently transfected with PRPS1-ALFA c-i) Western blot analysis of SEC fractions collected from native whole cell lysates of NIH3T3 P2/AP1 KO cells (c), P1/AP2 KO cells (d), P1 KO cells (e), P2 KO cells (f), AP2 KO cells (g), AP1 KO cells (h), and P1/AP1 KO cells (i). All the fractionation experiments were performed on Superose 6 Increase 3.2/300 column. The circular pictograms at the bottom of the SEC immunoblots illustrate the different configurations of the PRPS complex. A double circle means multiple copies of the protein are interacting within the heteromeric complex. A double circle with a dotted inner circle means multiple copies of the protein are forming homo-oligomers. A single circle means a single protein is interacting within the complex. A circle with lines inside indicates that the proteins may be forming a trimer or tetramer.

Techniques: Western Blot, Stable Transfection, Expressing, Transfection, Fractionation

a) Schematic representation of alternative start sites in mammalian AP1 and AP2 and their consequent translation into the short and long isoforms. Base positions for AP1 and AP2 correspond to the mouse homologs. TSS1 and TSS2 represents the upstream and downstream translation start sites, respectively. b) Phylogenetic distribution profiles of PRPS homologs (PRPS1, PRPS2, PRPSAP2, PRPSAP2 with N-terminal leader sequence, PRPSAP1, and PRPSAP1 with N-terminal leader sequence) in chordates (presence/absence) are noted across the tree. PRPSAP2 and PRPSAP1 isoforms with additional N-terminal leader sequences emerged in the ancestor of Amniota and Osteichthyes, respectively. c) and d) The N-terminal residues from a sequence alignment of PRPSAP2 (c) and PRPSAP1 (d) from representative organisms of Amniota and Osteichthyes, respectively. TSS1 and TSS2 represents the upstream and downstream translation start sites, respectively.

Journal: bioRxiv

Article Title: Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

doi: 10.1101/2024.10.01.616059

Figure Lengend Snippet: a) Schematic representation of alternative start sites in mammalian AP1 and AP2 and their consequent translation into the short and long isoforms. Base positions for AP1 and AP2 correspond to the mouse homologs. TSS1 and TSS2 represents the upstream and downstream translation start sites, respectively. b) Phylogenetic distribution profiles of PRPS homologs (PRPS1, PRPS2, PRPSAP2, PRPSAP2 with N-terminal leader sequence, PRPSAP1, and PRPSAP1 with N-terminal leader sequence) in chordates (presence/absence) are noted across the tree. PRPSAP2 and PRPSAP1 isoforms with additional N-terminal leader sequences emerged in the ancestor of Amniota and Osteichthyes, respectively. c) and d) The N-terminal residues from a sequence alignment of PRPSAP2 (c) and PRPSAP1 (d) from representative organisms of Amniota and Osteichthyes, respectively. TSS1 and TSS2 represents the upstream and downstream translation start sites, respectively.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

doi: 10.1101/2024.10.01.616059

Figure Lengend Snippet: a) The structure of trimeric human PRPS1 (PDB ID: 2HCR). The dashed box represents the allosteric site I, a zoom in of this site shows SO (which represents the phosphate of ADP) positioned at the trimeric interface and red color indicates the residues from each subunit contributing to the formation of allosteric site I. b) The amino acid sequence of the human PRPS1 was aligned with sequences of representative organisms from Opisthokonta PRPS1 (n = 44), Amoebozoa PrsA (n = 19), Opisthokonta PRPSAP2 (n = 46), jawed Vertebrata PRPSAP1 (n = 92), Amoebozoa PrsB (n = 20) and Class II PRPS (n = 53), and the corresponding allosteric site I residues were selected for generating the WebLogo. The numbers below the logo sequences indicate the corresponding residues positions of human PRPS1 and PRPSAP2 (for Opisthokonts), R. potamoides PrsA and PrsB (for Amoebozoa), human PRPSAP1 (for jawed Vertebrata), and A. parasiticum Class II PRPS annotated from SRX179384. X represents the absent amino acid residue at that position jawed Vertebrata AP1. Class II PRPS enzymes are shown as a reference since they are known to lack allosteric sites found in Class I PRPS (“classical” PRPS enzymes) . c) The structure of dimeric human PRPS1 (PDB ID: 2HCR). The dashed box represents the allosteric site II, a zoom in of this site shows SO positioned at the dimer interface and red color indicates the residues from each subunit contributing to the formation of allosteric site II. d) The amino acid sequence of the human PRPS1 was aligned with sequences from Opisthokonta PRPS1, Amoebozoa PrsA, Opisthokonta PRPSAP2, jawed Vertebrata PRPSAP1, Amoebozoa PrsB and Class II PRPS, and the corresponding allosteric site II residues were selected for generating the WebLogo similar to (b).

Techniques: Sequencing, Residue

PRPS2 regulates chemotaxis of tumor‐associated macrophages (TAM) and myeloid‐derived suppressor cells (MDSC) in tumor cells. (a) Relative mRNA and (b) protein levels of PRPS2 in control Lewis lung cancer (LLC), LLC‐shPRPS2, and LLC‐PRPS2 cells. (c) Number of migrated macrophages and (d) MDSC in a transwell chemotactic assay. Four wells per group. Statistical significance was analyzed using one‐way analysis of variance (ANOVA).

Journal: Thoracic Cancer

Article Title: PRPS2‐mediated modulation of the antitumor immune response in lung cancer through CCL2 ‐mediated tumor‐associated macrophages and myeloid‐derived suppressor cells

doi: 10.1111/1759-7714.15398

Figure Lengend Snippet: PRPS2 regulates chemotaxis of tumor‐associated macrophages (TAM) and myeloid‐derived suppressor cells (MDSC) in tumor cells. (a) Relative mRNA and (b) protein levels of PRPS2 in control Lewis lung cancer (LLC), LLC‐shPRPS2, and LLC‐PRPS2 cells. (c) Number of migrated macrophages and (d) MDSC in a transwell chemotactic assay. Four wells per group. Statistical significance was analyzed using one‐way analysis of variance (ANOVA).

Article Snippet: The following primary antibodies were used including PRPS2 antibody (ThermoFisher, cat no: PA5‐42007; at a dilution of 1:2000), CCL2 antibody (Abcam, cat no: ab25124; at a dilution of 1:1000), and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody (CST, cat no: 2118; at a dilution of 1:5000).

Techniques: Chemotaxis Assay, Derivative Assay, Control

PRPS2‐mediated CCL2 in tumor cells regulates chemotaxis of tumor‐associated macrophages (TAM) and myeloid‐derived suppressor cells (MDSC). (a) Relative mRNA and (b) protein levels of Ccl2 in control Lewis lung cancer (LLC), LLC‐shPRPS2, and LLC‐PRPS2 cells. (c) CCL2 protein level in the culture medium of control LLC, LLC‐shPRPS2, and LLC‐PRPS2 cells. (d) Number of migrated macrophages and (e) MDSC in a transwell chemotactic assay using isotype or anti‐CCL2 antibody. Four wells per group. Statistical significance was analyzed using one‐way analysis of variance (ANOVA).

Journal: Thoracic Cancer

Article Title: PRPS2‐mediated modulation of the antitumor immune response in lung cancer through CCL2 ‐mediated tumor‐associated macrophages and myeloid‐derived suppressor cells

doi: 10.1111/1759-7714.15398

Figure Lengend Snippet: PRPS2‐mediated CCL2 in tumor cells regulates chemotaxis of tumor‐associated macrophages (TAM) and myeloid‐derived suppressor cells (MDSC). (a) Relative mRNA and (b) protein levels of Ccl2 in control Lewis lung cancer (LLC), LLC‐shPRPS2, and LLC‐PRPS2 cells. (c) CCL2 protein level in the culture medium of control LLC, LLC‐shPRPS2, and LLC‐PRPS2 cells. (d) Number of migrated macrophages and (e) MDSC in a transwell chemotactic assay using isotype or anti‐CCL2 antibody. Four wells per group. Statistical significance was analyzed using one‐way analysis of variance (ANOVA).

Techniques: Chemotaxis Assay, Derivative Assay, Control

PRPS2 mediates CCL2 expression in vivo. (a) Lewis lung cancer (LLC) tumor volume at day 7, 10, and 14, and representative tumor images at day 14. Statistical significance was analyzed using two‐way analysis of variance (ANOVA). (b) Tumor weight at day 14. Eight mice per group. (c) The mRNA and (d) protein levels of CCL2 in control LLC, LLC‐shPRPS2, and LLC‐PRPS2 tumor tissues. Four mice per group. Statistical significance was analyzed using one‐way ANOVA.

Journal: Thoracic Cancer

Article Title: PRPS2‐mediated modulation of the antitumor immune response in lung cancer through CCL2 ‐mediated tumor‐associated macrophages and myeloid‐derived suppressor cells

doi: 10.1111/1759-7714.15398

Figure Lengend Snippet: PRPS2 mediates CCL2 expression in vivo. (a) Lewis lung cancer (LLC) tumor volume at day 7, 10, and 14, and representative tumor images at day 14. Statistical significance was analyzed using two‐way analysis of variance (ANOVA). (b) Tumor weight at day 14. Eight mice per group. (c) The mRNA and (d) protein levels of CCL2 in control LLC, LLC‐shPRPS2, and LLC‐PRPS2 tumor tissues. Four mice per group. Statistical significance was analyzed using one‐way ANOVA.

Techniques: Expressing, In Vivo, Control

PRPS2 regulates immune cell infiltration into tumors. Percentage of (a) CD4 T cells (CD3 + CD4 + ), (b) CD8 T cells (CD3 + CD8 + ), (c) tumor‐associated macrophages (TAM) (CD45 + F4/80 + CD206 + ), (d) M‐MDSC (CD45 + CD11b + Ly6C + ), and (e) PMN‐MDSC (CD45 + CD11b + Ly6G + ) in control Lewis lung cancer (LLC), LLC‐shPRPS2, and LLC‐PRPS2 tumor tissues at day 14. Relative mRNA levels of (f) S100A8, (g) S100A9, (h) Nos2, and (i) Arg1 in control LLC, LLC‐shPRPS2, and LLC‐PRPS2 tumor tissues at day 14. Four mice per group. Statistical significance was analyzed using one‐way analysis of variance (ANOVA).

Journal: Thoracic Cancer

Article Title: PRPS2‐mediated modulation of the antitumor immune response in lung cancer through CCL2 ‐mediated tumor‐associated macrophages and myeloid‐derived suppressor cells

doi: 10.1111/1759-7714.15398

Figure Lengend Snippet: PRPS2 regulates immune cell infiltration into tumors. Percentage of (a) CD4 T cells (CD3 + CD4 + ), (b) CD8 T cells (CD3 + CD8 + ), (c) tumor‐associated macrophages (TAM) (CD45 + F4/80 + CD206 + ), (d) M‐MDSC (CD45 + CD11b + Ly6C + ), and (e) PMN‐MDSC (CD45 + CD11b + Ly6G + ) in control Lewis lung cancer (LLC), LLC‐shPRPS2, and LLC‐PRPS2 tumor tissues at day 14. Relative mRNA levels of (f) S100A8, (g) S100A9, (h) Nos2, and (i) Arg1 in control LLC, LLC‐shPRPS2, and LLC‐PRPS2 tumor tissues at day 14. Four mice per group. Statistical significance was analyzed using one‐way analysis of variance (ANOVA).

Techniques: Control

Knockdown of CCL2 reverses the phenotype of PRPS2 overexpression. (a) Relative mRNA level of CCL2 in control Lewis lung cancer (LLC), LLC‐PRPS2‐shControl, and LLC‐PRPS2‐shCCL2 cells in vitro. (b) CCL2 protein level in the culture medium of control LLC, LLC‐PRPS2‐shControl, and LLC‐PRPS2‐shCCL2 cells. Four wells per group. Statistical significance was analyzed using one‐way analysis of variance (ANOVA). (c) LLC tumor volume at day 7, 10, and 14. Statistical significance was analyzed using two‐way ANOVA. (d) Tumor weight and representative tumor images at day 14. Eight mice per group. The (e) mRNA and (f) protein levels of Ccl2 in control LLC, LLC‐PRPS2‐shControl, and LLC‐PRPS2‐shCCL2 tumor tissues. (g) Percentage of CD4 T cells (CD3 + CD4 + ), (h) CD8 T cells (CD3 + CD8 + ), (i) tumor‐associated macrophages (TAM) (CD45 + F4/80 + CD206 + ), (j) M‐MDSC (CD45 + CD11b + Ly6C + ) and (k) PMN‐MDSC (CD45 + CD11b + Ly6G + ) in control LLC, LLC‐PRPS2‐shControl, and LLC‐PRPS2‐shCcl2 tumor tissues at day 14. Four mice per group. Statistical significance was analyzed using one‐way ANOVA.

Journal: Thoracic Cancer

Article Title: PRPS2‐mediated modulation of the antitumor immune response in lung cancer through CCL2 ‐mediated tumor‐associated macrophages and myeloid‐derived suppressor cells

doi: 10.1111/1759-7714.15398

Figure Lengend Snippet: Knockdown of CCL2 reverses the phenotype of PRPS2 overexpression. (a) Relative mRNA level of CCL2 in control Lewis lung cancer (LLC), LLC‐PRPS2‐shControl, and LLC‐PRPS2‐shCCL2 cells in vitro. (b) CCL2 protein level in the culture medium of control LLC, LLC‐PRPS2‐shControl, and LLC‐PRPS2‐shCCL2 cells. Four wells per group. Statistical significance was analyzed using one‐way analysis of variance (ANOVA). (c) LLC tumor volume at day 7, 10, and 14. Statistical significance was analyzed using two‐way ANOVA. (d) Tumor weight and representative tumor images at day 14. Eight mice per group. The (e) mRNA and (f) protein levels of Ccl2 in control LLC, LLC‐PRPS2‐shControl, and LLC‐PRPS2‐shCCL2 tumor tissues. (g) Percentage of CD4 T cells (CD3 + CD4 + ), (h) CD8 T cells (CD3 + CD8 + ), (i) tumor‐associated macrophages (TAM) (CD45 + F4/80 + CD206 + ), (j) M‐MDSC (CD45 + CD11b + Ly6C + ) and (k) PMN‐MDSC (CD45 + CD11b + Ly6G + ) in control LLC, LLC‐PRPS2‐shControl, and LLC‐PRPS2‐shCcl2 tumor tissues at day 14. Four mice per group. Statistical significance was analyzed using one‐way ANOVA.

Techniques: Knockdown, Over Expression, Control, In Vitro

Depletion of tumor‐associated macrophages (TAM) and myeloid‐derived suppressor cells (MDSC) reverses the phenotype of PRPS2 overexpression. (a) Lewis lung cancer (LLC) tumor volume at day 7, 10, and 14. (b) Tumor weight at day 14. Eight mice per group. (c) Percentage of CD4 T cells (CD3 + CD4 + ), (d) CD8 T cells (CD3 + CD8 + ), (e) TAM (CD45 + F4/80 + CD206 + ), (f) M‐MDSC (CD45 + CD11b + Ly6C + ) and (g) PMN‐MDSC (CD45 + CD11b + Ly6G + ) in LLC‐PRPS2 tumor tissues at day 14 from mice treated with control reagent (control liposomes and isotype antibody), clodronate liposomes (CL), or anti‐Gr‐1 antibody. Four mice per group. Statistical significance was analyzed using one‐way analysis of variance (ANOVA).

Journal: Thoracic Cancer

Article Title: PRPS2‐mediated modulation of the antitumor immune response in lung cancer through CCL2 ‐mediated tumor‐associated macrophages and myeloid‐derived suppressor cells

doi: 10.1111/1759-7714.15398

Figure Lengend Snippet: Depletion of tumor‐associated macrophages (TAM) and myeloid‐derived suppressor cells (MDSC) reverses the phenotype of PRPS2 overexpression. (a) Lewis lung cancer (LLC) tumor volume at day 7, 10, and 14. (b) Tumor weight at day 14. Eight mice per group. (c) Percentage of CD4 T cells (CD3 + CD4 + ), (d) CD8 T cells (CD3 + CD8 + ), (e) TAM (CD45 + F4/80 + CD206 + ), (f) M‐MDSC (CD45 + CD11b + Ly6C + ) and (g) PMN‐MDSC (CD45 + CD11b + Ly6G + ) in LLC‐PRPS2 tumor tissues at day 14 from mice treated with control reagent (control liposomes and isotype antibody), clodronate liposomes (CL), or anti‐Gr‐1 antibody. Four mice per group. Statistical significance was analyzed using one‐way analysis of variance (ANOVA).

Techniques: Derivative Assay, Over Expression, Control, Liposomes

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