Journal: Archives of biochemistry and biophysics

Article Title: UGDH promotes 5-fluorouracil resistance in colorectal cancer via the ROS-activated PI3K/AKT-EEF1A2-PRDX1 pathway.

doi: 10.1016/j.abb.2025.110445

Figure Lengend Snippet: Fig. 7. ROS activate the PI3K/AKT pathway, regulating the UGDH-EEF1A2-PRDX1 axis to promote chemoresistance. (A) KEGG pathway enrichment of DEGs in UGDH-knockdown SW620 cells (n = 3 replicates). (B) Western blot of PI3K/AKT pathway proteins in HCT8 cells ± LY294002 (10 μM, 48 h, n = 3). (C) PI3K/AKT phosphorylation in HCT8 control and shUGDH cells ± 5-FU (n = 3). (D) UGDH and PI3K/AKT activation in HCT8 cells treated with H2O2 (0–200 μM, 24 h, n = 3). (E) Time-dependent UGDH activation in HCT8 cells treated with 15 μM 5-FU (0–96 h). (n = 3). (F) Western blot of PI3K/AKT pathway proteins in HCT8 cells ± LY294002 (10 μM, n = 3)/± NAC (750 μМ, n = 3). Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The primary antibodies used were against UGDH (Proteintech), EEF1A2 (Proteintech), PRDX1 (Proteintech), PI3K (CST), phospho-PI3K (CST), Akt (CST), phospho-AKT (Ser473) (CST), FLAG (Proteintech), β-ACTIN (Proteintech), GAPDH (Beyotime), cleaved caspase 3 (CST), and cleaved PARP (CST).

Techniques: Knockdown, Western Blot, Phospho-proteomics, Control, Activation Assay

Journal: Hormones & cancer

Article Title: Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential.

doi: 10.1007/s12672-016-0268-z

Figure Lengend Snippet: Fig. 2 Re-expression of AR-activated genes UGDH and PSA is dose dependent following prolonged androgen depletion. LNCaP 33 and 81 cells were selected for 10 days in the indicated DHTconcentrations. Prior to harvest, media were removed and replaced with media containing vehicle or 10 nM DHT for 24 h. a Equivalent amounts of protein were immunoblotted for UGDH and tubulin. UGDH expression was quantified, normalized to tubulin, and plotted as the ratio of 24 h DHT- treated versus vehicle-treated expression to illustrate stimulated expression. Mean ± SEM is plotted; *p < 0.05 relative to LNCaP 33 at 10 nM DHT; ‡p < 0.05 for LNCaP 81 relative to LNCaP 33 at the indicated DHT treatment. b Blots in (a) were re-probed for PSA (34 kDa) and AR (102 kDa)

Article Snippet: To generate UGDH knockdown lines, LNCaP 33 cells were transfected with plasmids encoding short hairpin RNA (shRNA) targeted to UGDH or a scrambled non-targeting control (UGDH shRNA-pGFP-V-RS no. TG334012 and 29-mer oligo-pGFP-V-RS no. TR30008, respectively, from Origene Technologies, Rockville, MD).

Techniques: Expressing

Journal: Hormones & cancer

Article Title: Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential.

doi: 10.1007/s12672-016-0268-z

Figure Lengend Snippet: Fig. 7 UGDH knockdown reduces HSD3B1. LNCaP 33, 81, 22Rv1, and PC3 cell lines (a, c) or LNCaP 33 control and UGDH knockdown clones (b, d) were grown in standard steroid-replete media. Total cell lysates were normalized by protein content and analyzed in triplicate by immunoblots probed for HSD17B3 (a, b) or HSD3B1 (c, d). Densitometric quantification was normalized to tubulin and plotted as mean ± SEM of fold changes relative to LNCaP 33 or C1. *p < 0.05 relative to LNCaP 33; ‡p < 0.05 relative to C1

Article Snippet: To generate UGDH knockdown lines, LNCaP 33 cells were transfected with plasmids encoding short hairpin RNA (shRNA) targeted to UGDH or a scrambled non-targeting control (UGDH shRNA-pGFP-V-RS no. TG334012 and 29-mer oligo-pGFP-V-RS no. TR30008, respectively, from Origene Technologies, Rockville, MD).

Techniques: Knockdown, Control, Clone Assay, Western Blot

Journal:

Article Title: False-Positive Results Obtained by Following a Commonly Used Reverse Transcription-PCR Protocol for Detection of Influenza A Virus

doi: 10.1128/JCM.00916-06

Figure Lengend Snippet: Comparison of Gallus gallus clone AY006 28S rRNA gene and M52C and M253R oligonucleotides. Coincident nucleotides are shown in bold and in shaded boxes. *, M253R oligonucleotide reverse complementary sequence.

Article Snippet: A detailed study of the M52C and M253R primers and the Gallus gallus 28S clone AY006 rRNA sequence showed that the 3′ termini of the M52C and M253R primers shared 7 and 5 nucleotides, respectively, with the sequence of the Gallus gallus 28S rRNA gene (Fig. ).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Glycosaminoglycan-mediated lipoprotein uptake protects cancer cells from ferroptosis

doi: 10.1101/2024.05.13.593939

Figure Lengend Snippet: a. Schematic of parallel CRISPR screens in Karpas299 lymphoma cells transduced with a metabolism-focused sgRNA library (3,000 genes). First, a proliferation-based screen in the presence or absence of a GPX4 inhibitor (ML210) during 14 population doublings (left); second, a FACS-based screen where cells were incubated with DiI-LDL for 2 hours and subjected to sorting for 5% highest and 5% lowest fluorescent cells (right). b. Rank of most essential genes under ML210 treatment compared to untreated Karpas299 cells. Canonical anti-ferroptotic genes are shown in light blue, and glycosaminoglycan biosynthesis genes are shown in dark blue. c. Rank of most essential genes for DiI-LDL uptake in Karpas299 cells. Lipoprotein uptake genes are shown in orange and glycosaminoglycan biosynthesis pathway genes are shown in dark blue. d. Gene scores ranks for the GPX4 inhibition and DiI-LDL uptake screens in Karpas299. All genes essential in both screens are part of the glycosaminoglycan biosynthesis pathway (dark blue). e. Glycosaminoglycans, such as heparan sulfate and chondroitin sulfate, are formed by disaccharides repeats containing glucuronic acid (GlcUA) and other monosaccharides. The most upstream and rate-limiting enzyme of this pathway is UGDH. All genes in the pathway that scored in CRISPR screens are highlighted in blue. f. Quantification of total milligrams of heparan sulfate (HS, left) and chondroitin sulfate (CS, right) per grams of protein in the indicated UGDH _KO cell lines expressing a sgRNA resistant UGDH cDNA (blue) or an empty vector (grey). g. Number of doublings in 5 days (log 2 ) of the indicated UGDH _KO cell lines expressing a sgRNA resistant UGDH cDNA (blue) or an empty vector (grey) under the indicated concentrations of the GPX4 inhibitor, ML162. h. Representative flow cytometry plots showing the cellular uptake of DiI-LDL (left) or DiI-HDL (right) in A-498 UGDH _KO cells expressing a sgRNA resistant UGDH cDNA (blue) or an empty vector (grey). i. Immunoblot analysis of LDLR, SCARB1, and UGDH in HeLa cells transduced with a sgControl, sg LDLR , sg SCARB1 or sg UGDH . GAPDH is included as a loading control. j. Fold change in the uptake of DiI-LDL of the indicated HeLa cell lines relative to sgControl-transduced cells assessed by flow cytometry. k. Fold change in the uptake of DiI-HDL of the indicated HeLa cell lines relative to sgControl-transduced cells assessed by flow cytometry. f, g, j, k, Bars or lines represent mean ± s.d.; f, g, j, k, n=3 biologically independent samples. Statistical significance determined by two-tailed unpaired t-tests.

Article Snippet: Antibodies to ACSL3 (Ab151959), human GPX4 (Ab41787), LDLR (52818), SCARB1(52629) were purchased from Abcam; Heparan Sulfate (370255-S) from AMSBio; IRP2 (37135S), SLC7A11 (12691S), TFRC (13208S), and β-tubulin (2146S) from Cell Signaling Technology; GAPDH (GTX627408) and UGDH (GTX104993) from GeneTex; AIFM2 (20866-1-AP) and PAPSS1 (14708-1-AP) from ProteinTech; mouse GPX4 (sc-166570) from Santa Cruz Biotechnology; and Chondroitin Sulfate (C8035) from Sigma.

Techniques: CRISPR, Transduction, Incubation, Inhibition, Expressing, Plasmid Preparation, Flow Cytometry, Western Blot, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Glycosaminoglycan-mediated lipoprotein uptake protects cancer cells from ferroptosis

doi: 10.1101/2024.05.13.593939

Figure Lengend Snippet: a. Gene essentiality plot showing genes scores in untreated (x-axis) and ML210-treated (GPX4 inhibition) Karpas299 cells. Glycosaminoglycan (GAG) biosynthesis genes are highlighted (dark blue). b. Top-scoring genes under ML210 treatment. Negative scores represent genes whose loss potentiates ML210 toxicity; positive scores represent genes whose loss provides resistance to ML210. GAG biosynthesis genes are shown in dark blue, canonical anti-ferroptotic genes are shown in light blue, and lipid metabolism genes are shown in yellow. c. Top-scoring genes essential for DiI-LDL uptake. Negative scores represent genes whose loss reduce cellular DiI-LDL uptake. GAG biosynthesis genes are shown in dark blue and genes in canonical lipoprotein uptake pathways highlighted in yellow. d. Individual sgRNA scores for UGDH in untreated and ML210-treated conditions. e. Individual sgRNA scores for UGDH in high and low DiI-LDL uptake populations. f. Immunoblot analysis of UGDH in the indicated isogenic cell lines transduced with sg UGDH and expressing or not a sgRNA-resistant UGDH cDNA. GAPDH is included as a loading control. g. Number of doublings (log 2 ) in 5 days of indicated HeLa (left) or Caki-2 (right) cell lines expressing UGDH (blue) or not (grey) under the indicated concentrations of the GPX4 inhibitor ML162. h. Cellular lipid oxidation ratio using BODIPY-C11 in the indicated A-498 (left) or Karpas299 (right) UGDH _KO cell lines expressing a sgRNA resistant UGDH cDNA (blue) or an empty vector (grey) under the indicated conditions of GPX4 inhibition (ML162: 200 nM for Karpas299, 250 nM for A-498) and Fer-1 supplementation (1μM). i. Number of doublings (log 2 ) in 5 days of the indicated Karpas299 (top), A-498 (middle) and Tom2 (bottom) UGDH-deficient cell lines expressing a sgRNA resistant UGDH cDNA (dark blue) or an empty vector (grey) under the indicated concentrations of erastin. g-i, Bars represent mean ± s.d.; g-i, n=3 biologically independent samples. Statistical significance determined by two-tailed unpaired t-tests compared to empty vector transduced cells.

Article Snippet: Antibodies to ACSL3 (Ab151959), human GPX4 (Ab41787), LDLR (52818), SCARB1(52629) were purchased from Abcam; Heparan Sulfate (370255-S) from AMSBio; IRP2 (37135S), SLC7A11 (12691S), TFRC (13208S), and β-tubulin (2146S) from Cell Signaling Technology; GAPDH (GTX627408) and UGDH (GTX104993) from GeneTex; AIFM2 (20866-1-AP) and PAPSS1 (14708-1-AP) from ProteinTech; mouse GPX4 (sc-166570) from Santa Cruz Biotechnology; and Chondroitin Sulfate (C8035) from Sigma.

Techniques: Inhibition, Western Blot, Transduction, Expressing, Control, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Glycosaminoglycan-mediated lipoprotein uptake protects cancer cells from ferroptosis

doi: 10.1101/2024.05.13.593939

Figure Lengend Snippet: a. Number of doublings (log 2 ) in 5 days of the indicated Karpas299 (left) and A-498 (right) UGDH _KO cell lines expressing a sgRNA resistant UGDH cDNA (blue) or an empty vector (grey) under the indicated concentrations of ferrous ammonium citrate (FAC). b. Immunoblot analysis of TFRC (top) and IRP1 (bottom) in the indicated UGDH _KO cell lines expressing a sgRNA resistant UGDH cDNA or an empty vector treated or not with FAC (0.1 mg/mL). GAPDH is included as a loading control. c. Immunoblot analysis of GPX4 (left), SLC7A11 (center), and AIFM2 (right) in the indicated UGDH _KO cell lines expressing a sgRNA resistant UGDH cDNA or an empty vector. GAPDH is included as a loading control. d. Representative flow cytometry plot showing the uptake of DiI-LDL (PE median intensity) in Karpas299 UGDH _KO cells expressing a sgRNA resistant UGDH cDNA (blue) or an empty vector (grey). e. Cellular uptake of DiI-LDL measured as median PE intensity in the indicated Karpas299 (left), A-498 (center) and HeLa (right) UGDH _KO cells expressing a sgRNA resistant UGDH cDNA (blue) or an empty vector (grey). f. Cellular uptake of DiI-HDL measured as median PE intensity in the indicated A-498 (left) and HeLa (right) UGDH _KO cells expressing a sgRNA resistant UGDH cDNA (blue) or an empty vector (grey). a, e, f, Lines or bars represent mean ± s.d.; a, e, f, n=3 biologically independent samples. Statistical significance determined by two-tailed unpaired t-tests.

Article Snippet: Antibodies to ACSL3 (Ab151959), human GPX4 (Ab41787), LDLR (52818), SCARB1(52629) were purchased from Abcam; Heparan Sulfate (370255-S) from AMSBio; IRP2 (37135S), SLC7A11 (12691S), TFRC (13208S), and β-tubulin (2146S) from Cell Signaling Technology; GAPDH (GTX627408) and UGDH (GTX104993) from GeneTex; AIFM2 (20866-1-AP) and PAPSS1 (14708-1-AP) from ProteinTech; mouse GPX4 (sc-166570) from Santa Cruz Biotechnology; and Chondroitin Sulfate (C8035) from Sigma.

Techniques: Expressing, Plasmid Preparation, Western Blot, Control, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Glycosaminoglycan-mediated lipoprotein uptake protects cancer cells from ferroptosis

doi: 10.1101/2024.05.13.593939

Figure Lengend Snippet: a. Sulfation of glycosaminoglycans relies on the production of 3’-phosphoadenylylsulfate by PAPSS1 , and on its transports into the Golgi by SLC35B2 . Both genes (blue) scored in our screens. b. Individual sgRNA scores for PAPSS1 in the presence and absence of ML210 (left), or in high and low DiI-LDL populations (right). c. Immunoblot analysis of PAPSS1 in Karpas299 cells transduced with a sgControl (grey) or sg PAPSS1 (blue). GAPDH is included as a loading control. d. Quantification of total milligrams of heparan sulfate (HS) per grams of protein in Karpas299 cells transduced with a sgControl (grey) or sg PAPSS1 (blue). e. Quantification of total sulfate per glycosaminoglycan disaccharide in Karpas299 cells transduced with a sgControl (grey) or sg PAPSS1 (blue). f. Number of doublings (log 2 ) in 5 days of Karpas299 cells transduced with a sgControl (grey) or sg PAPSS1 (blue) under the indicated concentrations of the GPX4 inhibitor ML162. g. Glucuronic acid (GlcUA), formed by UGDH, is converted to UDP-Xylose via UXS1 . UDP-Xylose is the first monosaccharide used in the initiation of O-glycosylation that attaches GAG chains to serine-residues of proteoglycans. Genes involved in this process (blue) scored in our screens. h. Individual sgRNA scores for UXS1 in the presence and absence of ML210 (left), or in high and low DiI-LDL populations (right). i. Sanger sequencing of UXS1 gene exon 9 in Karpas299 parental cells (WT) or transduced with sg UXS1 . j. Quantification of total milligrams of heparan sulfate (HS) per grams of protein in Karpas299 cells transduced with a sgControl (grey) or sg UXS1 (blue). k. Number of doublings (log 2 ) in 5 days of Karpas299 cells transduced with a sgControl (grey) or sg UXS1 (blue) under the indicated concentrations of the GPX4 inhibitor ML162. i. Cellular uptake of DiI-LDL measured as median PE intensity in the indicated Karpas299 cells transduced with a sgControl, sg UGDH , sg PAPSS1 or sg UXS1 assessed by flow cytometry. d-f, j-l, Bars or lines represent mean ± s.d.; d-f, j-l, n=3 biologically independent samples. Statistical significance determined by two-tailed unpaired t-tests as indicated or compared to sgControl cells ( l ).

Article Snippet: Antibodies to ACSL3 (Ab151959), human GPX4 (Ab41787), LDLR (52818), SCARB1(52629) were purchased from Abcam; Heparan Sulfate (370255-S) from AMSBio; IRP2 (37135S), SLC7A11 (12691S), TFRC (13208S), and β-tubulin (2146S) from Cell Signaling Technology; GAPDH (GTX627408) and UGDH (GTX104993) from GeneTex; AIFM2 (20866-1-AP) and PAPSS1 (14708-1-AP) from ProteinTech; mouse GPX4 (sc-166570) from Santa Cruz Biotechnology; and Chondroitin Sulfate (C8035) from Sigma.

Techniques: Western Blot, Transduction, Control, Glycoproteomics, Sequencing, Flow Cytometry, Two Tailed Test