Journal: bioRxiv

Article Title: Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

doi: 10.1101/819961

Figure Lengend Snippet: (A) THZ-P1-2 inhibits off-target kinases identified in the DiscoverX KINOMEScan panel to varying degrees. In vitro kinase assays were performed by Invitrogen using the Adapta assay. (B) A streptavidin pulldown in HEK293T lysate shows little to no engagement of C-ABL and BRK by dtb-THZ-P1-2. (C) THZ-P1-2 shows mild antiproliferative activity on BCR-ABL addicted cell lines. Two cell lines containing BCR-ABL translocations, K562 and KU812F, were treated with THZ-P1-2 or known BCR-ABL inhibitors imatinib, nilotinib and dasatinib for 72h and assayed using Cell-Titer Glo. (D) THZ-P1-2 does not bind to and engage off-targets such as JNK, IRAK1, PKN3, CDK7 and CDK12 despite originating from the same core scaffold as inhibitors of these targets. Competitive streptavidin pulldowns in HEK293T cells were conducted with DMSO, 1 μ M THZ-P1-2 and 1 μ M of each target’s corresponding inhibitor, followed by pulldown in lysate with 1 μ M of the corresponding biotinylated or desthiobiotinylated inhibitor. (E) THZ-P1-2 causes a slight increase in LAMP1 and LC3B protein levels, as observed with positive control compounds bafilomycin A1 and chloroquine. HeLa cells were cultured for 24h in DMEM media supplemented with either 0.3% (serum-starved conditions) or 10% FBS. Cells were treated with DMSO, varying concentrations of THZ-P1-2 and single doses of bafilomycin A1 and chloroquine, harvested after 24h, and analyzed by Western blot. Related to .

Article Snippet: The following antibodies were used in this study: PI5P4Kα (5527; Cell Signaling), PI5P4Kβ (9694; Cell Signaling), PI5P4Kγ (HPA058551 ; Sigma-Aldrich),PI4P5Kα (9693; Cell Signaling), PI4P5Kβ (12541-1-AP; Proteintech), PI4P5Kγ (3296; Cell Signaling), PIKfyve (MABS522; EMD Millipore), c-Abl (sc-56887; Santa Cruz), Brk (ab137563; Santa Cruz), IRAK1 (4504; Cell Signaling), PKN3 (NBP1-30102; Novus Biologicals), SAP/JNK (9252; Cell Signaling), CDK7 (2916; Cell Signaling), CDK12 (11973; Cell Signaling), LAMP1 (9091; Cell Signaling), LC3B (2773; Cell Signaling), tubulin (3873; Cell Signaling).

Techniques: In Vitro, Activity Assay, Positive Control, Cell Culture, Western Blot

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A Venn diagram showing potential target genes of miR‐59 predicted by target prediction algorithms (TargetScan and miRanda) and GO analyze of miRNA sequence. B, C HGAFDFN003 cells were transfected with anti‐miR‐ctrl or anti‐miR‐59 for 5 days, the mRNA level of miR‐59‐targeting potential genes was assessed by qPCR (B). The protein expression of HMGA1 and HMGA2 was detected by western blot (C). D, E After transfection with anti‐miR‐ctrl or anti‐miR‐59 in progerin‐expressing CRL‐1474 cells, the protein expression (D) and mRNA level (E) of HMGAs were detected. F, G Western blot analysis of the expression of HMGAs (F) and qPCR analysis of the mRNA levels (G) in miR‐ctrl or miR‐59 mimics‐transfected CRL‐1474 cells. H, I Comparing HGPS patient cells (HGAFDFN003 p22 and HGAFDFN167 p20) with control (CRL‐1474 p26), the protein expression (H) and mRNA level (I) of HMGAs were assessed. J, K The protein (J) and mRNA (K) level of HMGAs were detected in 7, 88 and 92‐year‐old cells. L, M Transfected anti‐miR‐ctrl or anti‐miR‐59 in 88‐year‐old cells, the level of HMGAs was detected by Western blot (L) and qPCR (M). N, O The binding sites of miR‐59 on HMGAs 3′ UTR were analyzed by TargetScan and miRanda. Luciferase experiments with the wild‐type and the mutated 3′ UTR of HMGA1 (N)/HMGA2 (O). Data information: Each experiment was repeated at least three times. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Sequencing, Transfection, Expressing, Western Blot, Binding Assay, Luciferase

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A Western blot showing expression of HMGA1 and HMGA2 in progerin‐expressing CRL‐1474 cells relative to control cells. B The mRNA level of HMGA1 and HMGA2 was assessed in progerin expressed CRL‐1474 cells relative to control by qPCR. C, D Progerin‐expressing CRL‐1474 cells were transfected with HA‐control or HA‐HMGA2 in the same method to HMGA1. SA‐β‐gal staining was performed. Scale bars: 100 μm (C). The level of cyclin A2, lamin B1 was detected by western blot (D). E, F Transfected His‐HMGA1 or HA‐HMGA2 in progerin‐expressing IMR90 cells, the percentage of SA‐β‐gal positive cells was calculated, Scale bars: 100 μm (E). The level of cyclin A2 and lamin B1 was detected by Western blot (F). G CRL‐1474 cells were infected with control shRNA (shCtrl), HMGA1 shRNA (shHMGA1#1 or #2) or HMGA2 shRNA (shHMGA2#1 or #2), the efficiency of shRNA was detected by Western blot. H CRL‐1474 cells were infected with control shRNA (shCtrl), HMGA1 shRNA (shHMGA1) or HMGA2 shRNA (shHMGA2) for 5 days, the level of cyclin A2, lamin B1 was detected by Western blot. I HGAFDFN003 cells were transfected with anti‐miR‐59 or anti‐miR‐59/shHMGAs for 5 days, the mRNA level of miR‐59‐targeting potential genes was assessed. Data information: Each experiment was repeated at least three times. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Western Blot, Expressing, Transfection, Staining, Infection, shRNA

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A–C In Flag‐HMGA1 or Flag‐HMGA2 infected CRL‐1474 and HGPS cells (HGAFDFN003), the level of cyclin A2, lamin B1 was detected by western blot (A). SA‐β‐gal staining were performed, the percentage of SA‐β‐gal positive cells was calculated. Scale bars: 100 μm (B). Expression of Ki67 was analyzed by immunofluorescence. Scale bars: 50 μm (C). D, E Progerin‐expressing CRL‐1474 cells were transfected with His‐control or His‐HMGA1. SA‐β‐gal staining was performed. The percentage of SA‐β‐gal positive cells was calculated. Scale bars: 100 μm (D). The level of cyclin A2, lamin B1 was detected by Western blot (E). F–H Infection with control shRNA (shCtrl) or HMGA1/HMGA2 shRNA (shHMGA1 and shHMGA2) in anti‐miR‐59‐transfected HGPS cells (HGAFDFN003), cells were subjected to SA‐β‐gal staining. Scale bars: 100 μm (F). Cells were subjected to immunofluorescence using anti‐Ki67(red) antibody (left). The percentage of the Ki67 positive cells was calculated. Scale bars: 50 μm (G). The level of cyclin A2, lamin B1, HMGA1, and HMGA2 was detected by western blot (H). Data information: Each experiment was repeated at least three times. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Infection, Western Blot, Staining, Expressing, Immunofluorescence, Transfection, shRNA

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A Interaction of endogenous HMGA1 with HMGA2 and TFIIH4 complex was analyzed by Co‐IP assay using anti‐HMGA1 antibody in H1299 cells. Pulled‐down protein complexes were analyzed. Benzonase (B) was added in cell lysate. B Interaction of endogenous HMGA2 with HMGA1 and TFIIH4 complex was analyzed by Co‐IP assay using anti‐HMGA2 antibody in H1299 cells. Pulled‐down protein complexes were analyzed. C HMGA1‐associated proteins from HEK293T cells expressing FLAG‐HMGA1 were IP with anti‐Flag antibody. The protein bands were analyzed by mass spectrometry. Representative peptide fragments of HMGA1 associated proteins and peptide coverage of the indicated proteins were shown. D Interaction of endogenous CDK7 with HMGAs, XPB and TFIIH4 was analyzed by Co‐IP assay using anti‐CDK7 antibody in H1299 cells. Pulled‐down protein complexes were analyzed. E‐F HEK293T cells were transfected with Flag‐HMGA1 (E) or CDK7 (F). HMGA1 or CDK7 was IP with anti‐Flag antibody and anti‐CDK7 antibody. G HEK293T cell lysates were incubated with GST or GST‐HMGA1 sepharose beads. Pulled‐down protein complexes were analyzed. H Scheme of HMGA1 wild‐type structure, including AT hooks (AT) and acidic tail (C tail), and deletion mutants used for immunoprecipitation experiments below. I HEK293T cells were transfected with Flag‐HMGA1 deletion mutants. IP of Flag was performed. Pulled‐down protein complexes were analyzed. J HEK293T cell lysates were incubated with GST or GST‐HMGA1 deletion mutants sepharose beads. Recombinant proteins from pull‐down assays visualized by silver staining and Western blot. K, L Progerin‐expressing H1299 cells were transfected with His‐control or His‐HMGA1. Immunoprecipitation of RNA Pol II (K) or CDK7 (L) with anti‐RNAPII antibody or anti‐CDK7 antibody was performed, respectively. Pulled‐down protein complexes were analyzed. M, N RNAPII, the RNAPII Ser2P, and RNAPII Ser5P in HGAFDFN003 cells (M) and 88, 92‐year‐old cells (N) compared with CRL‐1474 cells were detected by Western blot. O Progerin‐expressing CRL‐1474 cells were transfected with His‐control or His‐HMGA1. Indicated proteins and modifications were detected by western blot. P Infection with siCtrl or siCDK7 in Flag‐HMGA1‐transfected HGPS cells (HGAFDFN003), cyclin A1, RNAPII, the RNAPII Ser2P and RNAPII Ser5P was detected. Q Infection with control shRNA (shCtrl) or HMGA1 shRNA (shHMGA1) in anti‐miR‐59‐transfected HGAFDFN003 cells. Indicated proteins and modifications were detected by western blot. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Co-Immunoprecipitation Assay, Expressing, Mass Spectrometry, Transfection, Incubation, Immunoprecipitation, Recombinant, Silver Staining, Western Blot, Infection, shRNA

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A Cell lysate used in endogenous IPs was run out on an ethidium bromide‐stained 1% agarose gel, respectively. Lanes: 1, no sample; 2, IP in the absence of Benzonase (Ben.); 3, IP in the presence of Ben. B Interactions between HMGA1 and HMGA2 were detected by co‐immunoprecipitation (Co‐IP) assay with anti‐Flag and anti‐HA in HEK‐293T cells which transfected with Flag‐HMGA1 or HA‐HMGA2, respectively. Pulled‐down protein complexes were analyzed. C HEK293T cells were transfected with Flag‐HMGA2. Immunoprecipitation of Flag with anti‐Flag antibody was performed. Pulled‐down protein complexes were analyzed. D HEK293T cells were transfected with Flag‐HMGA1, and Flag peptide was added in the Flag‐HMGA1 expression extracts. Immunoprecipitation of Flag with anti‐Flag antibody was performed. Pulled‐down protein complexes were analyzed. E HEK293T cell lysates were incubated with GST or GST‐HMGA1 sepharose beads, and GSH was added in the GST‐HMGA1 expression extracts. Pulled‐down protein complexes were analyzed. F HEK293T cell lysates were incubated with GST or GST‐HMGA2 sepharose beads. Pulled‐down protein complexes were analyzed. G, H Progerin‐expressing H1299 cells were transfected with HA‐control or HA‐HMGA2. Immunoprecipitation of RNAPII or CDK7 with anti‐RNAPII antibody or anti‐CDK7 antibody was performed. Pulled‐down protein complexes were analyzed by Western blot. I Western blot analysis of RNAPII, the RNAPII Ser2P and RNAPII Ser5P in progerin‐expressing CRL‐1474 cells. J In Flag‐HMGA1 or Flag‐HMGA2 infected HGPS cells (HGAFDFN003), indicated proteins and modifications was detected by Western blot. K Cells were subjected to immunofluorescence using anti‐RNAPII Ser5P (red) antibody and anti‐Flag (green) (left). The percentage of RNAPII Ser5P positive cells was calculated (right). Scale bars: 25 μm. L HEK293T cells were infected with control siCtrl, siCDK7#1 or #2, the efficiency of siRNA was detected. M Infection with siCtrl or siCDK7#1 in Flag‐HMGA1‐transfected HGPS cells (HGAFDFN003), the percentage of SA‐β‐gal positive cells was calculated. Data information: Each experiment was repeated at least three times. Data are presented as the mean ± SD (** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Staining, Agarose Gel Electrophoresis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Expressing, Incubation, Western Blot, Infection, Immunofluorescence

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A miR‐59 was detectehe d in heart, liver, lung, kidney, skin, muscle, and brain of Lmna G609G / G609G mice by qPCR at 12 weeks of life ( n = 5 for each group). B, C The mRNA level of Hmga1 (B) and Hmga2 (C) was detected in the heart, liver, lung, kidney, skin, muscle and brain of Lmna G609G / G609G mice by qPCR at 12 weeks of life ( n = 5 for each group). D Transfected anti‐miR‐Ctrl or anti‐miR‐59 in progerin‐expressing NIH3T3 cells. Western blot analysis of HMGAs. E, F The binding sites of miR‐59 on mice Hmgas 3′ UTR were analyzed. G, H Luciferase experiments with the wild‐type and the mutated 3′ UTR of Hmga1 and Hmga2. Data information: Each experiment was repeated at least three times. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Transfection, Expressing, Western Blot, Binding Assay, Luciferase

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: AAV9‐mCherry‐anti‐miR‐59 was injected into 8‐day‐old mice and detected at different points. A Running ability of male mice transduced with anti‐Ctrl versus anti‐miR‐59 at 12 weeks of life ( n = 5 for each group). B, C Masson staining showed the perivascular interstitial fibrosis in the heart and quadriceps muscle (B) (Scale bars, 50 μm), the adventitial fibrosis (blue areas), and the adventitial width of aorta (C). The boxes and arrows denoted the aortic adventitia. Scale bars, 200 μm (top), 50 μm (lower). D H&E staining in skin showed the epidermis and fat layer of skin. Scale bars, 150 μm. The boxes and arrows denoted epidermis and vertical bars indicated fat layer. E The level of miR‐59 was detected in the heart, liver, lung, kidney, skin, and muscle of Lmna G609G / G609G mice ( n = 5 for each group). F, G The mRNA level of Hmga1 (F) and Hmga2 (G) was detected in the heart, liver, lung, kidney, skin, and muscle of Lmna G609G / G609G mice ( n = 5 for each group). H Hmgas immunohistochemistry in skin of WT mice and Lmna G609G / G609G mice. Scale bars, 50 μm. Data information: Masson staining, H&E staining and immunohistochemistry were shown at 14 weeks of life, n = 5 for each group. Each experiment was repeated at least three times. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Injection, Transduction, Staining, Immunohistochemistry

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A Venn diagram showing potential target genes of miR‐59 predicted by target prediction algorithms (TargetScan and miRanda) and GO analyze of miRNA sequence. B, C HGAFDFN003 cells were transfected with anti‐miR‐ctrl or anti‐miR‐59 for 5 days, the mRNA level of miR‐59‐targeting potential genes was assessed by qPCR (B). The protein expression of HMGA1 and HMGA2 was detected by western blot (C). D, E After transfection with anti‐miR‐ctrl or anti‐miR‐59 in progerin‐expressing CRL‐1474 cells, the protein expression (D) and mRNA level (E) of HMGAs were detected. F, G Western blot analysis of the expression of HMGAs (F) and qPCR analysis of the mRNA levels (G) in miR‐ctrl or miR‐59 mimics‐transfected CRL‐1474 cells. H, I Comparing HGPS patient cells (HGAFDFN003 p22 and HGAFDFN167 p20) with control (CRL‐1474 p26), the protein expression (H) and mRNA level (I) of HMGAs were assessed. J, K The protein (J) and mRNA (K) level of HMGAs were detected in 7, 88 and 92‐year‐old cells. L, M Transfected anti‐miR‐ctrl or anti‐miR‐59 in 88‐year‐old cells, the level of HMGAs was detected by Western blot (L) and qPCR (M). N, O The binding sites of miR‐59 on HMGAs 3′ UTR were analyzed by TargetScan and miRanda. Luciferase experiments with the wild‐type and the mutated 3′ UTR of HMGA1 (N)/HMGA2 (O). Data information: Each experiment was repeated at least three times. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Sequencing, Transfection, Expressing, Western Blot, Binding Assay, Luciferase

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A Western blot showing expression of HMGA1 and HMGA2 in progerin‐expressing CRL‐1474 cells relative to control cells. B The mRNA level of HMGA1 and HMGA2 was assessed in progerin expressed CRL‐1474 cells relative to control by qPCR. C, D Progerin‐expressing CRL‐1474 cells were transfected with HA‐control or HA‐HMGA2 in the same method to HMGA1. SA‐β‐gal staining was performed. Scale bars: 100 μm (C). The level of cyclin A2, lamin B1 was detected by western blot (D). E, F Transfected His‐HMGA1 or HA‐HMGA2 in progerin‐expressing IMR90 cells, the percentage of SA‐β‐gal positive cells was calculated, Scale bars: 100 μm (E). The level of cyclin A2 and lamin B1 was detected by Western blot (F). G CRL‐1474 cells were infected with control shRNA (shCtrl), HMGA1 shRNA (shHMGA1#1 or #2) or HMGA2 shRNA (shHMGA2#1 or #2), the efficiency of shRNA was detected by Western blot. H CRL‐1474 cells were infected with control shRNA (shCtrl), HMGA1 shRNA (shHMGA1) or HMGA2 shRNA (shHMGA2) for 5 days, the level of cyclin A2, lamin B1 was detected by Western blot. I HGAFDFN003 cells were transfected with anti‐miR‐59 or anti‐miR‐59/shHMGAs for 5 days, the mRNA level of miR‐59‐targeting potential genes was assessed. Data information: Each experiment was repeated at least three times. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Western Blot, Expressing, Transfection, Staining, Infection, shRNA

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A–C In Flag‐HMGA1 or Flag‐HMGA2 infected CRL‐1474 and HGPS cells (HGAFDFN003), the level of cyclin A2, lamin B1 was detected by western blot (A). SA‐β‐gal staining were performed, the percentage of SA‐β‐gal positive cells was calculated. Scale bars: 100 μm (B). Expression of Ki67 was analyzed by immunofluorescence. Scale bars: 50 μm (C). D, E Progerin‐expressing CRL‐1474 cells were transfected with His‐control or His‐HMGA1. SA‐β‐gal staining was performed. The percentage of SA‐β‐gal positive cells was calculated. Scale bars: 100 μm (D). The level of cyclin A2, lamin B1 was detected by Western blot (E). F–H Infection with control shRNA (shCtrl) or HMGA1/HMGA2 shRNA (shHMGA1 and shHMGA2) in anti‐miR‐59‐transfected HGPS cells (HGAFDFN003), cells were subjected to SA‐β‐gal staining. Scale bars: 100 μm (F). Cells were subjected to immunofluorescence using anti‐Ki67(red) antibody (left). The percentage of the Ki67 positive cells was calculated. Scale bars: 50 μm (G). The level of cyclin A2, lamin B1, HMGA1, and HMGA2 was detected by western blot (H). Data information: Each experiment was repeated at least three times. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Infection, Western Blot, Staining, Expressing, Immunofluorescence, Transfection, shRNA

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A Interaction of endogenous HMGA1 with HMGA2 and TFIIH4 complex was analyzed by Co‐IP assay using anti‐HMGA1 antibody in H1299 cells. Pulled‐down protein complexes were analyzed. Benzonase (B) was added in cell lysate. B Interaction of endogenous HMGA2 with HMGA1 and TFIIH4 complex was analyzed by Co‐IP assay using anti‐HMGA2 antibody in H1299 cells. Pulled‐down protein complexes were analyzed. C HMGA1‐associated proteins from HEK293T cells expressing FLAG‐HMGA1 were IP with anti‐Flag antibody. The protein bands were analyzed by mass spectrometry. Representative peptide fragments of HMGA1 associated proteins and peptide coverage of the indicated proteins were shown. D Interaction of endogenous CDK7 with HMGAs, XPB and TFIIH4 was analyzed by Co‐IP assay using anti‐CDK7 antibody in H1299 cells. Pulled‐down protein complexes were analyzed. E‐F HEK293T cells were transfected with Flag‐HMGA1 (E) or CDK7 (F). HMGA1 or CDK7 was IP with anti‐Flag antibody and anti‐CDK7 antibody. G HEK293T cell lysates were incubated with GST or GST‐HMGA1 sepharose beads. Pulled‐down protein complexes were analyzed. H Scheme of HMGA1 wild‐type structure, including AT hooks (AT) and acidic tail (C tail), and deletion mutants used for immunoprecipitation experiments below. I HEK293T cells were transfected with Flag‐HMGA1 deletion mutants. IP of Flag was performed. Pulled‐down protein complexes were analyzed. J HEK293T cell lysates were incubated with GST or GST‐HMGA1 deletion mutants sepharose beads. Recombinant proteins from pull‐down assays visualized by silver staining and Western blot. K, L Progerin‐expressing H1299 cells were transfected with His‐control or His‐HMGA1. Immunoprecipitation of RNA Pol II (K) or CDK7 (L) with anti‐RNAPII antibody or anti‐CDK7 antibody was performed, respectively. Pulled‐down protein complexes were analyzed. M, N RNAPII, the RNAPII Ser2P, and RNAPII Ser5P in HGAFDFN003 cells (M) and 88, 92‐year‐old cells (N) compared with CRL‐1474 cells were detected by Western blot. O Progerin‐expressing CRL‐1474 cells were transfected with His‐control or His‐HMGA1. Indicated proteins and modifications were detected by western blot. P Infection with siCtrl or siCDK7 in Flag‐HMGA1‐transfected HGPS cells (HGAFDFN003), cyclin A1, RNAPII, the RNAPII Ser2P and RNAPII Ser5P was detected. Q Infection with control shRNA (shCtrl) or HMGA1 shRNA (shHMGA1) in anti‐miR‐59‐transfected HGAFDFN003 cells. Indicated proteins and modifications were detected by western blot. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Co-Immunoprecipitation Assay, Expressing, Mass Spectrometry, Transfection, Incubation, Immunoprecipitation, Recombinant, Silver Staining, Western Blot, Infection, shRNA

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A Cell lysate used in endogenous IPs was run out on an ethidium bromide‐stained 1% agarose gel, respectively. Lanes: 1, no sample; 2, IP in the absence of Benzonase (Ben.); 3, IP in the presence of Ben. B Interactions between HMGA1 and HMGA2 were detected by co‐immunoprecipitation (Co‐IP) assay with anti‐Flag and anti‐HA in HEK‐293T cells which transfected with Flag‐HMGA1 or HA‐HMGA2, respectively. Pulled‐down protein complexes were analyzed. C HEK293T cells were transfected with Flag‐HMGA2. Immunoprecipitation of Flag with anti‐Flag antibody was performed. Pulled‐down protein complexes were analyzed. D HEK293T cells were transfected with Flag‐HMGA1, and Flag peptide was added in the Flag‐HMGA1 expression extracts. Immunoprecipitation of Flag with anti‐Flag antibody was performed. Pulled‐down protein complexes were analyzed. E HEK293T cell lysates were incubated with GST or GST‐HMGA1 sepharose beads, and GSH was added in the GST‐HMGA1 expression extracts. Pulled‐down protein complexes were analyzed. F HEK293T cell lysates were incubated with GST or GST‐HMGA2 sepharose beads. Pulled‐down protein complexes were analyzed. G, H Progerin‐expressing H1299 cells were transfected with HA‐control or HA‐HMGA2. Immunoprecipitation of RNAPII or CDK7 with anti‐RNAPII antibody or anti‐CDK7 antibody was performed. Pulled‐down protein complexes were analyzed by Western blot. I Western blot analysis of RNAPII, the RNAPII Ser2P and RNAPII Ser5P in progerin‐expressing CRL‐1474 cells. J In Flag‐HMGA1 or Flag‐HMGA2 infected HGPS cells (HGAFDFN003), indicated proteins and modifications was detected by Western blot. K Cells were subjected to immunofluorescence using anti‐RNAPII Ser5P (red) antibody and anti‐Flag (green) (left). The percentage of RNAPII Ser5P positive cells was calculated (right). Scale bars: 25 μm. L HEK293T cells were infected with control siCtrl, siCDK7#1 or #2, the efficiency of siRNA was detected. M Infection with siCtrl or siCDK7#1 in Flag‐HMGA1‐transfected HGPS cells (HGAFDFN003), the percentage of SA‐β‐gal positive cells was calculated. Data information: Each experiment was repeated at least three times. Data are presented as the mean ± SD (** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Staining, Agarose Gel Electrophoresis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Expressing, Incubation, Western Blot, Infection, Immunofluorescence

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A–C Progerin‐expressing CRL‐1474 cells were transfected with His‐control or His‐HMGA1. Heatmap showing differential gene expression as determined by RNA‐seq in HGPS cells, P < 0.05. Lowly expressed genes are shown in blue, highly expressed ones in red (A). Venn diagram showing the number of overlapping genes between HGPS cells versus control cells and His‐HMGA1‐transfected HGPS cells versus His‐control‐transfected HGPS cells (B). GO enrichment analysis of HMGA1‐regulated genes (C). D, E The mRNA level of cell cycle genes in HGPS patient cells (HGAFDFN003 and HGAFDFN167) (D) and 88‐ and 92‐year‐old cells (E) was assessed by qPCR. F HGAFDFN003 cells were infected with His‐HMGA1 and the mRNA level of cell cycle genes was assessed by qPCR. G Infection with control shRNA (shCtrl) or HMGA1 shRNA (shHMGA1) in anti‐miR‐59‐transfected HGAFDFN003 cells. The mRNA level of cell cycle genes was assessed by qPCR. H–J Progerin‐expressing CRL‐1474 cells were transfected with His‐control or His‐HMGA1. ChIP–qPCR analyses for HMGA1 (H), CDK7 (I), ratio of RNAPII Ser5P/RNAPII (J). Data information: Each experiment was repeated at least three times. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Expressing, Transfection, RNA Sequencing Assay, Infection, shRNA

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: Progerin‐expressing CRL‐1474 cells were transfected with His‐control or His‐HMGA1. A A heatmap showing differential gene expression of the cell cycle by RNA‐seq in HGPS cells, P < 0.05. Lowly expressed genes are shown in blue, highly expressed ones in red. B The mRNA level of cell cycle genes was assessed by qPCR. C, D ChIP–qPCR analyses for RNAPII Ser5P (C) and RNAPII (D). Data information: Each experiment was repeated at least three times. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Expressing, Transfection, RNA Sequencing Assay

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: A miR‐59 was detectehe d in heart, liver, lung, kidney, skin, muscle, and brain of Lmna G609G / G609G mice by qPCR at 12 weeks of life ( n = 5 for each group). B, C The mRNA level of Hmga1 (B) and Hmga2 (C) was detected in the heart, liver, lung, kidney, skin, muscle and brain of Lmna G609G / G609G mice by qPCR at 12 weeks of life ( n = 5 for each group). D Transfected anti‐miR‐Ctrl or anti‐miR‐59 in progerin‐expressing NIH3T3 cells. Western blot analysis of HMGAs. E, F The binding sites of miR‐59 on mice Hmgas 3′ UTR were analyzed. G, H Luciferase experiments with the wild‐type and the mutated 3′ UTR of Hmga1 and Hmga2. Data information: Each experiment was repeated at least three times. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Transfection, Expressing, Western Blot, Binding Assay, Luciferase

Journal: The EMBO Journal

Article Title: Anti‐hsa‐miR ‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

doi: 10.15252/embj.2022110937

Figure Lengend Snippet: AAV9‐mCherry‐anti‐miR‐59 was injected into 8‐day‐old mice and detected at different points. A Running ability of male mice transduced with anti‐Ctrl versus anti‐miR‐59 at 12 weeks of life ( n = 5 for each group). B, C Masson staining showed the perivascular interstitial fibrosis in the heart and quadriceps muscle (B) (Scale bars, 50 μm), the adventitial fibrosis (blue areas), and the adventitial width of aorta (C). The boxes and arrows denoted the aortic adventitia. Scale bars, 200 μm (top), 50 μm (lower). D H&E staining in skin showed the epidermis and fat layer of skin. Scale bars, 150 μm. The boxes and arrows denoted epidermis and vertical bars indicated fat layer. E The level of miR‐59 was detected in the heart, liver, lung, kidney, skin, and muscle of Lmna G609G / G609G mice ( n = 5 for each group). F, G The mRNA level of Hmga1 (F) and Hmga2 (G) was detected in the heart, liver, lung, kidney, skin, and muscle of Lmna G609G / G609G mice ( n = 5 for each group). H Hmgas immunohistochemistry in skin of WT mice and Lmna G609G / G609G mice. Scale bars, 50 μm. Data information: Masson staining, H&E staining and immunohistochemistry were shown at 14 weeks of life, n = 5 for each group. Each experiment was repeated at least three times. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001). P ‐values were calculated by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: The following antibodies were used: antibodies against progerin (ab66587; Abcam); HMGA1 (#7777 for WB; Cell Signaling Technology, ab4078 for ChIP; and ab129153 for IHC; Abcam); HMGA2 (#8179 for WB; Cell Signaling Technology and GB111395 for IHC; Servicebio, Wuhan, China); β‐actin (A1978; Sigma, St. Louis, MO, USA); Ki67 (GTX16667; GeneTex); lamin B1 (ab16048; Abcam); cyclin A2 (ab181591; Abcam); Flag (M20008; Abmart); His (M30111; Abmart); HA ( SAB2702196 ; Sigma); RNAPII (ab817; Abcam); XPB (ab190698; Abcam); CDK7 (2916S; Cell Signaling Technology); TFIIH4 (DF8857; Affinity); RNAPII Ser2P (ab5095; Abcam); and RNAPII Ser5P (ab193467; Abcam).

Techniques: Injection, Transduction, Staining, Immunohistochemistry