Journal: bioRxiv

Article Title: Coordination of transcription-coupled repair and repair-independent release of stalled RNA polymerase II in response to transcription-blocking lesions

doi: 10.1101/2024.07.07.602436

Figure Lengend Snippet: a, Schematic representation of Damage-seq and PADD-seq. For Damage-seq, genomic DNA extracted from UV-irradiated cells are sonicated and ligated to the first adaptor, followed by immunoprecipitation with the lesion-specific antibody. The precise sites of the lesions are determined by the stalling of primer extension with high-fidelity DNA polymerase. After the ligation of the second adaptor, the primer extension products are PCR-amplified and sequenced. For PADD-seq, PolII-associated DNA are enriched by regular chromatin- immunoprecipitation (ChIP) with an anti-pan-RPB1 antibody. Purified DNA fragments are subjected to Damage-seq. b, Diagram of the experimental design for the measurement of TCR (by Damage-seq) and PolII-CPD interaction (by PADD-seq). c, Meta-gene analysis of PADD-seq signals around TSSs and TESs for active genes longer than 50 kb ( n = 2790) under indicated conditions. d, Quantification of PolII retention on damage sites by relative change of PADD-seq signals from 0.5 h to 2 h on each gene. Active genes longer than 20 kb were selected ( n = 4488). e, Meta-gene analysis of Damage-seq signals around TSSs for active genes longer than 50 kb ( n = 2790) under indicated conditions. Cells were collected immediately (0 h) or at 8 h after UV irradiation. Fraction CPDs remaining was calculated as the ratio of 8 h to 0 h. f, Violin plots of relative Damage-seq signals on each active gene ( n = 6406). Log2 value of the ratio of fraction CPDs remaining on TS to that on NTS was calculated. P value was calculated using two-tailed paired Student’s t -test for (d) and (f) . See also Supplementary Fig. 1 and 2. Source data are provided as a Source Data file.

Article Snippet: In brief, 167 μg of fragmented chromatin and 25 μg of anti-RPB1 antibody (Bethyl Laboratories, A304-405A) were incubated in RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% SDS and 0.1% Na-DOC) supplemented with protease inhibitors, 0.1 % BSA (Sigma) and 0.1 μg/μl tRNA (Sigma) for 2 h on a rotator at 4 °C.

Techniques: Irradiation, Sonication, Immunoprecipitation, Ligation, Amplification, Chromatin Immunoprecipitation, Purification, Two Tailed Test

Journal: bioRxiv

Article Title: Coordination of transcription-coupled repair and repair-independent release of stalled RNA polymerase II in response to transcription-blocking lesions

doi: 10.1101/2024.07.07.602436

Figure Lengend Snippet: a, Meta-gene analysis of Damage-seq signals around TSSs for active genes longer than 50 kb ( n = 2790) under indicated conditions. Cells were collected immediately (0 h) or at 8 h after UV irradiation. Fraction CPDs remaining was calculated as the ratio of 8 h to 0 h. Data of WT and CSB -KO cells are from our previous study . b, Violin plots of relative Damage-seq signals on each active gene ( n = 6406). Log2 value of the ratio of fraction CPDs remaining on TS to that on NTS was calculated. c, Meta-gene analysis of PADD-seq signals around TSSs and TESs for active genes longer than 50 kb ( n = 2790) under indicated conditions. d, Quantification of PolII retention on damage sites by relative change of PADD-seq signals from 0.5 h to 2 h on each gene. Active genes longer than 20 kb were selected ( n = 4488). P value was calculated using two-tailed paired Student’s t -test for (b) and (d) . e, Diagram showing that three possible fates of lesion-stalled PolII should co-exist in RPB1-K1268R cells. See also Supplementary Fig. 5. Source data are provided as a Source Data file.

Article Snippet: In brief, 167 μg of fragmented chromatin and 25 μg of anti-RPB1 antibody (Bethyl Laboratories, A304-405A) were incubated in RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% SDS and 0.1% Na-DOC) supplemented with protease inhibitors, 0.1 % BSA (Sigma) and 0.1 μg/μl tRNA (Sigma) for 2 h on a rotator at 4 °C.

Techniques: Irradiation, Two Tailed Test

Journal: Biochemical pharmacology

Article Title: m 6 A modification impacts hepatic drug and lipid metabolism properties by regulating carboxylesterase 2.

doi: 10.1016/j.bcp.2021.114766

Figure Lengend Snippet: Fig. 4. Posttranscriptional regulation of CES2 by RNA methylation via m6A reader protein YTHDC2. The stability of CES2 mRNA in siRNA-transfected HepG2 cells was examined (A-C). HepG2 cells were treated with 10 ng/µL α-amanitin 48 h after transfection with siMETTL3 and siMETTL14 (A), siFTO (B), siALKBH5 (C), or siControl (A-C). Total RNA was prepared after 0, 12, 24, and 36 h. The CES2 mRNA level was determined by using real-time RT-PCR. The CES2 mRNA levels at time 0 (the time of addition of α-amanitin) in each treatment were assigned values of 100%. YTHDC2 mRNA, YTHDF2 mRNA (D), CES2 mRNA (E), and CES2 protein (F) levels in siYTHDC2- or siYTHDF2-transfected HepG2 cells were determined by real-time RT-PCR and Western blotting. The mRNA and protein levels were normalized to β-actin levels. The values represent the levels relative to siControl. (G) Cell lysates from HepG2 cells were immunoprecipitated with an anti-human YTHDC2 antibody or normal rabbit IgG. An electropherogram of the PCR amplicon using primers for CES2 mRNA is shown. The length of the PCR product was 316 bp. Each point and column represent the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.01 compared with siControl.

Article Snippet: The rabbit anti-human YTHDC2 polyclonal antibody was from Bethyl Laboratories (Montgomery, TX).

Techniques: Methylation, Transfection, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Amplification

Journal: Biochemical pharmacology

Article Title: Trehalose protects against cisplatin-induced cochlear hair cell damage by activating TFEB-mediated autophagy.

doi: 10.1016/j.bcp.2021.114904

Figure Lengend Snippet: Fig. 8. Trehalose induces autophagy in HEI-OC1 cells and cochlear HCs. (a) HEI-OC1 cells were transfected with mRFP-GFP-LC3B plasmids and treated with trehalose. The yellow dots indicate autophagosomes, and the red dots indicate autolysosomes. n = 3. (b) Quantification of LC3B fluorescent puncta in (a). (c) qPCR showing changes in the transcription of the autophagy-related genes Atg5, Atg7, Becn1, TFEB, Lamp2 and p62 in HEI-OC1 cells. n = 3. (d) Immunofluorescence staining of myosin7a and LC3B in cochleae. n = 3. (e) Quantification of the LC3B intensity in (d). (f) Western blot results showing the expression of LC3II after trehalose treatment. n = 3. (g) Quantitative analysis of the data shown in the (f). n = 4. (h) RT-qPCR showing changes in the transcription of the autophagy-related genes Atg5, Atg7, Becn1, TFEB, Lamp2 and p62 in cochlear explants. n = 3. Scale bars: 10 µm. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control group; ###P < 0.001 versus the trehalose group.

Article Snippet: The following reagents and antibodies were used: TFEB (Bethyl, A303-673A, USA), LC3II (Novus, NB100-2220, USA), P65 (Novus, NBP1-42821, USA), LAMP1 (Sigma, SAB3500285, USA), LAMP2A (Abcam, ab125028, UK), a rabbit anti-myosin-VIIa antibody (Proteus Bioscience, 25–6790, UK), αII-spectrin (clone D8BI7, BioLegend, 803201, USA), a mouse anti-myosin-VIIa antibody (Santa Cruz Biotechnology, sc-74, 516AC, USA), cleaved caspase 3 (Cell Signaling Technology, 9664, USA), cleaved PARP (Cell Signaling Technology, 9544, USA), DAPI (Abcam, ab104139, UK), GAPDH (Cell Signaling Technology, 5174, USA), β-actin (Cell Signaling Technology, 4970, USA), cisplatin (Sigma, P4394, USA), trehalose (Sigma, T9531, USA), 3- methyladenine (3-MA; MCE, HY-19312, USA), rapamycin (MCE, HY10219, USA), hydroxychloroquine (HCQ) (MCE, HY-B1370, USA), and cyclosporin A (CsA) (MCE, HY-B0579, USA).

Techniques: Transfection, Immunofluorescence, Staining, Western Blot, Expressing, Quantitative RT-PCR, Control

Journal: Biochemical pharmacology

Article Title: Trehalose protects against cisplatin-induced cochlear hair cell damage by activating TFEB-mediated autophagy.

doi: 10.1016/j.bcp.2021.114904

Figure Lengend Snippet: Fig. 11. The protective effects of trehalose are attributed to TFEB in HEI-OC1 cells. (a) Western blot results showed the expression of TFEB transfected with tfeb- siRNA, n = 3. (b) Western blot results showing the expression of LC3II after TFEB silencing in the presence of trehalose. n = 3. (c) Western blot results showing the expression of cleaved caspase 3 and TFEB in the presence of cisplatin or trehalose treatment after TFEB silencing. n = 4. (d) Western blot results showing the expression of LC3II and TFEB in the presence or absence of cisplatin treatment after TFEB overexpression. n = 3. (e) Western blot results showing the expression of cleaved caspase 3 in the presence of cisplatin treatment after TFEB overexpression. n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control group; &P < 0.05, &&P < 0.01, and &&&P < 0.001 versus the trehalose plus cisplatin group; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the cisplatin group in (d) and (e) and versus the trehalose group in (b).

Article Snippet: The following reagents and antibodies were used: TFEB (Bethyl, A303-673A, USA), LC3II (Novus, NB100-2220, USA), P65 (Novus, NBP1-42821, USA), LAMP1 (Sigma, SAB3500285, USA), LAMP2A (Abcam, ab125028, UK), a rabbit anti-myosin-VIIa antibody (Proteus Bioscience, 25–6790, UK), αII-spectrin (clone D8BI7, BioLegend, 803201, USA), a mouse anti-myosin-VIIa antibody (Santa Cruz Biotechnology, sc-74, 516AC, USA), cleaved caspase 3 (Cell Signaling Technology, 9664, USA), cleaved PARP (Cell Signaling Technology, 9544, USA), DAPI (Abcam, ab104139, UK), GAPDH (Cell Signaling Technology, 5174, USA), β-actin (Cell Signaling Technology, 4970, USA), cisplatin (Sigma, P4394, USA), trehalose (Sigma, T9531, USA), 3- methyladenine (3-MA; MCE, HY-19312, USA), rapamycin (MCE, HY10219, USA), hydroxychloroquine (HCQ) (MCE, HY-B1370, USA), and cyclosporin A (CsA) (MCE, HY-B0579, USA).

Techniques: Western Blot, Expressing, Transfection, Over Expression, Control

Journal: Biochemical pharmacology

Article Title: Trehalose protects against cisplatin-induced cochlear hair cell damage by activating TFEB-mediated autophagy.

doi: 10.1016/j.bcp.2021.114904

Figure Lengend Snippet: Fig. 12. Calcineurin is involved in trehalose-induced TFEB nuclear translocation. (a) Immunoblot analysis of TFEB expression in nuclear and cytoplasmic sub fractions after trehalose treatment in the presence or absence of CsA. n = 3. (b) Quantitative analysis of the data shown in (a). (c) The expression of LC3II and P62 was analyzed by western blotting after CsA treatment. n = 4. (d) Quantitative analysis of the data shown in (c). (e) CCK-8 results showing the viability of HEI-OC1 cells after the different treatments. (f) Western blot results showing the expression of cleaved caspase 3 in the presence of cisplatin and trehalose treatment in the presence or absence of CsA. n = 3. (g) Quantitative analysis of the data shown in (f). *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control group; &P < 0.05, &&P < 0.01, and &&&P < 0.001 versus the trehalose group; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the cisplatin plus trehalose group.

Article Snippet: The following reagents and antibodies were used: TFEB (Bethyl, A303-673A, USA), LC3II (Novus, NB100-2220, USA), P65 (Novus, NBP1-42821, USA), LAMP1 (Sigma, SAB3500285, USA), LAMP2A (Abcam, ab125028, UK), a rabbit anti-myosin-VIIa antibody (Proteus Bioscience, 25–6790, UK), αII-spectrin (clone D8BI7, BioLegend, 803201, USA), a mouse anti-myosin-VIIa antibody (Santa Cruz Biotechnology, sc-74, 516AC, USA), cleaved caspase 3 (Cell Signaling Technology, 9664, USA), cleaved PARP (Cell Signaling Technology, 9544, USA), DAPI (Abcam, ab104139, UK), GAPDH (Cell Signaling Technology, 5174, USA), β-actin (Cell Signaling Technology, 4970, USA), cisplatin (Sigma, P4394, USA), trehalose (Sigma, T9531, USA), 3- methyladenine (3-MA; MCE, HY-19312, USA), rapamycin (MCE, HY10219, USA), hydroxychloroquine (HCQ) (MCE, HY-B1370, USA), and cyclosporin A (CsA) (MCE, HY-B0579, USA).

Techniques: Translocation Assay, Western Blot, Expressing, CCK-8 Assay, Control