negative shrna control shctrl  (Addgene inc)

Journal: Advanced Science

Article Title: A Mammalian Conserved Circular RNA CircLARP1B Regulates Hepatocellular Carcinoma Metastasis and Lipid Metabolism

doi: 10.1002/advs.202305902

Figure Lengend Snippet: CircLARP1B destabilizes LKB1 mRNA via perturbing HNRNPD. a–d) Stability assay of LKB1 mRNA and the steady levels of LKB1 protein (examined by western blotting) in human PLC cells (a,b) and murine Hepa1‐6 cells (c,d) treated siRNA against HNRNPD. siNC, negative control siRNA with scrambled sequences. e) Overall experimental strategy of eIF4E and eIF4G1 RIP assays. RNase I is introduced to digest unprotected RNAs across the IP procedure. Western blots showing HNRNPD knockdown efficiency and efficient IPs of eIF4E and eIF4G1 in PLC cells treated with siRNA against HNRNPD (right). ACTB protein acted as the loading control. f) Primers against various regions of LKB1 mRNA (top) and enrichment of LKB1 mRNA regions with eIF4E or eIF4G1 by RT‐qPCR (bottom). g,h) Stability assay of LKB1 mRNA (g) and the steady levels of LKB1 protein (h) in WT or circLARP1B‐Def PLC cells. i,j) Stability assay of Lkb1 mRNA (i) and the steady levels of Lkb1 protein (j) in Hepa1‐6 cells treated with shcircLARP1B or shCtrl. shCtrl, shRNA control that generates siRNA of scrambled sequences; shcircLARP1B, shRNA against the murine circLARP1B BSJ. k) In vitro competing assay of purified HNRNPD protein for equal moles of in vitro synthesized circLARP1B and LKB1 3′ UTR. An illustration of the assay was shown (top). Semiquantitative RT‐PCR gels and RT‐qPCR for circLARP1B and LKB1 3′ UTR were indicated (bottom). LKB1 3′ UTR, in vitro transcribed LKB1 3′ UTR fragments with the HNRNPD binding sequence. l) Association of LKB1 mRNA examined by RT‐qPCR of HNRNPD RIP in PLC cells treated with oligodeoxynucleotide antisense to two HNRNPD binding motifs in circLARP1B (ODN‐AS) or the control (ODN‐Ctrl, ODN with scrambled sequences). Two motifs with reverse complementary sequences were indicated with underlines. PS, phosphorothioate; 2′‐OMe, 2′‐ O ‐methyl. Western blot images indicate successful IP of HNRNPD protein. Enrichment, normalized to IgG. m) Western blotting and the corresponding quantification showing the steady level of LKB1 protein in PLC cells transfected with ODN‐AS or ODN‐Ctrl. (b,d,h,j,m) The grayscale statistics of western blotting were performed by ImageJ. (a–d,f–m) Data are shown as mean ± SD from three independent experiments. (a,c,g,i) P ‐values by two‐way ANOVA test. (b,d,f,h,j,l,m) P ‐values by two‐tailed unpaired Student's t ‐test.

Article Snippet: The shRNA against the BSJ of human or murine circLARP1B was cloned into the vector pLKO.1 (Sigma) and the negative‐control shRNA (shCtrl, MFCD07785395) was obtained from the MISSION shRNA Library (Sigma).

Techniques: Stability Assay, Western Blot, Negative Control, Quantitative RT-PCR, shRNA, In Vitro, Purification, Synthesized, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Sequencing, Protein Enrichment, Transfection, Two Tailed Test

Journal: Advanced Science

Article Title: A Mammalian Conserved Circular RNA CircLARP1B Regulates Hepatocellular Carcinoma Metastasis and Lipid Metabolism

doi: 10.1002/advs.202305902

Figure Lengend Snippet: CircLARP1B deficiency in mice causes liver changes attributable to Lkb1 surplus. a) Strategy of knockout (KO) reverse‐complementary sequence in mouse Larp1b intron 4 using CRISPR‐Cas9. PCR products of mouse genotyping are shown. WT, wildtype; circLARP1B −/− , KO of the intronic reverse‐complementary sequences; B1, mouse B1 repeat. b) RT‐qPCR analysis of the steady levels and nascent levels (with nuclear run‐on assay) of circLARP1B and Larp1b mRNA in WT and circLARP1B −/− mouse liver. Data are from three independent experiments. c) Representative Oil Red O staining and the quantification in WT and circLARP1B −/− mouse liver ( N = 5 per group). Nuclei stained with hematoxylin (blue). Scale bar: 50 µm. d) Representative IHC staining and quantification of the proteins in liver from WT and circLARP1B −/− mice ( N = 5 per group). Scale bar: 50 µm. AOD, average optical density. e) Western blot of the indicated proteins in livers from WT or circLARP1B −/− mice. f) Representative Lkb1 IHC staining in livers from WT or circLARP1B −/− mice with the intravenous tail injection of AAV8‐shCtrl or AAV8‐shLkb1 for three weeks ( N = 5 per group). Scale bar: 50 µm. g) Representative Oil Red O staining in liver from WT or circLARP1B −/− mice with AAV8‐shCtrl or AAV8‐shLkb1 injection ( N = 5 per group). shCtrl, negative control shRNA construct that gives rise to siRNA with scrambled sequences. Nuclei stained with hematoxylin (blue). Scale bar: 50 µm. h) Representative IHC staining of the indicated proteins in liver from WT or circLARP1B −/− mice with AAV8‐shCtrl or AAV8‐shLkb1 injection ( N = 5 per group). Scale bar: 50 µm. i) Western blot of the indicated proteins in livers from WT or circLARP1B −/− mice with AAV8‐shCtrl or AAV8‐shLkb1 injection. Data are shown as mean ± SD. (c,g) Lipid level is defined as the percentage of Oil Red positive area calculated by Image‐Pro plus. (d,f,h) The IHC signal is defined as the average optical density (AOD) calculated by ImageJ. (e,i) The grayscale statistics of western blotting was performed by ImageJ. (c–i) All animals were kept in a controlled environment (23–25 °C with a 12‐h light‐dark cycle and lights on at 8:00 AM), on normal diet feeding with free access to water; all data were from mice at 9:00 AM. (b–d,f–h) P ‐values by two‐tailed unpaired Student's t ‐test.

Article Snippet: The shRNA against the BSJ of human or murine circLARP1B was cloned into the vector pLKO.1 (Sigma) and the negative‐control shRNA (shCtrl, MFCD07785395) was obtained from the MISSION shRNA Library (Sigma).

Techniques: Knock-Out, Sequencing, CRISPR, Quantitative RT-PCR, Nuclear Run-on Assay, Staining, Immunohistochemistry, Western Blot, Injection, Negative Control, shRNA, Construct, Two Tailed Test

Journal: Signal transduction and targeted therapy

Article Title: A20 promotes colorectal cancer immune evasion by upregulating STC1 expression to block "eat-me" signal.

doi: 10.1038/s41392-023-01545-x

Figure Lengend Snippet: Fig. 3 A20 inhibited antitumor immune response in vivo. a The expression of A20 in mice CT26-luc-GFP cells. b The cell proliferation of CT26- luc-GFP cells detected by CCK8 kit, n = 4. c The experimental scheme of the animal study. d The in vivo images of mice tumors with different treatments were detected by the IVIS bioluminescence imaging system, n = 7. e Statistical analysis of total flux from the IVIS bioluminescence images. f The representative images of the metastatic nodes in lung from BALB/C mice. g The survival curve of mice, n = 7. h The experimental scheme of the animal study. i The images of tumors excised from BALB/C mice at the end of experiments, n = 6. j Tumor weights of the four treatment groups, n = 6. k Tumor growth curves of the four treatment groups, n = 6. l The images of tumors excised from BALB/C mice at the end of experiments, n = 8. m Tumor weights of the four treatment groups, n = 8. n Tumor growth curves of the four treatment groups, n = 8. o–v The infiltration of CD3 (+) (X400), CD8 (+) (X400), CD4 (+) (X200), and Granzyme B(+) (X400) T cells detected by immunohistochemical staining. p-value was calculated by one-way ANOVA analysis. Shctrl+IgG, n = 7, Shctrl+αPD-1, n = 5, A20sh4+IgG, n = 7, A20sh4+αPD-1, n = 3. w–y Flow cytometry analysis of CD8 (+) and CD4 ( + ) T cells of mice spleens (n = 4 per group). Data was represented as the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant

Article Snippet: The shRNAs of A20 or STC1 and a negative shRNA control (shctrl) were cloned into pSIH-H1-puro lentivector (Addgene, USA).

Techniques: In Vivo, Expressing, Imaging, Immunohistochemical staining, Staining, Flow Cytometry