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β tubulin  (Abcam)


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    Abcam β tubulin
    Decreased expression of NMNAT3 protein in the caudate nucleus of Parkinson’s disease patient brain. ( A ) Representative western blot of four non-disease control and four Parkinson’s disease subjects. Caudate nucleus and cerebellum samples were lysed in RIPA buffer and NMNATs −1, −2, −3, SARM1 <t>and</t> <t>β-tubulin</t> loading control were subsequently detected with specific antibodies. Bands were visualized using electrochemiluminescence detection. ( B ) Quantification of NMNAT3 protein expression. NMNAT3 band intensities were quantified by densitometric analysis using FIJI ImageJ v.1.53a and were normalized for β-tubulin. Results were expressed as ratio ± SEM ( n = 19 for non-disease controls, n = 19 for Parkinson’s disease subjects). Statistical analysis comprised Student’s t -test with Welch correction and was performed using Prism v8.3. Abbreviations: NDC, non-disease control subjects; PD, Parkinson’s disease subjects; NMNAT, nicotinamide mononucleotide adenylyltransferase; SARM1, sterile alpha and TIR motif containing 1.
    β Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ab180271/Recombinant+Dengue+virus+2+Dengue+Virus+2+envelope+protein/pmc09433734-50-0-5
    Average 99 stars, based on 24 article reviews
    β tubulin - by Bioz Stars, 2026-09
    99/100 stars

    Images

    1) Product Images from "Alpha-synucleinopathy reduces NMNAT3 protein levels and neurite formation that can be rescued by targeting the NAD+ pathway"

    Article Title: Alpha-synucleinopathy reduces NMNAT3 protein levels and neurite formation that can be rescued by targeting the NAD+ pathway

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddac077

    Decreased expression of NMNAT3 protein in the caudate nucleus of Parkinson’s disease patient brain. ( A ) Representative western blot of four non-disease control and four Parkinson’s disease subjects. Caudate nucleus and cerebellum samples were lysed in RIPA buffer and NMNATs −1, −2, −3, SARM1 and β-tubulin loading control were subsequently detected with specific antibodies. Bands were visualized using electrochemiluminescence detection. ( B ) Quantification of NMNAT3 protein expression. NMNAT3 band intensities were quantified by densitometric analysis using FIJI ImageJ v.1.53a and were normalized for β-tubulin. Results were expressed as ratio ± SEM ( n = 19 for non-disease controls, n = 19 for Parkinson’s disease subjects). Statistical analysis comprised Student’s t -test with Welch correction and was performed using Prism v8.3. Abbreviations: NDC, non-disease control subjects; PD, Parkinson’s disease subjects; NMNAT, nicotinamide mononucleotide adenylyltransferase; SARM1, sterile alpha and TIR motif containing 1.
    Figure Legend Snippet: Decreased expression of NMNAT3 protein in the caudate nucleus of Parkinson’s disease patient brain. ( A ) Representative western blot of four non-disease control and four Parkinson’s disease subjects. Caudate nucleus and cerebellum samples were lysed in RIPA buffer and NMNATs −1, −2, −3, SARM1 and β-tubulin loading control were subsequently detected with specific antibodies. Bands were visualized using electrochemiluminescence detection. ( B ) Quantification of NMNAT3 protein expression. NMNAT3 band intensities were quantified by densitometric analysis using FIJI ImageJ v.1.53a and were normalized for β-tubulin. Results were expressed as ratio ± SEM ( n = 19 for non-disease controls, n = 19 for Parkinson’s disease subjects). Statistical analysis comprised Student’s t -test with Welch correction and was performed using Prism v8.3. Abbreviations: NDC, non-disease control subjects; PD, Parkinson’s disease subjects; NMNAT, nicotinamide mononucleotide adenylyltransferase; SARM1, sterile alpha and TIR motif containing 1.

    Techniques Used: Expressing, Western Blot, Electrochemiluminescence

    Inverse correlation of NMNAT3 and monomeric α-syn expression levels in caudate nucleus of Parkinson’s disease brain. ( A ) Western blot detection of α-syn in caudate nucleus RIPA lysates of Parkinson’s disease patients. α-syn was detected using anti-α-syn primary antibody and bands were visualized using electrochemiluminescence detection. Soluble oligomers of α-syn are identified by * . ( B ) Correlation between tubulin-normalized NMNAT3 and monomeric α-syn expressions was performed by the Spearman correlation coefficient analysis using Prism v.8.3. For all panels: 1–8 = Parkinson’s disease sample. Abbreviations: NMNAT3, nicotinamide mononucleotide adenylyltransferase 3; SYN, α-synuclein; kDa, kilodaltons; M, molecular weight markers; 1–8, sample lanes.
    Figure Legend Snippet: Inverse correlation of NMNAT3 and monomeric α-syn expression levels in caudate nucleus of Parkinson’s disease brain. ( A ) Western blot detection of α-syn in caudate nucleus RIPA lysates of Parkinson’s disease patients. α-syn was detected using anti-α-syn primary antibody and bands were visualized using electrochemiluminescence detection. Soluble oligomers of α-syn are identified by * . ( B ) Correlation between tubulin-normalized NMNAT3 and monomeric α-syn expressions was performed by the Spearman correlation coefficient analysis using Prism v.8.3. For all panels: 1–8 = Parkinson’s disease sample. Abbreviations: NMNAT3, nicotinamide mononucleotide adenylyltransferase 3; SYN, α-synuclein; kDa, kilodaltons; M, molecular weight markers; 1–8, sample lanes.

    Techniques Used: Expressing, Western Blot, Electrochemiluminescence, Molecular Weight

    The expression of wild-type α-SYN decreased NMNAT3 protein expression in differentiated, DA SH-SY5Y cells. Cells stably expressing green fluorescent protein or wild-type α-SYN N -terminally fused to EGFP were differentiated with 10 μM retinoic acid for 7 days. ( A ) Cells were imaged using an Axiovert C40 microscope under phase-contrast illumination. Arrows indicate cell bodies; * indicates neurite projections. Scale bar = 50 μm. ( B ) Cells were lysed using RIPA and the expressions of α-SYN, the neuronal marker NeuN, TH and β-tubulin proteins were detected using specific antibodies by western blotting. Bands were visualized using electrochemiluminescence detection and quantified using FIJI ImageJ v.1.53a. ( C ) α-syn aggregates were assessed using an antibody raised against monomeric α-syn (top image) and an antibody raised against aggregate forms of α-syn (bottom image) using western blotting. ( D ) The localizations of EGFP, α-SYN-EGFP and NMNAT3 were imaged using confocal microscopy, with EGFP and α-SYN-EGFP imaged using facile fluorescence and NMNAT3 imaged using a combination of mouse-anti-NMNAT3 and chicken–anti-mouse AlexaFluor594. Nuclei were counterstained using DAPI. Cells were imaged as Z-stacks and shown as maximum projection images. Areas of interest are magnified. Arrows show examples of colocalization between α-SYN-EGFP and NMNAT3. All images were imaged using a x40 objective lens, with WT (x2) panels imaged at 2-fold magnification using a x40 lens. Scale bar = 10 μm. ( E ) NMNAT3 protein expression was detected in cell RIPA lysates by western blotting using an anti-NMNAT3 primary antibody. Bands were visualized using electrochemiluminescence detection. ( F ) Quantification of NMNAT3 protein expressions in cell RIPA lysates was performed by densitometric analysis using FIJI ImageJ v.1.53a, normalized for β-tubulin expression and expressed as ratio ± SD ( n = 3). Statistical analysis comprised Student’s t -test with the Welch correction using Prism v.8.3. For all panels: MOCK, SH-SY5Y transfected with empty vector; WT, SH-SY5Y expressing wild-type α-SYN; UM, undifferentiated parental SH-SY5Y cells; DM, differentiated parental SH-SY5Y cells; UW, undifferentiated wild-type α-SYN-expressing cells; DW, differentiated SH-SY5Y wild-type α-SYN-expressing cells; M, molecular weight markers; kDa, kilodaltons. Abbreviations: SYN, synuclein; TH, tyrosine hydroxylase; NMNAT3, nicotinamide mononucleotide adenylyltransferase 3.
    Figure Legend Snippet: The expression of wild-type α-SYN decreased NMNAT3 protein expression in differentiated, DA SH-SY5Y cells. Cells stably expressing green fluorescent protein or wild-type α-SYN N -terminally fused to EGFP were differentiated with 10 μM retinoic acid for 7 days. ( A ) Cells were imaged using an Axiovert C40 microscope under phase-contrast illumination. Arrows indicate cell bodies; * indicates neurite projections. Scale bar = 50 μm. ( B ) Cells were lysed using RIPA and the expressions of α-SYN, the neuronal marker NeuN, TH and β-tubulin proteins were detected using specific antibodies by western blotting. Bands were visualized using electrochemiluminescence detection and quantified using FIJI ImageJ v.1.53a. ( C ) α-syn aggregates were assessed using an antibody raised against monomeric α-syn (top image) and an antibody raised against aggregate forms of α-syn (bottom image) using western blotting. ( D ) The localizations of EGFP, α-SYN-EGFP and NMNAT3 were imaged using confocal microscopy, with EGFP and α-SYN-EGFP imaged using facile fluorescence and NMNAT3 imaged using a combination of mouse-anti-NMNAT3 and chicken–anti-mouse AlexaFluor594. Nuclei were counterstained using DAPI. Cells were imaged as Z-stacks and shown as maximum projection images. Areas of interest are magnified. Arrows show examples of colocalization between α-SYN-EGFP and NMNAT3. All images were imaged using a x40 objective lens, with WT (x2) panels imaged at 2-fold magnification using a x40 lens. Scale bar = 10 μm. ( E ) NMNAT3 protein expression was detected in cell RIPA lysates by western blotting using an anti-NMNAT3 primary antibody. Bands were visualized using electrochemiluminescence detection. ( F ) Quantification of NMNAT3 protein expressions in cell RIPA lysates was performed by densitometric analysis using FIJI ImageJ v.1.53a, normalized for β-tubulin expression and expressed as ratio ± SD ( n = 3). Statistical analysis comprised Student’s t -test with the Welch correction using Prism v.8.3. For all panels: MOCK, SH-SY5Y transfected with empty vector; WT, SH-SY5Y expressing wild-type α-SYN; UM, undifferentiated parental SH-SY5Y cells; DM, differentiated parental SH-SY5Y cells; UW, undifferentiated wild-type α-SYN-expressing cells; DW, differentiated SH-SY5Y wild-type α-SYN-expressing cells; M, molecular weight markers; kDa, kilodaltons. Abbreviations: SYN, synuclein; TH, tyrosine hydroxylase; NMNAT3, nicotinamide mononucleotide adenylyltransferase 3.

    Techniques Used: Expressing, Stable Transfection, Microscopy, Marker, Western Blot, Electrochemiluminescence, Confocal Microscopy, Fluorescence, Transfection, Plasmid Preparation, Molecular Weight

    Primary and secondary antibodies used in the study
    Figure Legend Snippet: Primary and secondary antibodies used in the study

    Techniques Used: Confocal Microscopy

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    Article Snippet: .. The following primary antibodies were used: rabbit anti-BVR (Abcam ab180208; 1:1,000), rabbit anti-HO-2 (generated as previously described ( Zakhary et al., 1996 ); 1:1,000), mouse anti-total OXPHOS cocktail (Abcam ab110413; 1:5,000), mouse anti-pS473 AKT (Cell Signaling 587F11; 1:1000), rabbit anti-AKT (Cell Signaling 11E7; 1:1000), rabbit anti-pERK (Cell Signaling 9101; 1:1000), rabbit anti-tERK (Cell Signaling 9102; 1:1000), and mouse anti-β-actin (Santa Cruz Biotech sc-47778 HRP; 1:10,000). .. The following primary antibodies were used: rabbit anti-BVR (Abcam ab180208; 1:1,000), rabbit anti-HO-2 (generated as previously described (Zakhary et al., 1996); 1:1,000), mouse anti-total OXPHOS cocktail (Abcam ab110413; 1:5,000), mouse anti-pS473 AKT (Cell Signaling 587F11; 1:1000), rabbit anti-AKT (Cell Signaling 11E7; 1:1000), rabbit anti-pERK (Cell Signaling 9101; 1:1000), rabbit anti-tERK (Cell Signaling 9102; 1:1000), and mouse anti-β-actin (Santa Cruz Biotech sc-47778 HRP; 1:10,000).

    Western Blot:

    Article Title: Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction
    Article Snippet: MIN6 cells were cultured in DMEM (Invitrogen) containing 4.5g/L glucose supplemented with 15% v/v heat-inactivated FCS, 50 μMβ-mercaptoethanol, and 50 mg/mL penicillin and 100 mg/ml streptomycin and insulin release was performed as described ( Poy et al ., 2004 ). .. The following primary antibodies were used for Western blots at 1:1000 dilution: PITPNA (Abcam, ab180234), Cadm1 (MBL, CM004-3), Gephyrin (BD Biosciences, 610585), CHOP (Cell Signaling, 2895S), BiP/GRP78 (Cell Signaling, 3177S), DRP1 (Proteintech, 12957-1-AP), β-Actin (Cell Signaling, 3700S), and γ-Tubulin (Sigma, T6557). .. The following primary antibodies were used for immunofluorescence: PITPNA (1:200, Sigma, SAB1400211).

    Blocking Assay:

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1
    Article Snippet: .. After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control. .. After washed with TBST three times, membranes were then incubated with the matching goat anti-rabbit (dilution at 1:3,000; cat. no. A0208; Beyotime) for 2 h at room temperature.

    Saline:

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1
    Article Snippet: .. After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control. .. After washed with TBST three times, membranes were then incubated with the matching goat anti-rabbit (dilution at 1:3,000; cat. no. A0208; Beyotime) for 2 h at room temperature.

    Incubation:

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1
    Article Snippet: .. After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control. .. After washed with TBST three times, membranes were then incubated with the matching goat anti-rabbit (dilution at 1:3,000; cat. no. A0208; Beyotime) for 2 h at room temperature.

    Control:

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1
    Article Snippet: .. After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control. .. After washed with TBST three times, membranes were then incubated with the matching goat anti-rabbit (dilution at 1:3,000; cat. no. A0208; Beyotime) for 2 h at room temperature.

    other:

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    Article Snippet: rabbit anti-BVR , Abcam , ab180208.



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    pole2  (Abcam)
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    Abcam pole2
    <t>POLE2</t> level is upregulated and associated with various clinicopathological parameters in RCC. The POLE2 mRNA level in RCC was obtained from The Cancer Genome Atlas Kidney Clear Cell Carcinoma dataset (TCGA-KIRC; containing 72 normal tissues and 530 RCC tissues) and International Cancer Genome Consortium (ICGC; containing 44 normal tissues and 91 RCC tissues). The POLE2 mRNA levels were compared in different clinicopathological parameters: (A,C) Cancer vs. para-cancer, (B) Carcinoma vs. paired para-cancer, (D) TNM stage, and (E) G stage. (F) Kaplan-Meier curves of the overall survival of RCC patients with high or low POLE2 expression in TCGA-KIRC cohort. (G) Representative immunohistochemical staining for POLE2 in RCC and adjacent normal tissues. (H) Kaplan-Meier curves of the overall survival of RCC patients with high or low POLE2 expression. ** P < 0.01, *** P < 0.001.
    Pole2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ab180271/Recombinant+Dengue+virus+2+Dengue+Virus+2+envelope+protein/pmc07905105-75-25-32
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    pole2 - by Bioz Stars, 2026-09
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    Image Search Results


    a Immunostaining in paraffin-embedded pancreata from 8-week-old female Ins-Cre, Pitpna flox/flox , and littermate control wild-type (WT) mice was performed to assess: insulin (magenta), glucagon (Gcg, green) and apoptotic marker TUNEL (cyan). White arrowheads point to TUNEL-positive and insulin-positive cells in ( b ), TUNEL-positive beta cell number ( n = 6), P = 0.0007. c TUNEL-positive alpha cell number ( n = 6). d Western blot analysis of Pitpna, BiP/GRP78, and CHOP after treatment of hydrogen peroxide (H 2 O 2 ) in isolated islets of 8-week-old male Ins-Cre, Pitpna flox/flox , and WT mice. e qRT-PCR analysis of Pitpna , PC1/3 , PC2 , CPE , CGA , and CGB mRNA expression in islets of male WT and Ins-Cre, Pitpna flox/flox mice at age 10 weeks ( n = 5), P Pitpna < 0.0001, P PC1/3 < 0.0001, P PC2 = 0.0441; P CPE = 0.0488, P CGA = 0.1313, P CGB = 0.0585. Data are presented as mean values ± SEM for ( b ), ( c ), ( e ). * P < 0.05, *** P < 0.001 and n.s. denotes not significant. Two-tailed unpaired Student t-test were used in for ( b ), ( c ), ( e ). All primary source data are reported in the Source data file.

    Journal: Nature Communications

    Article Title: Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction

    doi: 10.1038/s41467-023-39978-1

    Figure Lengend Snippet: a Immunostaining in paraffin-embedded pancreata from 8-week-old female Ins-Cre, Pitpna flox/flox , and littermate control wild-type (WT) mice was performed to assess: insulin (magenta), glucagon (Gcg, green) and apoptotic marker TUNEL (cyan). White arrowheads point to TUNEL-positive and insulin-positive cells in ( b ), TUNEL-positive beta cell number ( n = 6), P = 0.0007. c TUNEL-positive alpha cell number ( n = 6). d Western blot analysis of Pitpna, BiP/GRP78, and CHOP after treatment of hydrogen peroxide (H 2 O 2 ) in isolated islets of 8-week-old male Ins-Cre, Pitpna flox/flox , and WT mice. e qRT-PCR analysis of Pitpna , PC1/3 , PC2 , CPE , CGA , and CGB mRNA expression in islets of male WT and Ins-Cre, Pitpna flox/flox mice at age 10 weeks ( n = 5), P Pitpna < 0.0001, P PC1/3 < 0.0001, P PC2 = 0.0441; P CPE = 0.0488, P CGA = 0.1313, P CGB = 0.0585. Data are presented as mean values ± SEM for ( b ), ( c ), ( e ). * P < 0.05, *** P < 0.001 and n.s. denotes not significant. Two-tailed unpaired Student t-test were used in for ( b ), ( c ), ( e ). All primary source data are reported in the Source data file.

    Article Snippet: The following primary antibodies were used for Western blots at 1:1000 dilution: PITPNA (Abcam, ab180234), Cadm1 (MBL, CM004-3), Gephyrin (BD Biosciences, 610585), CHOP (Cell Signaling, 2895S), BiP/GRP78 (Cell Signaling, 3177S), DRP1 (Proteintech, 12957-1-AP), β-Actin (Cell Signaling, 3700S), and γ-Tubulin (Sigma, T6557).

    Techniques: Immunostaining, Control, Marker, TUNEL Assay, Western Blot, Isolation, Quantitative RT-PCR, Expressing, Two Tailed Test

    a Representative transmission electron micrographs of pancreatic beta-cells from non-diabetic human donors after lentiviral-mediated over-expression of PITPNA (OE- PITPNA ) or inhibition of PITPNA (sh- PITPNA ) in comparison to respective control lentiviruses (OE- Ctrl or sh- Ctrl ). The second raw image represents a zoom-in view of the white box from the first raw image. Granule profile: immature secretory granules (blue box), mature secretory granules (red box), crystal-containing granules (yellow box), and empty secretory granules (orange box). b Quantification of granule density in beta-cells of lentiviral-treated human islets shown in panel ( a ) ( n = 9). P sh-Ctrl vs sh-PITPNA = 0.0199, P OE-Ctrl vs OE-PITPNA < 0.0001. c Quantifi c ation of docked vesicles in beta-cells of lentiviral-treated human islets shown in panel ( a ) ( n = 9). P sh-Ctrl vs sh-PITPNA = 0.0004, P OE-Ctrl vs OE-PITPNA = 0.00421. d Quantification of immature secretory granules (ISG, n = 7, 8, 5, 6 for sh- Ctrl , sh- PITPNA , OE- Ctrl , OE- PITPNA , respectively), mature secretory granules (MSG, n = 8, 8, 8, 6 for sh- Ctrl , sh- PITPNA , OE- Ctrl , OE- PITPNA , respectively), crystal-containing granules (CCG, n = 6, 8, 8, 8 for sh- Ctrl , sh- PITPNA , OE- Ctrl , OE- PITPNA , respectively), and empty secretory granules (ESG, n = 7, 7, 7, 7 for sh- Ctrl , sh- PITPNA , OE- Ctrl , OE- PITPNA , respectively) in beta-cells of isolated human islets after lentiviral treatments shown in panel ( a ). ( P sh-Ctrl vs sh-PITPNA < 0.0001, P OE-Ctrl vs OE-PITPNA = 0.0128 for ISG), ( P sh-Ctrl vs sh-PITPNA < 0.0001, P OE-Ctrl vs OE-PITPNA = 0.0008 for MSG), ( P sh-Ctrl vs sh-PITPNA = 0.0068, P OE-Ctrl vs OE-PITPNA = 0.6874 for CCG), ( P sh-Ctrl vs sh-PITPNA = 0.0207, P OE-Ctrl vs OE-PITPNA = 0.4262 for ESG). e Proinsulin expression in isolated human islets after lentiviral-mediated over-expression of PITPNA (OE- PITPNA , n = 5 biologically independent samples), knockdown of PITPNA (sh- PITPNA , n = 7 biologically independent samples) or control lentivirus (sh- Ctrl , n = 7 biologically independent samples). f Quantification of proinsulin in isolated human islets after densitometric analysis of western blots shown in panel ( f ). g Western blot analysis of PITPNA, and ER stress/unfolded protein response (UPR) proteins IRE1α, ERO1, PDI, and CHOP in human islets after lentiviral-mediated over-expression of PITPNA (OE- PITPNA ), knockdown of PITPNA (sh- PITPNA ) or control lentivirus (sh- Ctrl ). Data are presented as mean values ± SEM for ( b ), ( c ), ( d ), e ). * P < 0.05, ** P < 0.01, *** P < 0.001. Ordinary one-way ANOVA with Turkey’s multiple comparisons test was used for ( b ), ( c ), ( d ), ( e ). All primary source data are reported in the Source data file.

    Journal: Nature Communications

    Article Title: Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction

    doi: 10.1038/s41467-023-39978-1

    Figure Lengend Snippet: a Representative transmission electron micrographs of pancreatic beta-cells from non-diabetic human donors after lentiviral-mediated over-expression of PITPNA (OE- PITPNA ) or inhibition of PITPNA (sh- PITPNA ) in comparison to respective control lentiviruses (OE- Ctrl or sh- Ctrl ). The second raw image represents a zoom-in view of the white box from the first raw image. Granule profile: immature secretory granules (blue box), mature secretory granules (red box), crystal-containing granules (yellow box), and empty secretory granules (orange box). b Quantification of granule density in beta-cells of lentiviral-treated human islets shown in panel ( a ) ( n = 9). P sh-Ctrl vs sh-PITPNA = 0.0199, P OE-Ctrl vs OE-PITPNA < 0.0001. c Quantifi c ation of docked vesicles in beta-cells of lentiviral-treated human islets shown in panel ( a ) ( n = 9). P sh-Ctrl vs sh-PITPNA = 0.0004, P OE-Ctrl vs OE-PITPNA = 0.00421. d Quantification of immature secretory granules (ISG, n = 7, 8, 5, 6 for sh- Ctrl , sh- PITPNA , OE- Ctrl , OE- PITPNA , respectively), mature secretory granules (MSG, n = 8, 8, 8, 6 for sh- Ctrl , sh- PITPNA , OE- Ctrl , OE- PITPNA , respectively), crystal-containing granules (CCG, n = 6, 8, 8, 8 for sh- Ctrl , sh- PITPNA , OE- Ctrl , OE- PITPNA , respectively), and empty secretory granules (ESG, n = 7, 7, 7, 7 for sh- Ctrl , sh- PITPNA , OE- Ctrl , OE- PITPNA , respectively) in beta-cells of isolated human islets after lentiviral treatments shown in panel ( a ). ( P sh-Ctrl vs sh-PITPNA < 0.0001, P OE-Ctrl vs OE-PITPNA = 0.0128 for ISG), ( P sh-Ctrl vs sh-PITPNA < 0.0001, P OE-Ctrl vs OE-PITPNA = 0.0008 for MSG), ( P sh-Ctrl vs sh-PITPNA = 0.0068, P OE-Ctrl vs OE-PITPNA = 0.6874 for CCG), ( P sh-Ctrl vs sh-PITPNA = 0.0207, P OE-Ctrl vs OE-PITPNA = 0.4262 for ESG). e Proinsulin expression in isolated human islets after lentiviral-mediated over-expression of PITPNA (OE- PITPNA , n = 5 biologically independent samples), knockdown of PITPNA (sh- PITPNA , n = 7 biologically independent samples) or control lentivirus (sh- Ctrl , n = 7 biologically independent samples). f Quantification of proinsulin in isolated human islets after densitometric analysis of western blots shown in panel ( f ). g Western blot analysis of PITPNA, and ER stress/unfolded protein response (UPR) proteins IRE1α, ERO1, PDI, and CHOP in human islets after lentiviral-mediated over-expression of PITPNA (OE- PITPNA ), knockdown of PITPNA (sh- PITPNA ) or control lentivirus (sh- Ctrl ). Data are presented as mean values ± SEM for ( b ), ( c ), ( d ), e ). * P < 0.05, ** P < 0.01, *** P < 0.001. Ordinary one-way ANOVA with Turkey’s multiple comparisons test was used for ( b ), ( c ), ( d ), ( e ). All primary source data are reported in the Source data file.

    Article Snippet: The following primary antibodies were used for Western blots at 1:1000 dilution: PITPNA (Abcam, ab180234), Cadm1 (MBL, CM004-3), Gephyrin (BD Biosciences, 610585), CHOP (Cell Signaling, 2895S), BiP/GRP78 (Cell Signaling, 3177S), DRP1 (Proteintech, 12957-1-AP), β-Actin (Cell Signaling, 3700S), and γ-Tubulin (Sigma, T6557).

    Techniques: Transmission Assay, Over Expression, Inhibition, Comparison, Control, Isolation, Expressing, Knockdown, Western Blot

    BDNF effect on the BDNF-TrkB signalling pathway in Q140 striatal neurons. ( A ). Representative immunoblots of TuJ1, TrkB, TrkB (pY490), Akt, pAkt (pS473), CREB and pCREB (pS133), and in WT and Q140 cells with and without BDNF (25 ng/mL) for 30 min. We detect reduced levels of pAkt (p8473) and pCREB (pS133) in Q140 (lane 3 compared to lane 1 in WT. Repeated in triplicates. ( B ). Striatal neuron staining with FITC-tubulin CREB and pCREB (pSer133) confirms that the activation of CREB into pCREB in WT occurs with BDNF after 30 min. ( C ). Staining with CREB and pCREB (pSer133) shows alterations of pCREB in WT and Q140 after 30 min inductions, with or without BDNF (25 ng/mL). Repeated in triplicates. Scale bar is 100 μm.

    Journal: Biology

    Article Title: Failure to Thrive: Impaired BDNF Transport along the Cortical–Striatal Axis in Mouse Q140 Neurons of Huntington’s Disease

    doi: 10.3390/biology12020157

    Figure Lengend Snippet: BDNF effect on the BDNF-TrkB signalling pathway in Q140 striatal neurons. ( A ). Representative immunoblots of TuJ1, TrkB, TrkB (pY490), Akt, pAkt (pS473), CREB and pCREB (pS133), and in WT and Q140 cells with and without BDNF (25 ng/mL) for 30 min. We detect reduced levels of pAkt (p8473) and pCREB (pS133) in Q140 (lane 3 compared to lane 1 in WT. Repeated in triplicates. ( B ). Striatal neuron staining with FITC-tubulin CREB and pCREB (pSer133) confirms that the activation of CREB into pCREB in WT occurs with BDNF after 30 min. ( C ). Staining with CREB and pCREB (pSer133) shows alterations of pCREB in WT and Q140 after 30 min inductions, with or without BDNF (25 ng/mL). Repeated in triplicates. Scale bar is 100 μm.

    Article Snippet: Rabbit anti- β-Tubulin iso3 (TuJ1) , AbCam, Cambridge, UK , Ab18027 , (WB, 1:1000–2000) .

    Techniques: Western Blot, Staining, Activation Assay

    Design and Fabrication of Cortical–Striatal Neuronal Microfluidic Chamber. ( A ). Photograph of the chambers’ AutoCAD design with detailed numbers. ( B ). Photograph of the master template, a 100 mm silicon wafer of crosslinked SU8 negative resists, containing microfluidic channel features. Inset from ( B ) showing detail of long (750 μm, for axon isolation) and short (100 μm, for dendrite isolation) microgrooves separated by a central chamber. ( C ). Low power (5 ×) image of cut PDMS microfluidic chamber. Inset from ( C ) showing cell culture areas in blue and microgrooves in white. Microfluidic chamber with directional cortico-striatal co-culture. ( D ). Differential interference contrast (DIC) to show neurons growing in chamber. ( E ). Further confirmation of neuron in chamber via immunostaining. Microtubule (TuJ1) staining reveals axons growing through the chamber grooves (Top). The DARPP-32 striatal neuron marker confirms the cortex and striatum separation across the left and right chambers (Bottom). Scale bar is 100 μm.

    Journal: Biology

    Article Title: Failure to Thrive: Impaired BDNF Transport along the Cortical–Striatal Axis in Mouse Q140 Neurons of Huntington’s Disease

    doi: 10.3390/biology12020157

    Figure Lengend Snippet: Design and Fabrication of Cortical–Striatal Neuronal Microfluidic Chamber. ( A ). Photograph of the chambers’ AutoCAD design with detailed numbers. ( B ). Photograph of the master template, a 100 mm silicon wafer of crosslinked SU8 negative resists, containing microfluidic channel features. Inset from ( B ) showing detail of long (750 μm, for axon isolation) and short (100 μm, for dendrite isolation) microgrooves separated by a central chamber. ( C ). Low power (5 ×) image of cut PDMS microfluidic chamber. Inset from ( C ) showing cell culture areas in blue and microgrooves in white. Microfluidic chamber with directional cortico-striatal co-culture. ( D ). Differential interference contrast (DIC) to show neurons growing in chamber. ( E ). Further confirmation of neuron in chamber via immunostaining. Microtubule (TuJ1) staining reveals axons growing through the chamber grooves (Top). The DARPP-32 striatal neuron marker confirms the cortex and striatum separation across the left and right chambers (Bottom). Scale bar is 100 μm.

    Article Snippet: Rabbit anti- β-Tubulin iso3 (TuJ1) , AbCam, Cambridge, UK , Ab18027 , (WB, 1:1000–2000) .

    Techniques: Isolation, Cell Culture, Co-Culture Assay, Immunostaining, Staining, Marker

    List of all antibodies used along with their catalog number.

    Journal: Biology

    Article Title: Failure to Thrive: Impaired BDNF Transport along the Cortical–Striatal Axis in Mouse Q140 Neurons of Huntington’s Disease

    doi: 10.3390/biology12020157

    Figure Lengend Snippet: List of all antibodies used along with their catalog number.

    Article Snippet: Rabbit anti- β-Tubulin iso3 (TuJ1) , AbCam, Cambridge, UK , Ab18027 , (WB, 1:1000–2000) .

    Techniques:

    Primary and secondary antibodies used in the study

    Journal: Human Molecular Genetics

    Article Title: Alpha-synucleinopathy reduces NMNAT3 protein levels and neurite formation that can be rescued by targeting the NAD+ pathway

    doi: 10.1093/hmg/ddac077

    Figure Lengend Snippet: Primary and secondary antibodies used in the study

    Article Snippet: β-Tubulin , 1:1000 , ab180207 (Abcam) , 1:5000 , A4416 (Sigma).

    Techniques: Confocal Microscopy

    Decreased expression of NMNAT3 protein in the caudate nucleus of Parkinson’s disease patient brain. ( A ) Representative western blot of four non-disease control and four Parkinson’s disease subjects. Caudate nucleus and cerebellum samples were lysed in RIPA buffer and NMNATs −1, −2, −3, SARM1 and β-tubulin loading control were subsequently detected with specific antibodies. Bands were visualized using electrochemiluminescence detection. ( B ) Quantification of NMNAT3 protein expression. NMNAT3 band intensities were quantified by densitometric analysis using FIJI ImageJ v.1.53a and were normalized for β-tubulin. Results were expressed as ratio ± SEM ( n = 19 for non-disease controls, n = 19 for Parkinson’s disease subjects). Statistical analysis comprised Student’s t -test with Welch correction and was performed using Prism v8.3. Abbreviations: NDC, non-disease control subjects; PD, Parkinson’s disease subjects; NMNAT, nicotinamide mononucleotide adenylyltransferase; SARM1, sterile alpha and TIR motif containing 1.

    Journal: Human Molecular Genetics

    Article Title: Alpha-synucleinopathy reduces NMNAT3 protein levels and neurite formation that can be rescued by targeting the NAD+ pathway

    doi: 10.1093/hmg/ddac077

    Figure Lengend Snippet: Decreased expression of NMNAT3 protein in the caudate nucleus of Parkinson’s disease patient brain. ( A ) Representative western blot of four non-disease control and four Parkinson’s disease subjects. Caudate nucleus and cerebellum samples were lysed in RIPA buffer and NMNATs −1, −2, −3, SARM1 and β-tubulin loading control were subsequently detected with specific antibodies. Bands were visualized using electrochemiluminescence detection. ( B ) Quantification of NMNAT3 protein expression. NMNAT3 band intensities were quantified by densitometric analysis using FIJI ImageJ v.1.53a and were normalized for β-tubulin. Results were expressed as ratio ± SEM ( n = 19 for non-disease controls, n = 19 for Parkinson’s disease subjects). Statistical analysis comprised Student’s t -test with Welch correction and was performed using Prism v8.3. Abbreviations: NDC, non-disease control subjects; PD, Parkinson’s disease subjects; NMNAT, nicotinamide mononucleotide adenylyltransferase; SARM1, sterile alpha and TIR motif containing 1.

    Article Snippet: β-Tubulin , 1:1000 , ab180207 (Abcam) , 1:5000 , A4416 (Sigma).

    Techniques: Expressing, Western Blot, Electrochemiluminescence

    Inverse correlation of NMNAT3 and monomeric α-syn expression levels in caudate nucleus of Parkinson’s disease brain. ( A ) Western blot detection of α-syn in caudate nucleus RIPA lysates of Parkinson’s disease patients. α-syn was detected using anti-α-syn primary antibody and bands were visualized using electrochemiluminescence detection. Soluble oligomers of α-syn are identified by * . ( B ) Correlation between tubulin-normalized NMNAT3 and monomeric α-syn expressions was performed by the Spearman correlation coefficient analysis using Prism v.8.3. For all panels: 1–8 = Parkinson’s disease sample. Abbreviations: NMNAT3, nicotinamide mononucleotide adenylyltransferase 3; SYN, α-synuclein; kDa, kilodaltons; M, molecular weight markers; 1–8, sample lanes.

    Journal: Human Molecular Genetics

    Article Title: Alpha-synucleinopathy reduces NMNAT3 protein levels and neurite formation that can be rescued by targeting the NAD+ pathway

    doi: 10.1093/hmg/ddac077

    Figure Lengend Snippet: Inverse correlation of NMNAT3 and monomeric α-syn expression levels in caudate nucleus of Parkinson’s disease brain. ( A ) Western blot detection of α-syn in caudate nucleus RIPA lysates of Parkinson’s disease patients. α-syn was detected using anti-α-syn primary antibody and bands were visualized using electrochemiluminescence detection. Soluble oligomers of α-syn are identified by * . ( B ) Correlation between tubulin-normalized NMNAT3 and monomeric α-syn expressions was performed by the Spearman correlation coefficient analysis using Prism v.8.3. For all panels: 1–8 = Parkinson’s disease sample. Abbreviations: NMNAT3, nicotinamide mononucleotide adenylyltransferase 3; SYN, α-synuclein; kDa, kilodaltons; M, molecular weight markers; 1–8, sample lanes.

    Article Snippet: β-Tubulin , 1:1000 , ab180207 (Abcam) , 1:5000 , A4416 (Sigma).

    Techniques: Expressing, Western Blot, Electrochemiluminescence, Molecular Weight

    The expression of wild-type α-SYN decreased NMNAT3 protein expression in differentiated, DA SH-SY5Y cells. Cells stably expressing green fluorescent protein or wild-type α-SYN N -terminally fused to EGFP were differentiated with 10 μM retinoic acid for 7 days. ( A ) Cells were imaged using an Axiovert C40 microscope under phase-contrast illumination. Arrows indicate cell bodies; * indicates neurite projections. Scale bar = 50 μm. ( B ) Cells were lysed using RIPA and the expressions of α-SYN, the neuronal marker NeuN, TH and β-tubulin proteins were detected using specific antibodies by western blotting. Bands were visualized using electrochemiluminescence detection and quantified using FIJI ImageJ v.1.53a. ( C ) α-syn aggregates were assessed using an antibody raised against monomeric α-syn (top image) and an antibody raised against aggregate forms of α-syn (bottom image) using western blotting. ( D ) The localizations of EGFP, α-SYN-EGFP and NMNAT3 were imaged using confocal microscopy, with EGFP and α-SYN-EGFP imaged using facile fluorescence and NMNAT3 imaged using a combination of mouse-anti-NMNAT3 and chicken–anti-mouse AlexaFluor594. Nuclei were counterstained using DAPI. Cells were imaged as Z-stacks and shown as maximum projection images. Areas of interest are magnified. Arrows show examples of colocalization between α-SYN-EGFP and NMNAT3. All images were imaged using a x40 objective lens, with WT (x2) panels imaged at 2-fold magnification using a x40 lens. Scale bar = 10 μm. ( E ) NMNAT3 protein expression was detected in cell RIPA lysates by western blotting using an anti-NMNAT3 primary antibody. Bands were visualized using electrochemiluminescence detection. ( F ) Quantification of NMNAT3 protein expressions in cell RIPA lysates was performed by densitometric analysis using FIJI ImageJ v.1.53a, normalized for β-tubulin expression and expressed as ratio ± SD ( n = 3). Statistical analysis comprised Student’s t -test with the Welch correction using Prism v.8.3. For all panels: MOCK, SH-SY5Y transfected with empty vector; WT, SH-SY5Y expressing wild-type α-SYN; UM, undifferentiated parental SH-SY5Y cells; DM, differentiated parental SH-SY5Y cells; UW, undifferentiated wild-type α-SYN-expressing cells; DW, differentiated SH-SY5Y wild-type α-SYN-expressing cells; M, molecular weight markers; kDa, kilodaltons. Abbreviations: SYN, synuclein; TH, tyrosine hydroxylase; NMNAT3, nicotinamide mononucleotide adenylyltransferase 3.

    Journal: Human Molecular Genetics

    Article Title: Alpha-synucleinopathy reduces NMNAT3 protein levels and neurite formation that can be rescued by targeting the NAD+ pathway

    doi: 10.1093/hmg/ddac077

    Figure Lengend Snippet: The expression of wild-type α-SYN decreased NMNAT3 protein expression in differentiated, DA SH-SY5Y cells. Cells stably expressing green fluorescent protein or wild-type α-SYN N -terminally fused to EGFP were differentiated with 10 μM retinoic acid for 7 days. ( A ) Cells were imaged using an Axiovert C40 microscope under phase-contrast illumination. Arrows indicate cell bodies; * indicates neurite projections. Scale bar = 50 μm. ( B ) Cells were lysed using RIPA and the expressions of α-SYN, the neuronal marker NeuN, TH and β-tubulin proteins were detected using specific antibodies by western blotting. Bands were visualized using electrochemiluminescence detection and quantified using FIJI ImageJ v.1.53a. ( C ) α-syn aggregates were assessed using an antibody raised against monomeric α-syn (top image) and an antibody raised against aggregate forms of α-syn (bottom image) using western blotting. ( D ) The localizations of EGFP, α-SYN-EGFP and NMNAT3 were imaged using confocal microscopy, with EGFP and α-SYN-EGFP imaged using facile fluorescence and NMNAT3 imaged using a combination of mouse-anti-NMNAT3 and chicken–anti-mouse AlexaFluor594. Nuclei were counterstained using DAPI. Cells were imaged as Z-stacks and shown as maximum projection images. Areas of interest are magnified. Arrows show examples of colocalization between α-SYN-EGFP and NMNAT3. All images were imaged using a x40 objective lens, with WT (x2) panels imaged at 2-fold magnification using a x40 lens. Scale bar = 10 μm. ( E ) NMNAT3 protein expression was detected in cell RIPA lysates by western blotting using an anti-NMNAT3 primary antibody. Bands were visualized using electrochemiluminescence detection. ( F ) Quantification of NMNAT3 protein expressions in cell RIPA lysates was performed by densitometric analysis using FIJI ImageJ v.1.53a, normalized for β-tubulin expression and expressed as ratio ± SD ( n = 3). Statistical analysis comprised Student’s t -test with the Welch correction using Prism v.8.3. For all panels: MOCK, SH-SY5Y transfected with empty vector; WT, SH-SY5Y expressing wild-type α-SYN; UM, undifferentiated parental SH-SY5Y cells; DM, differentiated parental SH-SY5Y cells; UW, undifferentiated wild-type α-SYN-expressing cells; DW, differentiated SH-SY5Y wild-type α-SYN-expressing cells; M, molecular weight markers; kDa, kilodaltons. Abbreviations: SYN, synuclein; TH, tyrosine hydroxylase; NMNAT3, nicotinamide mononucleotide adenylyltransferase 3.

    Article Snippet: β-Tubulin , 1:1000 , ab180207 (Abcam) , 1:5000 , A4416 (Sigma).

    Techniques: Expressing, Stable Transfection, Microscopy, Marker, Western Blot, Electrochemiluminescence, Confocal Microscopy, Fluorescence, Transfection, Plasmid Preparation, Molecular Weight

    Primary and secondary antibodies used in the study

    Journal: Human Molecular Genetics

    Article Title: Alpha-synucleinopathy reduces NMNAT3 protein levels and neurite formation that can be rescued by targeting the NAD+ pathway

    doi: 10.1093/hmg/ddac077

    Figure Lengend Snippet: Primary and secondary antibodies used in the study

    Article Snippet: β-Tubulin , 1:1000 , ab180207 (Abcam) , 1:5000 , A4416 (Sigma).

    Techniques: Confocal Microscopy

    Immunostaining in paraffin-embedded pancreata from 10-week-old Ins -Cre, Pitpna flox/flox and littermate control wild-type (WT) mice was performed to assess: A , insulin (red), glucagon (Gcg, magenta) and apoptotic marker (TUNEL, green) Scale bar= 50μm. In far-right panel, scale bar= 20μm, B , TUNEL-positive beta cell number (n=6), and C , TUNEL-positive alpha cell number (n=6). D , Western blot analysis of Pitpna, BiP/GRP78, and CHOP after treatment of hydrogen peroxide (H 2 O 2 ) in isolated islets of 10-week-old Ins -Cre, Pitpna flox/flox and WT mice. E , qRT-PCR analysis of Pitpna, PC1/3, PC2, CPE, CGA and CGB mRNA expression in islets of WT and Ins -Cre, Pitpna flox/flox mice at age 10 weeks (n=5). Results presented as mean ± SEM. * P < 0.05, *** P <0.001, and n.s. denotes not significant.

    Journal: bioRxiv

    Article Title: Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction

    doi: 10.1101/2022.07.06.498991

    Figure Lengend Snippet: Immunostaining in paraffin-embedded pancreata from 10-week-old Ins -Cre, Pitpna flox/flox and littermate control wild-type (WT) mice was performed to assess: A , insulin (red), glucagon (Gcg, magenta) and apoptotic marker (TUNEL, green) Scale bar= 50μm. In far-right panel, scale bar= 20μm, B , TUNEL-positive beta cell number (n=6), and C , TUNEL-positive alpha cell number (n=6). D , Western blot analysis of Pitpna, BiP/GRP78, and CHOP after treatment of hydrogen peroxide (H 2 O 2 ) in isolated islets of 10-week-old Ins -Cre, Pitpna flox/flox and WT mice. E , qRT-PCR analysis of Pitpna, PC1/3, PC2, CPE, CGA and CGB mRNA expression in islets of WT and Ins -Cre, Pitpna flox/flox mice at age 10 weeks (n=5). Results presented as mean ± SEM. * P < 0.05, *** P <0.001, and n.s. denotes not significant.

    Article Snippet: The following primary antibodies were used for Western blots at 1:1000 dilution: PITPNA (Abcam, ab180234), Cadm1 (MBL, CM004-3), Gephyrin (BD Biosciences, 610585), CHOP (Cell Signaling, 2895S), BiP/GRP78 (Cell Signaling, 3177S), DRP1 (Proteintech, 12957-1-AP), β-Actin (Cell Signaling, 3700S), and γ-Tubulin (Sigma, T6557).

    Techniques: Immunostaining, Marker, TUNEL Assay, Western Blot, Isolation, Quantitative RT-PCR, Expressing

    A , Representative transmission electron micrographs of pancreatic beta-cells from non-diabetic human donors after lentiviral-mediated over-expression of PITPNA (OE- PITPNA ) or inhibition of PITPNA (sh- PITPNA ) in comparison to control lentivirus ( sh-Ctrl ); granule profile: immature secretory granule (blue box), mature secretory granules (red box), crystal-containing granules (yellow box), and empty secretory granules (orange box). B , C , Quantification of granule density and docked vesicles in beta-cells of lentiviral - treated human islets shown in panel ( A ) (n=4). D , Quantification of immature secretory granule (ISG), mature secretory granules (MSG), crystal-containing granules (CCG), and empty secretory granules (ESG) in beta-cells of isolated human islets after lentiviral treatments shown in panel ( A ) (n=8-9). E , F , Quantification of proinsulin in isolated human islets after densitometric analysis of western blots shown in panel ( F ). G, Western blot analysis of PITPNA, and ER stress/unfolded protein response (UPR) proteins IRE1α, ERO1, PDI, and CHOP in human islets after lentiviral-mediated over-expression of PITPNA (OE- PITPNA ), knockdown of PITPNA (sh- PITPNA ) or control lentivirus (sh- Ctrl ). Results presented as mean ± SEM. * P < 0.05; ** P – 0.01; *** p <0.001, and n.s. denotes not significant. See also Figure S4.

    Journal: bioRxiv

    Article Title: Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction

    doi: 10.1101/2022.07.06.498991

    Figure Lengend Snippet: A , Representative transmission electron micrographs of pancreatic beta-cells from non-diabetic human donors after lentiviral-mediated over-expression of PITPNA (OE- PITPNA ) or inhibition of PITPNA (sh- PITPNA ) in comparison to control lentivirus ( sh-Ctrl ); granule profile: immature secretory granule (blue box), mature secretory granules (red box), crystal-containing granules (yellow box), and empty secretory granules (orange box). B , C , Quantification of granule density and docked vesicles in beta-cells of lentiviral - treated human islets shown in panel ( A ) (n=4). D , Quantification of immature secretory granule (ISG), mature secretory granules (MSG), crystal-containing granules (CCG), and empty secretory granules (ESG) in beta-cells of isolated human islets after lentiviral treatments shown in panel ( A ) (n=8-9). E , F , Quantification of proinsulin in isolated human islets after densitometric analysis of western blots shown in panel ( F ). G, Western blot analysis of PITPNA, and ER stress/unfolded protein response (UPR) proteins IRE1α, ERO1, PDI, and CHOP in human islets after lentiviral-mediated over-expression of PITPNA (OE- PITPNA ), knockdown of PITPNA (sh- PITPNA ) or control lentivirus (sh- Ctrl ). Results presented as mean ± SEM. * P < 0.05; ** P – 0.01; *** p <0.001, and n.s. denotes not significant. See also Figure S4.

    Article Snippet: The following primary antibodies were used for Western blots at 1:1000 dilution: PITPNA (Abcam, ab180234), Cadm1 (MBL, CM004-3), Gephyrin (BD Biosciences, 610585), CHOP (Cell Signaling, 2895S), BiP/GRP78 (Cell Signaling, 3177S), DRP1 (Proteintech, 12957-1-AP), β-Actin (Cell Signaling, 3700S), and γ-Tubulin (Sigma, T6557).

    Techniques: Transmission Assay, Over Expression, Inhibition, Isolation, Western Blot

    Primer sequences used for RT-qPCR.

    Journal: Frontiers in Pharmacology

    Article Title: LINC00586 Represses ASXL1 Expression Thus Inducing Epithelial-To-Mesenchymal Transition of Colorectal Cancer Cells Through LSD1-Mediated H3K4me2 Demethylation

    doi: 10.3389/fphar.2022.887822

    Figure Lengend Snippet: Primer sequences used for RT-qPCR.

    Article Snippet: After SDS-PAGE separation and membrane transfer, the protein was probed with the following primary antibodies (Abcam, United Kingdom): Anti-ASXL1 antibody (ab228009), anti-SEMA6B antibody (ab180215), anti-E-cadherin antibody (ab15148), anti-N-cadherin antibody (ab18203), anti-vimentin antibody (ab137321), and anti-GAPDH antibody (ab9485).

    Techniques:

    High expression of LINC00586 inhibits the expression of ASXL1 in CRC cells. (A) The mRNA and protein expression of ASXL1 was analyzed by real-time qPCR and immunoblotting analysis in CRC tissues ( n = 54) and surrounding non-neoplastic mucosa ( n = 54). (B) The mRNA and protein expression of ASXL1 was analyzed by real-time qPCR and immunoblotting analysis in a panel of CRC cell lines (HCTl16, LoVo, HT-29, SW480, and SW620) and FHC. (C,D) , The mRNA and protein expression of ASXL1 was analyzed by real-time qPCR (C) and immunoblotting analysis (D) in HCTl16 and LoVo cells upon LINC00586 knockdown or overexpression. * p < 0.05 by unpaired t test or by one-way ANOVA with Tukey’s test [only for (B) ].

    Journal: Frontiers in Pharmacology

    Article Title: LINC00586 Represses ASXL1 Expression Thus Inducing Epithelial-To-Mesenchymal Transition of Colorectal Cancer Cells Through LSD1-Mediated H3K4me2 Demethylation

    doi: 10.3389/fphar.2022.887822

    Figure Lengend Snippet: High expression of LINC00586 inhibits the expression of ASXL1 in CRC cells. (A) The mRNA and protein expression of ASXL1 was analyzed by real-time qPCR and immunoblotting analysis in CRC tissues ( n = 54) and surrounding non-neoplastic mucosa ( n = 54). (B) The mRNA and protein expression of ASXL1 was analyzed by real-time qPCR and immunoblotting analysis in a panel of CRC cell lines (HCTl16, LoVo, HT-29, SW480, and SW620) and FHC. (C,D) , The mRNA and protein expression of ASXL1 was analyzed by real-time qPCR (C) and immunoblotting analysis (D) in HCTl16 and LoVo cells upon LINC00586 knockdown or overexpression. * p < 0.05 by unpaired t test or by one-way ANOVA with Tukey’s test [only for (B) ].

    Article Snippet: After SDS-PAGE separation and membrane transfer, the protein was probed with the following primary antibodies (Abcam, United Kingdom): Anti-ASXL1 antibody (ab228009), anti-SEMA6B antibody (ab180215), anti-E-cadherin antibody (ab15148), anti-N-cadherin antibody (ab18203), anti-vimentin antibody (ab137321), and anti-GAPDH antibody (ab9485).

    Techniques: Expressing, Western Blot, Knockdown, Over Expression

    LINC00586 epigenetically silenced ASXL1 transcription through LSD1-mediated H3K4me2 demethylation. (A) Relative LINC00586 expression levels in nuclear and cytosolic fractions of HCTl16 and LoVo cells were determined by real-time qPCR. Nuclear controls: U6; Cytosolic controls: GAPDH. (B) RIP assay was performed to determine LINC00586 binding with LSD1. A significant enrichment of endogenous LINC00586 was detected in the anti-LSD1 RIP fraction (relative to nonspecific IgG control) in HCTl16 and LoVo cells. (C) HCTl16 and LoVo cells were subject to ChIP-PCR assays using anti-LSD1, anti-H3K4me2 or nonspecific IgG control for ASXL1 enrichment. (D) ChIP-PCR assays of ASXL1 enrichment in HCTl16 and LoVo cells following LSD1 knockdown and/or LINC00586 overexpression. * p < 0.05 by unpaired t test or by one-way ANOVA with Tukey’s test.

    Journal: Frontiers in Pharmacology

    Article Title: LINC00586 Represses ASXL1 Expression Thus Inducing Epithelial-To-Mesenchymal Transition of Colorectal Cancer Cells Through LSD1-Mediated H3K4me2 Demethylation

    doi: 10.3389/fphar.2022.887822

    Figure Lengend Snippet: LINC00586 epigenetically silenced ASXL1 transcription through LSD1-mediated H3K4me2 demethylation. (A) Relative LINC00586 expression levels in nuclear and cytosolic fractions of HCTl16 and LoVo cells were determined by real-time qPCR. Nuclear controls: U6; Cytosolic controls: GAPDH. (B) RIP assay was performed to determine LINC00586 binding with LSD1. A significant enrichment of endogenous LINC00586 was detected in the anti-LSD1 RIP fraction (relative to nonspecific IgG control) in HCTl16 and LoVo cells. (C) HCTl16 and LoVo cells were subject to ChIP-PCR assays using anti-LSD1, anti-H3K4me2 or nonspecific IgG control for ASXL1 enrichment. (D) ChIP-PCR assays of ASXL1 enrichment in HCTl16 and LoVo cells following LSD1 knockdown and/or LINC00586 overexpression. * p < 0.05 by unpaired t test or by one-way ANOVA with Tukey’s test.

    Article Snippet: After SDS-PAGE separation and membrane transfer, the protein was probed with the following primary antibodies (Abcam, United Kingdom): Anti-ASXL1 antibody (ab228009), anti-SEMA6B antibody (ab180215), anti-E-cadherin antibody (ab15148), anti-N-cadherin antibody (ab18203), anti-vimentin antibody (ab137321), and anti-GAPDH antibody (ab9485).

    Techniques: Expressing, Binding Assay, Control, Knockdown, Over Expression

    LINC00586 modulates CRC cell viability, invasion, EMT, and tumorigenicity by inhibiting ASXL1. (A) The mRNA and protein expression of ASXL1 was determined in HCTl16 and LoVo cells upon ASXL1 overexpression and/or LINC00586 overexpression by real-time qPCR and immunoblotting analysis. (B) The viability of HCTl16 and LoVo cells was examined by CCK-8 test upon ASXL1 overexpression and/or LINC00586 overexpression. (C) Representative view (× 200) of HCTl16 and LoVo cells migrating from upper transwell chambers into lower ones and statistics of migrating cells upon ASXL1 overexpression and/or LINC00586 overexpression. (D) The mRNA expression of E-Cadherin, N-Cadherin, and vimentin by real-time qPCR in HCTl16 and LoVo cells upon ASXL1 overexpression and/or LINC00586 overexpression. (E) Immunoblots of E-Cadherin, N-Cadherin, vimentin, and their quantitative analysis in HCTl16 and LoVo cells upon ASXL1 overexpression and/or LINC00586 overexpression. (F) HCTl16 cells were infected with the recombinant lentivirus harboring the full length of human ASXL1 gene alone or in combination with the recombinant lentivirus harboring human LINC00586 transcripts and injected into the nude mice ( n = 5), and the tumors were obtained 2 weeks after xenograft implantation and weighed. (G) The mRNA expression of E-Cadherin, N-Cadherin, and vimentin by real-time qPCR in CRC xenograft tissue sections upon ASXL1 overexpression and/or LINC00586 overexpression. (H) Immunoblots of E-Cadherin, N-Cadherin, vimentin, and their quantitative analysis in CRC xenograft tissue sections upon ASXL1 overexpression and/or LINC00586 overexpression.

    Journal: Frontiers in Pharmacology

    Article Title: LINC00586 Represses ASXL1 Expression Thus Inducing Epithelial-To-Mesenchymal Transition of Colorectal Cancer Cells Through LSD1-Mediated H3K4me2 Demethylation

    doi: 10.3389/fphar.2022.887822

    Figure Lengend Snippet: LINC00586 modulates CRC cell viability, invasion, EMT, and tumorigenicity by inhibiting ASXL1. (A) The mRNA and protein expression of ASXL1 was determined in HCTl16 and LoVo cells upon ASXL1 overexpression and/or LINC00586 overexpression by real-time qPCR and immunoblotting analysis. (B) The viability of HCTl16 and LoVo cells was examined by CCK-8 test upon ASXL1 overexpression and/or LINC00586 overexpression. (C) Representative view (× 200) of HCTl16 and LoVo cells migrating from upper transwell chambers into lower ones and statistics of migrating cells upon ASXL1 overexpression and/or LINC00586 overexpression. (D) The mRNA expression of E-Cadherin, N-Cadherin, and vimentin by real-time qPCR in HCTl16 and LoVo cells upon ASXL1 overexpression and/or LINC00586 overexpression. (E) Immunoblots of E-Cadherin, N-Cadherin, vimentin, and their quantitative analysis in HCTl16 and LoVo cells upon ASXL1 overexpression and/or LINC00586 overexpression. (F) HCTl16 cells were infected with the recombinant lentivirus harboring the full length of human ASXL1 gene alone or in combination with the recombinant lentivirus harboring human LINC00586 transcripts and injected into the nude mice ( n = 5), and the tumors were obtained 2 weeks after xenograft implantation and weighed. (G) The mRNA expression of E-Cadherin, N-Cadherin, and vimentin by real-time qPCR in CRC xenograft tissue sections upon ASXL1 overexpression and/or LINC00586 overexpression. (H) Immunoblots of E-Cadherin, N-Cadherin, vimentin, and their quantitative analysis in CRC xenograft tissue sections upon ASXL1 overexpression and/or LINC00586 overexpression.

    Article Snippet: After SDS-PAGE separation and membrane transfer, the protein was probed with the following primary antibodies (Abcam, United Kingdom): Anti-ASXL1 antibody (ab228009), anti-SEMA6B antibody (ab180215), anti-E-cadherin antibody (ab15148), anti-N-cadherin antibody (ab18203), anti-vimentin antibody (ab137321), and anti-GAPDH antibody (ab9485).

    Techniques: Expressing, Over Expression, Western Blot, CCK-8 Assay, Infection, Recombinant, Injection

    The graphical summary of the mechanism of LINC00586 in regulating CRC progression. LINC00586 epigenetically silenced ASXL1 transcription through LSD1-mediated H3K4me2 demethylation, thereby leading to the development and progression of CRC.

    Journal: Frontiers in Pharmacology

    Article Title: LINC00586 Represses ASXL1 Expression Thus Inducing Epithelial-To-Mesenchymal Transition of Colorectal Cancer Cells Through LSD1-Mediated H3K4me2 Demethylation

    doi: 10.3389/fphar.2022.887822

    Figure Lengend Snippet: The graphical summary of the mechanism of LINC00586 in regulating CRC progression. LINC00586 epigenetically silenced ASXL1 transcription through LSD1-mediated H3K4me2 demethylation, thereby leading to the development and progression of CRC.

    Article Snippet: After SDS-PAGE separation and membrane transfer, the protein was probed with the following primary antibodies (Abcam, United Kingdom): Anti-ASXL1 antibody (ab228009), anti-SEMA6B antibody (ab180215), anti-E-cadherin antibody (ab15148), anti-N-cadherin antibody (ab18203), anti-vimentin antibody (ab137321), and anti-GAPDH antibody (ab9485).

    Techniques:

    Autophagy downregulates G9a protein level through calpain activation. a, Autophagy induced by elevated cytoplasmic Ca 2+ (1 µM A23167), nutrient starvation (EBSS), and metabolic stress (1 µM Torin1) decreased the G9a protein level. For starvation-induced autophagy, Hela cells were washed twice with PBS and incubated with EBSS for the indicated time. An mTOR inhibitor (Torin1) and a Ca 2+ selective ionophore (A23187) were solubilized in dimethyl sulfoxide and diluted with DMEM containing 10% FBS to achieve the indicated final concentrations. b, Confocal immunofluorescence (IF) revealed that the endogenous G9a protein level was diminished during starvation in HEPES and EBSS media and metabolic stress (Torin1)-induced autophagy. However, sequestration of Ca 2+ by 5 µM BAPTA/AM prevented the reduction in G9a protein in Hela cells. The intensity of the G9a protein was quantified using the mean fluorescence intensity (MFI) of individual cells (n = 3 [40–80 per group]). c, The G9a protein level was diminished during serum starvation (HEPES)-induced autophagy in Huh7 cells. However, in the presence of a Ca 2+ -chelating agent (10 µM BAPTA/AM), the G9a protein level was significantly elevated. d, Calpain 1 expression downregulated the endogenous G9a protein in 293T cells. e, Calpain 1 and 2 expression downregulated exogenous G9a-GFP in 293T cells. f, Inhibition of calpains by calpeptin, a cell permeable calpain inhibitor, elevated the endogenous G9a protein level in 293T cells under starvation in EBSS medium. g, Expression of CAST/Calpastatin, an endogenous calpain inhibitory protein, prevented a reduction in the G9a protein level in Hela cells under growth and starvation (HEPES) conditions. All experiments were repeated at least three times, and statistical analyses were performed using student’s t tests to compare groups (mean ± SEM of n = 3 replicates; *p < 0.05; **p < 0.01; ***p < 0.001).

    Journal: bioRxiv

    Article Title: G9a/GLP methyltransferases inhibit autophagy by methylation-mediated ATG12 protein degradation

    doi: 10.1101/2021.02.05.430008

    Figure Lengend Snippet: Autophagy downregulates G9a protein level through calpain activation. a, Autophagy induced by elevated cytoplasmic Ca 2+ (1 µM A23167), nutrient starvation (EBSS), and metabolic stress (1 µM Torin1) decreased the G9a protein level. For starvation-induced autophagy, Hela cells were washed twice with PBS and incubated with EBSS for the indicated time. An mTOR inhibitor (Torin1) and a Ca 2+ selective ionophore (A23187) were solubilized in dimethyl sulfoxide and diluted with DMEM containing 10% FBS to achieve the indicated final concentrations. b, Confocal immunofluorescence (IF) revealed that the endogenous G9a protein level was diminished during starvation in HEPES and EBSS media and metabolic stress (Torin1)-induced autophagy. However, sequestration of Ca 2+ by 5 µM BAPTA/AM prevented the reduction in G9a protein in Hela cells. The intensity of the G9a protein was quantified using the mean fluorescence intensity (MFI) of individual cells (n = 3 [40–80 per group]). c, The G9a protein level was diminished during serum starvation (HEPES)-induced autophagy in Huh7 cells. However, in the presence of a Ca 2+ -chelating agent (10 µM BAPTA/AM), the G9a protein level was significantly elevated. d, Calpain 1 expression downregulated the endogenous G9a protein in 293T cells. e, Calpain 1 and 2 expression downregulated exogenous G9a-GFP in 293T cells. f, Inhibition of calpains by calpeptin, a cell permeable calpain inhibitor, elevated the endogenous G9a protein level in 293T cells under starvation in EBSS medium. g, Expression of CAST/Calpastatin, an endogenous calpain inhibitory protein, prevented a reduction in the G9a protein level in Hela cells under growth and starvation (HEPES) conditions. All experiments were repeated at least three times, and statistical analyses were performed using student’s t tests to compare groups (mean ± SEM of n = 3 replicates; *p < 0.05; **p < 0.01; ***p < 0.001).

    Article Snippet: Recombinant Flag-tagged human G9a (Active Motif, 31410), human calpain 1 (Novus, P4041), and calpain S1 (Abcam, ab180298) were incubated at 37°C for 10 min in calpain buffer (20 mM HEPES pH 7.5, 50 mM KCl, 2 mM MgCl 2 , 5 mM CaCl 2 , 1 mM DTT).

    Techniques: Activation Assay, Incubation, Immunofluorescence, Fluorescence, Expressing, Inhibition

    (a) Calpain 1 expression downregulated the endogenous GLP protein levels in 293T cells. The samples were manipulated as described in (mean ± SEM of n = 3 replicates; *p < 0.05; **p < 0.01). (b) HA-GLP was cleaved and degraded by calpain 1 and 2 expression in 293T cells. (c) A calpain inhibitor, calpeptin, elevated the endogenous GLP protein level in 293T cells under starvation in EBSS medium (mean ± SEM of n = 3 replicates). NS no significant difference (Student’s t -test). (d) Expression of CAST/Calpastatin, a calpain inhibitory protein, in Hela cells significantly stabilized endogenous GLP under starvation in HEPES medium (mean ± SEM of n = 3 replicates; **p < 0.01). (e) Cleaved HA-GLP was degraded by the UPS pathway. MG132, a proteasome inhibitor, led to the accumulation of the cleaved GLP intermediates produced by elevated Ca 2+ influx (0.5 µM A23187) and the expression of calpains 1 and 2 in 293T cells.

    Journal: bioRxiv

    Article Title: G9a/GLP methyltransferases inhibit autophagy by methylation-mediated ATG12 protein degradation

    doi: 10.1101/2021.02.05.430008

    Figure Lengend Snippet: (a) Calpain 1 expression downregulated the endogenous GLP protein levels in 293T cells. The samples were manipulated as described in (mean ± SEM of n = 3 replicates; *p < 0.05; **p < 0.01). (b) HA-GLP was cleaved and degraded by calpain 1 and 2 expression in 293T cells. (c) A calpain inhibitor, calpeptin, elevated the endogenous GLP protein level in 293T cells under starvation in EBSS medium (mean ± SEM of n = 3 replicates). NS no significant difference (Student’s t -test). (d) Expression of CAST/Calpastatin, a calpain inhibitory protein, in Hela cells significantly stabilized endogenous GLP under starvation in HEPES medium (mean ± SEM of n = 3 replicates; **p < 0.01). (e) Cleaved HA-GLP was degraded by the UPS pathway. MG132, a proteasome inhibitor, led to the accumulation of the cleaved GLP intermediates produced by elevated Ca 2+ influx (0.5 µM A23187) and the expression of calpains 1 and 2 in 293T cells.

    Article Snippet: Recombinant Flag-tagged human G9a (Active Motif, 31410), human calpain 1 (Novus, P4041), and calpain S1 (Abcam, ab180298) were incubated at 37°C for 10 min in calpain buffer (20 mM HEPES pH 7.5, 50 mM KCl, 2 mM MgCl 2 , 5 mM CaCl 2 , 1 mM DTT).

    Techniques: Expressing, Produced

    Calpains cleave G9a protein for ubiquitin-mediated degradation. a, Immunoprecipitation (IP) assays show that G9a bound to calpain 1 and 2. 293T cells were co-transfected with 5xMyc-calpain and G9a-GFP plasmids, and samples were prepared as described in the STAR Methods. To prevent G9a cleavage by calpains, the calpain inhibitor calpeptin (10 µM), protease inhibitors, and EGTA (1 mM) were added. b, G9a was cleaved by elevated Ca 2+ influx (A23187) and the expression of calpains in 293T cells. The accumulation of cleaved G9a fragments by MG132 indicates that the cleaved G9a was degraded by the UPS. c, Expression of calpain 1 or calpain 2 promoted G9a ubiquitination in 293T cells. 293T cells were co-transfected with the indicated constructs. d, Elevated Ca 2+ influx by A23187 promoted G9a ubiquitination, whereas Ca 2+ chelation by BAPTA/AM reduced G9a ubiquitination under starvation in EBSS medium. GST pull-down assays were performed to monitor the ubiquitination level of G9a-GST after 12 hours of EBSS starvation. e, Calpain cleavage sites in G9a are located between aa 600 and aa 700. f, The Δ600–700 G9a-GFP mutant lacking the calpain cleavage sites was more resistant than WT G9a-GFP to calpain 1, as indicated by the lack of cleavage products (red arrow) and a higher level of intact G9a protein. The cleavage products of the Δ600–700 G9a (black arrow) suggest the presence of an additional calpain cleavage site at the N-terminus of G9a. g, An in vitro calpain cleavage assay confirmed that G9a has two cleavage sites. The main site is between aa 600 and aa 700. An additional N-terminal cleavage site is near aa 300. Recombinant Flag-tagged hG9a, calpain 1, and calpain S1 (a calpain 1 co-activator) were incubated for 10 min at 37°C, and the reaction products were subjected to Western blot analysis. h, A schematic diagram of the G9a cleavage sites and fragments in and . The calpain cleavage sequences in G9a are highly conserved across vertebrates.

    Journal: bioRxiv

    Article Title: G9a/GLP methyltransferases inhibit autophagy by methylation-mediated ATG12 protein degradation

    doi: 10.1101/2021.02.05.430008

    Figure Lengend Snippet: Calpains cleave G9a protein for ubiquitin-mediated degradation. a, Immunoprecipitation (IP) assays show that G9a bound to calpain 1 and 2. 293T cells were co-transfected with 5xMyc-calpain and G9a-GFP plasmids, and samples were prepared as described in the STAR Methods. To prevent G9a cleavage by calpains, the calpain inhibitor calpeptin (10 µM), protease inhibitors, and EGTA (1 mM) were added. b, G9a was cleaved by elevated Ca 2+ influx (A23187) and the expression of calpains in 293T cells. The accumulation of cleaved G9a fragments by MG132 indicates that the cleaved G9a was degraded by the UPS. c, Expression of calpain 1 or calpain 2 promoted G9a ubiquitination in 293T cells. 293T cells were co-transfected with the indicated constructs. d, Elevated Ca 2+ influx by A23187 promoted G9a ubiquitination, whereas Ca 2+ chelation by BAPTA/AM reduced G9a ubiquitination under starvation in EBSS medium. GST pull-down assays were performed to monitor the ubiquitination level of G9a-GST after 12 hours of EBSS starvation. e, Calpain cleavage sites in G9a are located between aa 600 and aa 700. f, The Δ600–700 G9a-GFP mutant lacking the calpain cleavage sites was more resistant than WT G9a-GFP to calpain 1, as indicated by the lack of cleavage products (red arrow) and a higher level of intact G9a protein. The cleavage products of the Δ600–700 G9a (black arrow) suggest the presence of an additional calpain cleavage site at the N-terminus of G9a. g, An in vitro calpain cleavage assay confirmed that G9a has two cleavage sites. The main site is between aa 600 and aa 700. An additional N-terminal cleavage site is near aa 300. Recombinant Flag-tagged hG9a, calpain 1, and calpain S1 (a calpain 1 co-activator) were incubated for 10 min at 37°C, and the reaction products were subjected to Western blot analysis. h, A schematic diagram of the G9a cleavage sites and fragments in and . The calpain cleavage sequences in G9a are highly conserved across vertebrates.

    Article Snippet: Recombinant Flag-tagged human G9a (Active Motif, 31410), human calpain 1 (Novus, P4041), and calpain S1 (Abcam, ab180298) were incubated at 37°C for 10 min in calpain buffer (20 mM HEPES pH 7.5, 50 mM KCl, 2 mM MgCl 2 , 5 mM CaCl 2 , 1 mM DTT).

    Techniques: Ubiquitin Proteomics, Immunoprecipitation, Transfection, Expressing, Construct, Mutagenesis, In Vitro, Cleavage Assay, Recombinant, Incubation, Western Blot

    Fading fluorescence intensity from the truncation G9a mutants fused to a C-terminal GFP indicates that calpain 1 cleaved between aa 600 and aa 700 in G9a. Constructs expressing truncated G9a-GFP with or without 5xMyc-calpain 1 were transfected into Hela cells, and GFP signals were acquired using a Leica DM5000 B.

    Journal: bioRxiv

    Article Title: G9a/GLP methyltransferases inhibit autophagy by methylation-mediated ATG12 protein degradation

    doi: 10.1101/2021.02.05.430008

    Figure Lengend Snippet: Fading fluorescence intensity from the truncation G9a mutants fused to a C-terminal GFP indicates that calpain 1 cleaved between aa 600 and aa 700 in G9a. Constructs expressing truncated G9a-GFP with or without 5xMyc-calpain 1 were transfected into Hela cells, and GFP signals were acquired using a Leica DM5000 B.

    Article Snippet: Recombinant Flag-tagged human G9a (Active Motif, 31410), human calpain 1 (Novus, P4041), and calpain S1 (Abcam, ab180298) were incubated at 37°C for 10 min in calpain buffer (20 mM HEPES pH 7.5, 50 mM KCl, 2 mM MgCl 2 , 5 mM CaCl 2 , 1 mM DTT).

    Techniques: Fluorescence, Construct, Expressing, Transfection

    Peptide mapping by LC-MS/MS indicates that calpain 1 cleaved at the 615 position in G9a starting with a GGCLSAV sequence (green). In addition, LC-MS/MS data reveal three other calpain cleavage sites (blue). G9a-GST and 5xMyc-calpain 1 were transfected into 293T cells. The cleaved G9a-GST was purified using a glutathione Sepharose column as described in the STAR Methods and digested with trypsin prior to analysis with LC-MS/MS.

    Journal: bioRxiv

    Article Title: G9a/GLP methyltransferases inhibit autophagy by methylation-mediated ATG12 protein degradation

    doi: 10.1101/2021.02.05.430008

    Figure Lengend Snippet: Peptide mapping by LC-MS/MS indicates that calpain 1 cleaved at the 615 position in G9a starting with a GGCLSAV sequence (green). In addition, LC-MS/MS data reveal three other calpain cleavage sites (blue). G9a-GST and 5xMyc-calpain 1 were transfected into 293T cells. The cleaved G9a-GST was purified using a glutathione Sepharose column as described in the STAR Methods and digested with trypsin prior to analysis with LC-MS/MS.

    Article Snippet: Recombinant Flag-tagged human G9a (Active Motif, 31410), human calpain 1 (Novus, P4041), and calpain S1 (Abcam, ab180298) were incubated at 37°C for 10 min in calpain buffer (20 mM HEPES pH 7.5, 50 mM KCl, 2 mM MgCl 2 , 5 mM CaCl 2 , 1 mM DTT).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Sequencing, Transfection, Purification

    (a) Both GST-WT G9a and GST-Δ600–700 G9a expression downregulated the ATG12-ATG5 conjugate level under starvation in EBSS medium. The expression of the calpain-resistant Δ600–700 G9a in 293T cells suppressed the formation of the ATG12-ATG5 conjugate and the conversion of LC3BI to LC3BII more noticeably than the expression of WT G9a while increasing the p62 level under starvation conditions. (b) HA-GLP expression in Hela cells also reduced the ATG12-ATG5 conjugate level under growth and starvation (HEPES) conditions. (c) The expression of calpains 1 and 2 promoted the conversion of LC3BI to LC3BII under starvation in HEPES medium. (d) Autophagy induction by G9a inhibition (2 µM UNC0638) required ATG5. Only when ATG5 was reconstituted in ATG5−/− MEFs, UNC0638-mediated LC3BII conversion occur. Thus, ATG12-ATG5 conjugate formation was necessary for UNC0638-induced autophagy.

    Journal: bioRxiv

    Article Title: G9a/GLP methyltransferases inhibit autophagy by methylation-mediated ATG12 protein degradation

    doi: 10.1101/2021.02.05.430008

    Figure Lengend Snippet: (a) Both GST-WT G9a and GST-Δ600–700 G9a expression downregulated the ATG12-ATG5 conjugate level under starvation in EBSS medium. The expression of the calpain-resistant Δ600–700 G9a in 293T cells suppressed the formation of the ATG12-ATG5 conjugate and the conversion of LC3BI to LC3BII more noticeably than the expression of WT G9a while increasing the p62 level under starvation conditions. (b) HA-GLP expression in Hela cells also reduced the ATG12-ATG5 conjugate level under growth and starvation (HEPES) conditions. (c) The expression of calpains 1 and 2 promoted the conversion of LC3BI to LC3BII under starvation in HEPES medium. (d) Autophagy induction by G9a inhibition (2 µM UNC0638) required ATG5. Only when ATG5 was reconstituted in ATG5−/− MEFs, UNC0638-mediated LC3BII conversion occur. Thus, ATG12-ATG5 conjugate formation was necessary for UNC0638-induced autophagy.

    Article Snippet: Recombinant Flag-tagged human G9a (Active Motif, 31410), human calpain 1 (Novus, P4041), and calpain S1 (Abcam, ab180298) were incubated at 37°C for 10 min in calpain buffer (20 mM HEPES pH 7.5, 50 mM KCl, 2 mM MgCl 2 , 5 mM CaCl 2 , 1 mM DTT).

    Techniques: Expressing, Inhibition

    G9a regulates autophagy and apoptosis through K72 methylation in ATG12 a, Reconstitution of a methylation-resistant mutant of ATG12 (K72R) in ATG12-deficient MEFs promoted basal autophagy, whereas a hyper-methylation mutant of ATG12 (K71R) was less effective than WT ATG12 in inducing basal autophagy. b, Reconstitution of K72R ATG12 mutant in ATG12 −/− MEFs promoted autophagy when mTOR was inhibited (1 µM Torin1). c, Reconstitution of the methylation-resistant K72R ATG12 in ATG12-deficient MEFs conferred resistance to 10 µM UNC0368-induced apoptosis, whereas the expression of the hyper-methylated K71R ATG12 mutant in ATG12-deficient MEFs produced the opposite effect. d, Our model proposes that G9a/GLP inhibit autophagy by directly methylating ATG12. The methylated ATG12 subsequently undergoes methylation-mediated protein degradation under non-stress conditions. Under stress conditions, the Ca 2+ -activated calpain system downregulates the G9a/GLP proteins, thereby allowing the accumulation of ATG12, ATG12-ATG5 conjugate, and LC3II. Subsequent activation of lysosomal activity removes the ATG12-ATG5-ATG16L1 complex at a later stage of autophagy. All experiments were repeated at least three times, and statistical analyses were performed using student’s t tests to compare groups (mean ± SEM of n = 3 replicates; *p < 0.05; **p < 0.01; ***p < 0.001).

    Journal: bioRxiv

    Article Title: G9a/GLP methyltransferases inhibit autophagy by methylation-mediated ATG12 protein degradation

    doi: 10.1101/2021.02.05.430008

    Figure Lengend Snippet: G9a regulates autophagy and apoptosis through K72 methylation in ATG12 a, Reconstitution of a methylation-resistant mutant of ATG12 (K72R) in ATG12-deficient MEFs promoted basal autophagy, whereas a hyper-methylation mutant of ATG12 (K71R) was less effective than WT ATG12 in inducing basal autophagy. b, Reconstitution of K72R ATG12 mutant in ATG12 −/− MEFs promoted autophagy when mTOR was inhibited (1 µM Torin1). c, Reconstitution of the methylation-resistant K72R ATG12 in ATG12-deficient MEFs conferred resistance to 10 µM UNC0368-induced apoptosis, whereas the expression of the hyper-methylated K71R ATG12 mutant in ATG12-deficient MEFs produced the opposite effect. d, Our model proposes that G9a/GLP inhibit autophagy by directly methylating ATG12. The methylated ATG12 subsequently undergoes methylation-mediated protein degradation under non-stress conditions. Under stress conditions, the Ca 2+ -activated calpain system downregulates the G9a/GLP proteins, thereby allowing the accumulation of ATG12, ATG12-ATG5 conjugate, and LC3II. Subsequent activation of lysosomal activity removes the ATG12-ATG5-ATG16L1 complex at a later stage of autophagy. All experiments were repeated at least three times, and statistical analyses were performed using student’s t tests to compare groups (mean ± SEM of n = 3 replicates; *p < 0.05; **p < 0.01; ***p < 0.001).

    Article Snippet: Recombinant Flag-tagged human G9a (Active Motif, 31410), human calpain 1 (Novus, P4041), and calpain S1 (Abcam, ab180298) were incubated at 37°C for 10 min in calpain buffer (20 mM HEPES pH 7.5, 50 mM KCl, 2 mM MgCl 2 , 5 mM CaCl 2 , 1 mM DTT).

    Techniques: Methylation, Mutagenesis, Expressing, Produced, Activation Assay, Activity Assay

    POLE2 level is upregulated and associated with various clinicopathological parameters in RCC. The POLE2 mRNA level in RCC was obtained from The Cancer Genome Atlas Kidney Clear Cell Carcinoma dataset (TCGA-KIRC; containing 72 normal tissues and 530 RCC tissues) and International Cancer Genome Consortium (ICGC; containing 44 normal tissues and 91 RCC tissues). The POLE2 mRNA levels were compared in different clinicopathological parameters: (A,C) Cancer vs. para-cancer, (B) Carcinoma vs. paired para-cancer, (D) TNM stage, and (E) G stage. (F) Kaplan-Meier curves of the overall survival of RCC patients with high or low POLE2 expression in TCGA-KIRC cohort. (G) Representative immunohistochemical staining for POLE2 in RCC and adjacent normal tissues. (H) Kaplan-Meier curves of the overall survival of RCC patients with high or low POLE2 expression. ** P < 0.01, *** P < 0.001.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1

    doi: 10.3389/fcell.2021.622344

    Figure Lengend Snippet: POLE2 level is upregulated and associated with various clinicopathological parameters in RCC. The POLE2 mRNA level in RCC was obtained from The Cancer Genome Atlas Kidney Clear Cell Carcinoma dataset (TCGA-KIRC; containing 72 normal tissues and 530 RCC tissues) and International Cancer Genome Consortium (ICGC; containing 44 normal tissues and 91 RCC tissues). The POLE2 mRNA levels were compared in different clinicopathological parameters: (A,C) Cancer vs. para-cancer, (B) Carcinoma vs. paired para-cancer, (D) TNM stage, and (E) G stage. (F) Kaplan-Meier curves of the overall survival of RCC patients with high or low POLE2 expression in TCGA-KIRC cohort. (G) Representative immunohistochemical staining for POLE2 in RCC and adjacent normal tissues. (H) Kaplan-Meier curves of the overall survival of RCC patients with high or low POLE2 expression. ** P < 0.01, *** P < 0.001.

    Article Snippet: After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control.

    Techniques: Expressing, Immunohistochemical staining, Staining

    POLE2 expression in renal cell carcinoma and para-carcinoma tissues detected by immunohistochemistry.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1

    doi: 10.3389/fcell.2021.622344

    Figure Lengend Snippet: POLE2 expression in renal cell carcinoma and para-carcinoma tissues detected by immunohistochemistry.

    Article Snippet: After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control.

    Techniques: Expressing, Immunohistochemistry

    Relationship between POLE2 expression and tumor characteristics in patients with renal cell carcinoma.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1

    doi: 10.3389/fcell.2021.622344

    Figure Lengend Snippet: Relationship between POLE2 expression and tumor characteristics in patients with renal cell carcinoma.

    Article Snippet: After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control.

    Techniques: Expressing

    Relationship between POLE2 expression and tumor characteristics in patients with renal cell carcinoma.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1

    doi: 10.3389/fcell.2021.622344

    Figure Lengend Snippet: Relationship between POLE2 expression and tumor characteristics in patients with renal cell carcinoma.

    Article Snippet: After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control.

    Techniques: Expressing

    Knockdown of POLE2 expression inhibits RCC cell proliferation and migration. (A) The cell proliferation activity of A498 and ACHN cells infected with shCtrl or shPOLE2 lentivirus was detected by MTT assays. (B) The clone formation ability of A498 and ACHN cells infected with shCtrl or shPOLE2 lentivirus was detected by (B) clone formation assays. (C) The migration ability of A498 and ACHN cells infected with shCtrl or shPOLE2 lentivirus was detected by Transwell assays. (D) The apoptosis rate of A498 and ACHN cells infected with shCtrl or shPOLE2 lentivirus was measured using flow cytometry. (E) The clone formation ability of A498 and ACHN cells infected with shCtrl or shPOLE2 lentivirus was detected by soft agar colony formation assays. (F) The protein expression levels of N-cadherin, Vimentin and Snail in A498 and ACHN cells infected with shCtrl or shPOLE2 lentivirus was detected by Western blot, GAPDH served as loading control. (G) PDOs were generated from biopsies of RCC patients (red circle) and cultured. (H) Representative images of three different RCC organoids transfected with shCtrl or shPOLE2 lentivirus and quantification of organoid diameters. *** P < 0.001. The red arrow points to the organoid.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1

    doi: 10.3389/fcell.2021.622344

    Figure Lengend Snippet: Knockdown of POLE2 expression inhibits RCC cell proliferation and migration. (A) The cell proliferation activity of A498 and ACHN cells infected with shCtrl or shPOLE2 lentivirus was detected by MTT assays. (B) The clone formation ability of A498 and ACHN cells infected with shCtrl or shPOLE2 lentivirus was detected by (B) clone formation assays. (C) The migration ability of A498 and ACHN cells infected with shCtrl or shPOLE2 lentivirus was detected by Transwell assays. (D) The apoptosis rate of A498 and ACHN cells infected with shCtrl or shPOLE2 lentivirus was measured using flow cytometry. (E) The clone formation ability of A498 and ACHN cells infected with shCtrl or shPOLE2 lentivirus was detected by soft agar colony formation assays. (F) The protein expression levels of N-cadherin, Vimentin and Snail in A498 and ACHN cells infected with shCtrl or shPOLE2 lentivirus was detected by Western blot, GAPDH served as loading control. (G) PDOs were generated from biopsies of RCC patients (red circle) and cultured. (H) Representative images of three different RCC organoids transfected with shCtrl or shPOLE2 lentivirus and quantification of organoid diameters. *** P < 0.001. The red arrow points to the organoid.

    Article Snippet: After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control.

    Techniques: Expressing, Migration, Activity Assay, Infection, Flow Cytometry, Western Blot, Generated, Cell Culture, Transfection

    Knockdown of POLE2 attenuated tumorigenesis and tumor growth of ACHN cells in vivo . (A) The growth curves of xenografts from ACHN cells infected with shCtrl or shPOLE2 lentivirus, and the tumor weight examined at the ending point. (B) Fluorescent intensity of the xenograft model at the ending point. (C) Immunohistochemical staining for proliferation marker protein Ki-67 protein in the xenograft tumors. (D) HE staining in the xenograft tumors. *P < 0.05, **P < 0.01.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1

    doi: 10.3389/fcell.2021.622344

    Figure Lengend Snippet: Knockdown of POLE2 attenuated tumorigenesis and tumor growth of ACHN cells in vivo . (A) The growth curves of xenografts from ACHN cells infected with shCtrl or shPOLE2 lentivirus, and the tumor weight examined at the ending point. (B) Fluorescent intensity of the xenograft model at the ending point. (C) Immunohistochemical staining for proliferation marker protein Ki-67 protein in the xenograft tumors. (D) HE staining in the xenograft tumors. *P < 0.05, **P < 0.01.

    Article Snippet: After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control.

    Techniques: In Vivo, Infection, Immunohistochemical staining, Staining, Marker

    POLE2 interacts with STC1. (A) The volcano map of DEGs between normal ACHN cells and knock-down POLE2 ACHN cells. (B) Classic pathway enrichment analysis. (C) Ingenuity Pathway Analysis (IPA)-identified interaction network between POLE2 and the pathways p53 Signaling, B Cell Receptor Signaling, and HGF Signaling. (D,E) The expression levels of downstream genes related to POLE2 were detected by (D) qRT-PCR and (E) Western blot. (F) The cell proliferation activity of ACHN cells infected with shCtrl, sh CDC42EP3, shEZR, shGDAP1, shRDM1 or shSTC1 lentivirus was detected by HCS Cell Proliferation Assay. (G) The interaction of POLE2 and STC1 in ACHN cells were examined by Co-IP experiment. (H) Representative immunohistochemical staining for STC1 in RCC and adjacent normal tissues. * P < 0.05, ** P < 0.01, *** P < 0.001.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1

    doi: 10.3389/fcell.2021.622344

    Figure Lengend Snippet: POLE2 interacts with STC1. (A) The volcano map of DEGs between normal ACHN cells and knock-down POLE2 ACHN cells. (B) Classic pathway enrichment analysis. (C) Ingenuity Pathway Analysis (IPA)-identified interaction network between POLE2 and the pathways p53 Signaling, B Cell Receptor Signaling, and HGF Signaling. (D,E) The expression levels of downstream genes related to POLE2 were detected by (D) qRT-PCR and (E) Western blot. (F) The cell proliferation activity of ACHN cells infected with shCtrl, sh CDC42EP3, shEZR, shGDAP1, shRDM1 or shSTC1 lentivirus was detected by HCS Cell Proliferation Assay. (G) The interaction of POLE2 and STC1 in ACHN cells were examined by Co-IP experiment. (H) Representative immunohistochemical staining for STC1 in RCC and adjacent normal tissues. * P < 0.05, ** P < 0.01, *** P < 0.001.

    Article Snippet: After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Infection, Proliferation Assay, Co-Immunoprecipitation Assay, Immunohistochemical staining, Staining

    POLE2 promotes RCC cell proliferation and migration by regulating STC1. (A) The cell proliferation activity of control, POLE2, NC(OE + KD), POLE2 + shSTC1 groups was detected by HCS Cell Proliferation Assay. (B) The clone formation ability of NC(OE + KD) and POLE2 + shSTC1 groups was detected by clone formation assays. (C,D) The migration ability of NC(OE + KD) and POLE2 + shSTC1 groups was evaluated by (C) Transwell assays and (D) wound-healing assay. (E) The apoptosis rate of control, POLE2, NC(OE + KD), POLE2 + shSTC1 group was measured using flow cytometry. ** P < 0.01, *** P < 0.001.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1

    doi: 10.3389/fcell.2021.622344

    Figure Lengend Snippet: POLE2 promotes RCC cell proliferation and migration by regulating STC1. (A) The cell proliferation activity of control, POLE2, NC(OE + KD), POLE2 + shSTC1 groups was detected by HCS Cell Proliferation Assay. (B) The clone formation ability of NC(OE + KD) and POLE2 + shSTC1 groups was detected by clone formation assays. (C,D) The migration ability of NC(OE + KD) and POLE2 + shSTC1 groups was evaluated by (C) Transwell assays and (D) wound-healing assay. (E) The apoptosis rate of control, POLE2, NC(OE + KD), POLE2 + shSTC1 group was measured using flow cytometry. ** P < 0.01, *** P < 0.001.

    Article Snippet: After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control.

    Techniques: Migration, Activity Assay, Proliferation Assay, Wound Healing Assay, Flow Cytometry

    The underlying mechanism of POLE2 on apoptosis of A498 cells. (A) The apoptosis relative proteins were detected by human apoptosis antibody array in A498 cells with or without POLE2 knockdown. (B) Differences in human apoptotic antibody array were analyzed in A498 cells with or without POLE2 knockdown. (C) The gray values of differentially expressed proteins in A498 cells with or without POLE2 knockdown. (D) Cancer-related pathway proteins were confirmed by western blot in A498 cells with or without POLE2 knockdown, GAPDH served as loading control. * P < 0.05, ** P < 0.01.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1

    doi: 10.3389/fcell.2021.622344

    Figure Lengend Snippet: The underlying mechanism of POLE2 on apoptosis of A498 cells. (A) The apoptosis relative proteins were detected by human apoptosis antibody array in A498 cells with or without POLE2 knockdown. (B) Differences in human apoptotic antibody array were analyzed in A498 cells with or without POLE2 knockdown. (C) The gray values of differentially expressed proteins in A498 cells with or without POLE2 knockdown. (D) Cancer-related pathway proteins were confirmed by western blot in A498 cells with or without POLE2 knockdown, GAPDH served as loading control. * P < 0.05, ** P < 0.01.

    Article Snippet: After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control.

    Techniques: Ab Array, Western Blot

    The mechanism diagram of POLE2 involved in the biological function of RCC.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1

    doi: 10.3389/fcell.2021.622344

    Figure Lengend Snippet: The mechanism diagram of POLE2 involved in the biological function of RCC.

    Article Snippet: After blocking with 5% skim milk-Tris-based saline-Tween 20 (TBST) at room temperature for 1 h, membranes were incubated overnight at 4°C with primary antibodies to POLE2 (dilution at 1:1000; cat. no. ab180214; Abcam), N-cadherin (dilution at 1:1000; cat. no. ab18203; Abcam), Vimentin (dilution at 1:1000; cat. no. ab92547; Abcam), Snail (dilution at 1:1000; cat. no. ab194583; CST), BCL-2 (dilution at 1:1000; cat. no. 3879S; Abcam), CDC42EP3 (dilution at 1:1000; cat. no. NBP1-88382; NOVUS), EZR (dilution at 1:2000; cat. no. ab40839; abcam), GDAP1 (dilution at 1:1000; cat. no. orb39831; biorbyt), RDM1 (dilution at 1:1000; cat. no. orb352658; biorbyt), and STC1 (dilution at 1:1000; cat. no. ab229477; abcam), GAPDH (dilution at 1:3000; cat. no. AP0063; Bioworld) served as loading control.

    Techniques: